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Involvement of BMP6 and E2F5 in Skeletal Myogenesis


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1 INVOLVEMENT OF BMP6 AND E2F5 IN SKELETAL MYOGENESIS By JENNELLE ROBIN MCQUOWN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Jennelle Robin McQuown

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3 To Bill and my family for all of their love and support. I would be lost without you guys! All of my love, J.

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4 ACKNOWLEDGMENTS I would like to thank my major professor, Dr Sally Johnson, for servi ng as my supervisory committee chair. I would also like to thank my committee members (Dr S. Paul Oh, Dr. Lori Warren, and Dr. Lokenga Badinga) for their suppor t and advice. I could not have asked for a more supportive and encouraging group. I would al so like to thank the members of Dr. Sally Johnsons lab for their assistance with my dissertation project. I also count myself lucky to have a great group of friends and family who served as my sounding board, my cheering squad, and my source of inspiration. The completion of my Ph.D. has definitely b een a family goal. Without the love and support of my family, especially my mom and dad, Linda and Dan McQuown, I might have not achieved this accomplishment. Another huge so urce of encouragement and support was William Pittsley. I feel that I share this degree with him since he has gone through every triumph, every failure, and every fear and hope al ong with me, and I am eternally grateful. I also send prayers up to my Grandpa Cossentine and Grandpa McQuown whom werent able to see me finish this degree but I know that theyre both smiling down and sending me their love. For my Grandma McQuown and Grandma Cossentine, I am so thankf ul that they are here to encourage me, send me their love, and call me the first Dr. McQuown of the family! Finally, I would like to thank all of my wonderful friends. I f eel lucky to have such a great cheering squad!

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 LITERATURE REVIEW.......................................................................................................13 Introduction................................................................................................................... ..........13 Skeletal Muscle Biology........................................................................................................ .13 The SR and Transverse Tubule Sy stems within Skeletal M uscle...................................14 Major Fiber Type Classification......................................................................................15 Muscle Architecture........................................................................................................16 Skeletal Muscle Function................................................................................................19 Skeletal Muscle Development................................................................................................22 Myogenic Regulatory Factors (MRFs)............................................................................25 Satellite Cells................................................................................................................ ..........27 Satellite Cell Marker Proteins.........................................................................................28 c-Met.......................................................................................................................... ......29 Syndecans 3 and 4...........................................................................................................30 M-cadherin..................................................................................................................... .30 CD34........................................................................................................................... .....30 Pax7........................................................................................................................... ......31 Myocyte nuclear factor (MNF).......................................................................................32 Notch.......................................................................................................................... .....32 Growth Factor Effects on Satellite Cells................................................................................34 Insulin-like Growth Factor I (IGF-I)...............................................................................35 Fibroblast Growth Factors (FGFs)..................................................................................36 Hepatocyte Growth Factor (HGF)...................................................................................37 Transforming Growth Factor Beta (TGF ) Superfamily................................................38 Myostatin...................................................................................................................... ...39 Bone Morphogenic Proteins (BMPs)..............................................................................40 BMP Function in vivo : Lessons Learned from Knockouts............................................44 Current BMP6 Studies.....................................................................................................45 Ras/Raf in Skeletal Muscle..................................................................................................... 46 The Raf Family................................................................................................................. ......47

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6 E2F Family..................................................................................................................... ........48 E2F Signaling Mechanism and Involvement of Pocket Proteins....................................49 Cell Cycle Repression.....................................................................................................50 E2Fs and Pocket Proteins in Skeletal Muscle.................................................................51 2 MATERIALS AND METHODS...........................................................................................56 Myoblast Cell Culture.......................................................................................................... ...56 Growth Factor Treatment, a nd BrdU Pulsing and Fixation....................................................57 Plasmids and Transfections....................................................................................................5 7 BrdU Staining and BrdU Incorporation..................................................................................58 Immunofluorescent E2F5 Staining.........................................................................................58 RNA Isolation and Nylon Arrays...........................................................................................59 Western Blots.................................................................................................................. ........59 Alkaline Phosphatase Staining...............................................................................................60 p38 Inhibition Assays.......................................................................................................... ...61 Apoptosis Analysis............................................................................................................. ....61 Statistics..................................................................................................................... .............61 3 DIFFERENTIAL EXPRESSION OF TGF SUPERFAMILY MEMBERS DURING SKELETAL MYOGENESIS.................................................................................................63 Objective...................................................................................................................... ...........63 Differential Transcriptional Activity in Myoblasts versus Myofibers...................................63 Differentiation TGF Gene Expression in Myoblasts and Myofibers...................................64 Discussion..................................................................................................................... ..........65 4 IMPACT OF BMP6 ON SKELETAL MYOGENESIS.........................................................75 Objective...................................................................................................................... ...........75 Inhibition of Skeletal Myoge nic Differentiation by BMP6....................................................75 Dose-Dependent Effects of Recombinant BMP6 on Myoblasts............................................76 Induction of ALP Activity in Response to BMP6..................................................................76 BMP6 Induces Rapid Transdi fferentiation in Myoblasts.......................................................77 BMP6 Does Not Alter Proliferation Rates of Myoblasts.......................................................77 BMP6 is not Anti-Apoptotic...................................................................................................77 Discussion..................................................................................................................... ..........78 5 BMP6 SIGNALING DURI NG SKELETAL MYOGENESIS...............................................96 Objective...................................................................................................................... ...........96 Analysis of BMP Signaling Systems in Myoblasts................................................................96 Impact of Notch Inhibitor on BMP6-Med iated Inhibition of Differentiation........................98 Discussion..................................................................................................................... ..........99

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7 6 IMPACT OF E2F5 ON SKELETAL MYOGENESIS.........................................................113 Objective...................................................................................................................... .........113 BMP6 Treatment does not cause E2F5 Nuclear Accumulation in Myoblasts.....................113 Presence of E2F5 in Satellite Cell Position..........................................................................113 E2F5 Does Not Inhibit Myofiber Differentiation.................................................................114 pRb does not interact with E2F5 to Exert In hibitory Effects on Muscle Specific Activity.115 E2F5 is Transcriptionally Active..........................................................................................115 Discussion..................................................................................................................... ........116 7 SUMMARY AND CONCLUSIONS...................................................................................126 APPENDIX A GENE ARRAY LAYOUT AND TABLE............................................................................130 B SUMMARY OF ABBREVIATIONS..................................................................................137 LITERATURE CITED............................................................................................................... .141 BIOGRAPHICAL SKETCH.......................................................................................................180

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8 LIST OF TABLES Table page 1-1. Receptor and R-Smad specificity for TGF superfamily members......................................53 1-2. TGF superfamily transgenic knockouts..............................................................................54

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9 LIST OF FIGURES Figure page 3-1. Differential transcriptional activ ity in myoblasts versus myofibers......................................69 3-2. Intact RNA and cDNA probe synthesis.................................................................................70 3-3. Gene expression profiles in embr yonic and satellite cell myofibers......................................71 3-4. Differential gene expressi on in myoblasts and myofibers.....................................................72 3-5. Relative BMP gene expressi on in myoblasts and myofibers................................................73 3-6. BMP6 does not undergo autocrine gene regulation...............................................................74 4-1. Biochemical inhibition of skeletal myogenesis by BMP6.....................................................88 4-2. Inhibition of skel etal myogenesis by BMP6..........................................................................89 4-3. BMP6 dose response curve................................................................................................. ...90 4-4. BMP6 induction of alka line phosphatase activity.................................................................91 4-5. BMP6 does not induce alkaline phosphata se (ALP) activity in fibroblasts. .......................92 4-6. BMP6 induces rapid tran sdifferentiation in myoblasts.........................................................93 4-7. BMP6 treatment does not alter myoblast pr oliferation.........................................................94 4-8. BMP6 is not anti-apoptotic................................................................................................ .....95 5-1. Potential BMP6 signaling pathwa ys affecting skeletal myogenesis...................................106 5-2. Verification of BMP and TGF signaling axis...................................................................107 5-3. BMP6 treatment increases p38 phosphorylation..................................................................108 5-4. p38 inhibition and BMP6 treatment result in additive inhibition of muscle specific reporter activity.............................................................................................................. ..109 5-5. BMP6 and p38 signal through independe nt pathways to influence myogenic differentiation................................................................................................................ ...110 5-6. p38 signaling does not play a signi ficant role in transdifferentiation..................................111 5-7. BMP6 inhibition of differentiati on is mediated in part by Notch........................................112 6-1. BMP6 treatment does not cause E2F5 nuclear accumulation in myoblasts........................121

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10 6-2. Presence of E2F5 in satellite cell position...........................................................................122 6-3. E2F5 does not inhibit myogenic differentiation..................................................................123 6-4. pRb does not interact with E2F5 to exert inhibitory effects on mu scle specific activity....124 6-5. E2F5 is transcriptionally active......................................................................................... ..125 7-1. Proposed model of BMP6 on myogenic di fferentiation and inter action with Notch..........129

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INVOLVEMENT OF BMP6 AND E2F5 IN SKELETAL MYOGENESIS By Jennelle Robin McQuown May 2007 Chair: Sally E. Johnson Major: Animal Sciences An amazing dynamic exists within skeletal muscle that is required for the development and maintenance of the musculature system in res ponse to stimuli. A differential BMPand TGF responsiveness and gene expressi on profile in myoblasts versus myofibers, suggests that TGF superfamily members play distinct regulatory roles at specific st ages of myogenesis. Treatment of myoblasts with BMP6 results in an increase in alkaline phosphata se activity in a dosedependent and time-dependent manner. Exogenous BMP6 treatment results in inhibition of myoblast differentiation as observe d by significant inhibi tion of muscle repor ter activity, muscle specific protein synthesis, and myoblast fusion. BMP6 treatment does not alter proliferation rates or play an anti-apoptotic role in myoblasts. Inhibition of p38 activity and BMP6 treatment caused significant inhibition of TnI-Luc activity and muscle specific protein expression markers suggesting an additive effect through independent signaling ca scades. Inte restingly, the combination of BMP6 and Notch inhibition partiall y restores MyHC expression in fibers. This suggests that the myogenic inhibitory effect observe d in the presence of BMP6 is not a cell cycle effect and not a direct target of the Raf/MEK/ ERK signaling axis, but is partially mediated by functional Notch signaling. Therefore, BMP6-med iated inhibition of differentiation is regulated or partially controlled by the Notch signaling pa thway. Ectopic BMP6 treatment did not induce

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12 E2F5 nuclear accumulation in myoblasts further suggesting BMP6 is not causing a cell cycle effect. E2F5 is present in non-dividing cells in vivo outside the dystrophin border in the putative satellite cell position, although E2F5 did not imp act myogenic differentiation. Additionally, the inability of E2F5 to inhibit differentiation is not due to insufficient pocket protein function. Multiple signaling axes are key factors during Raf-imposed block to myogenic differentiation. Further understanding of the regene rative ability in skeletal muscle in response to stimuli would be useful for patients or animals that experience severe muscle trauma, and for individuals with skeletal muscle disorders, and would provide additional information for human therapeutic and agricultural applications that would be nefit from enhanced muscle mass.

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13 CHAPTER 1 LITERATURE REVIEW Introduction Within an organism, skeletal muscle functi ons in locomotor activity, postural behavior, and breathing. Skeletal muscle will undergo injury in response to direct trauma or as an indirect result of neurological dysfuncti on or genetic defects, and if not repaired, muscle mass and locomotion abilities may be lost, and death can possibly occur (Charg and Rudnicki, 2004). An amazing dynamic exists within skeletal muscle that is required for th e maintenance of the musculature in regards to the states of atrophy, injury, and subsequent repair. Understanding the regenerative ability of skeletal muscle in response to these stimuli are im portant areas of research. For instance, patients who experience se vere muscle trauma, individuals with genetic skeletal muscle disorders like muscular dystr ophy, and improving the recovery of astronauts experiencing a weightless environment would be nefit from such studies. Many changes to muscle physiology occur in an adaptive response to decreased or increas ed usage of skeletal muscle (Stein and Wade, 2005). The adaptive res ponses of decreased usag e includes a shift in myosin isoforms from slow to fast fiber type, replacement of protein with fat within the muscle, shifts in energy source for metabolism away from lipids to glucose, a loss of bone mineral density, and an increase in bone resorption (Sha ckelford et al., 2004; St ein and Wade, 2005). Skeletal Muscle Biology The mechanics of skeletal muscle contraction begin with a neural stimulus for contraction. This results in a generation of an action potential in a moto r neuron causing the release of acetylcholine into the synaptic cleft of the neuromuscular ju nction (NMJ). This released acetylcholine binds with the receptors on the moto r end-plate, producing an end plate potential, which leads to the depolarization that is conduct ed down the transverse tubules deep into the

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14 muscle fiber. Depolarization re sults in calcium being released from the sarcoplasmic reticulum (SR) (Crouch, 1985). In the resting state, myosin cross-bridges remain connected to actin in a weak binding state. When depolarization signa l reaches the SR, calcium is released into the sarcoplasm, which binds to troponin, causing a shift in troponin pos ition to uncover the active sites on actin. The energized or cocked myosin cross-bridge then forms a strong bond at the active sites on actin. Inorganic phosphate released from the myosin cross-bridge energizes the cross-bridge to allow it the ability to pull the actin molecule s. Cross-bridge movement is completed by the release of ADP from the myosin cross bridge. At this point, the myosin cross-bridges are in what is considered the strong bi nding state with actin. Attachment of ATP to myosin allows the cross-bridge to break the strong binding state and form a weak binding state. ATP is then broken down to ADP + Pi + energy. The energy is released and used to energize the myosin cross-bridges. This cycle can repeat as long as calcium and ATP are present. The cycle is stopped when the SR actively removes calci um from the sarcoplasm (Lieber, 2002). The SR and Transverse Tubule Syst ems within Skeletal Muscle Within the muscle, two membrane systems are pr esent to activate the filaments. These two systems are called the transverse tubule (T) system and the SR system. The T-system consists of invaginations in the surface membrane physically contiguous with the sarcolemma, or holes that extend into the fiber and transver sely crosses the long axis of th e fiber. The T-system conveys activation signals received from the motor neuron into the myofibrils. Since motor neurons are not in direct contact, the T-system helps in crease activation time ve rsus a simple diffusion system. The release of calcium switches on musc le activation and the removal of calcium from the myofilament cause muscle relaxation. Embe dded in the SR membrane are specific calcium channels and pumps that contro l this calcium release and upta ke. The SR envelops each

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15 myofibril to provide contact between the activatio n and force generation systems. The SR also contacts the T-system, thus acti ng as a middle man in skeletal mu scle activation and relaxation. The T and SR systems are arranged in a structur e called a triad, which is a single T tubule surrounded by two SR tubules located at each Z disk and the tria d functions as the interface between the extracellular and intracellular surfaces of th e fiber (Lieber, 2002). Additionally, the SR and T systems are linked by junctional feet structur es. Junctional feet are composed primarily of two components: ryanodine receptors (R yR) and dihydropyridine receptors (DHP). RyRs are voltage sensing pr oteins embedded in the SR that sense action potential traveling across the T system. DHPs are calcium release channels through which the myofilament receives activati ng calcium (Lieber, 2002). Major Fiber Type Classification Skeletal muscles can be classi fied as either red or white based on the major proportion of red or white fibers they contain. Few muscles are composed solely of one fiber type; most are of a heterogeneous composition. The red colorati on of red fibers is due to higher myoglobin content as compared to white fibers and the myoglobin allows for oxygen storage required for oxidative metabolism. Conversely, white fibers have a high content of glycolytic enzymes and low levels of oxidative enzyme activity (Smith, 1972). Another method of skeletal muscle fiber desi gnation is based upon cont raction rates: slowtwitch vs. fast-twitch fibers or t ype I vs. type II. There are four different adult myosin isoforms in skeletal muscle: type I, IIA, IIX(D), and IIB with type I and IIA designated as red muscle fibers and type IIX(D) and IIB designated as white muscle fibers (Brny, 1967; Roy et al., 1984). Slow-twitch fibers, whic h are also called Type I fibe rs, are characterized by higher number of mitochondria, larger mitochondria, a nd the mitochondria are located in two general areas: the subsarcolemma, and the intermyofibrillar. The mitochondr ia within Type I fibers are

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16 more efficient than in Type II fibers. For exam ple, in the subsarcolemma, the mitochondria are nine times more efficient in Type I and in the myofib rillar, they are five times more efficient in I. These fibers are surrounded by numerous capillaries and have a higher concentration of myoglobin, which gives the red muscle fiber types thei r visual color. Type I fibers have a large capacity for aerobic metabolism and can use ATP mo re efficiently than Type II without lactic acid buildup. The lipid c ontent is greater in red muscle fibers which serves as a metabolic fuel source and while these fibers contra ct slower, they can contract for a longer time. These fibers play a role in posture because they are less eas ily fatigued, as long as oxygen is available and due to their higher resistance to fatigue and allowance for a higher maximal oxygen uptake (VO2 max) they are selected for in endurance type training (Evans et al., 1994; Van Swearingen and Lance-Jones, 1995; Lieber, 2002). Fast twitch or Type II fibers are glycolytic and can produce for ce at a higher rate. This is called a phasic mode of action sin ce contraction occurs in short bur sts and these fibers are more easily fatigued. White fibers have a more exte nsively developed SR and T-tubule system along with more narrow Z disks versus white fibers. They also have fewer and smaller mitochondria than Type I and the glycolytic metabolism that predominates in these fibers can occur both aerobically or anaerobically. White fibers have a lower capillary density than red fibers. Functional implications demonstrate that Type II fibers are increas ed during strength training due to their ability to produce fo rce at a higher rate (Zhang and McLennan, 1998; Lieber, 2002). Muscle Architecture The anatomical and biomechanical properties of skeletal muscle determine the efficiency and overall ability of the muscle to generate fo rce and movement. The la rgest functional unit of contractile filaments is the myofibril, which is a string of sarcomeres. The muscle fiber is

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17 composed of myofibrils arranged side-by-side an d the groups of muscle fibers are sheathed in connective tissue termed the perimysium (Lieber, 2002). The number of sarcomeres within a fiber depe nds on the muscle fiber length and diameter of the fiber. They are the most important determinant of muscle fiber function. The total distance of myofibrillar shortening is equal to the sum of the shortening distance of each sarcomere. This allows for whole muscles to be able to shorten up to several centimeters even though each sarcomere can only shorten ~1 m giving the muscle a tremendous ability to adapt. Within the sarcomere are two major types of contractile filaments, the thick filaments and the thin filaments. These filaments are large polym ers of myosin and actin proteins. In thick filaments, which are the myosin-containing filame nt, a feathered appearance is observed with projections coming out at eith er end of the filament becau se one molecule rotates ~60 relative to molecules on either side. These filaments genera te the tension during musc le contraction and the thin filaments or actin-containing filament regul ates the tension generated (Gordon et al., 1966a; Gordon et al., 1996b; Lieber, 2002). Tension generation is also a function of the magnitude of the overlap between the actin and myosin filament s. Passive tension plays a role in providing resistance in the absence of muscle activation. Th e source of passive tensi on is due to the protein titin, which connects the thick myosin filaments end to end (Magid and Reedy, 1980; Labeit and Kolmerer, 1995). Striated skeletal muscle is composed of bundl es of enormous, multi-nucleated cells and the striations arise from a repeating pattern in th e myofibrils. Active interdigitations of these filaments produce muscle shortening and give muscle its striated appearance. Some characteristics of striated muscle are its ability to contract and generate tension when activated and its ability to return to its original length and form after contracti on or stretching ceases.

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18 These properties are primarily the result of separate sets of filament systems: contractile actin and myosin filaments and viscoelastic titin a nd intermediate filament s (Vigoreaux, 1994; Knupp et al., 2002). Within the thin filaments, approximately 5% of the myofibrillar protein is composed of troponin molecules, which are located approximate ly every seven or eight actin molecules along the actin filament. Troponins ar e the regulatory proteins responsib le for turning on contraction. Troponin is composed of three s ubunits: Tn-I, Tn-C, and Tn-T. Tn-I exerts an inhibitory influence on tropomyosin when calcium is not pres ent. Tn-C: binds calcium during contraction. Tn-T: binds troponin to tropomyosin. Tropomyosin is a long, rigid, and insoluble rod-shaped molecule that stretches along in close contact with each strand of the thin filament. Within each groove of the actin super-helix lays a strand of tropomyosin and a single molecule extends the length of seven G-actin molecule s (Lieber, 2002). The dark striations observed in skeletal muscle are a lattice of thick filaments termed the A band because it appears dark under a microscope. The light striations are termed I bands, which are regions of the myofibril containing only thin filaments and Z disks. The Z disks are the structure that attach to the thin filaments to act as actin-anchoring stru ctures and function as a common link to mechanically integrate contractile and elastic elements (S chroeter et al., 1996). They are also involved in transmission of activ e and passive forces a nd differences in Z band structure have been described for distinct cla sses of muscle and fiber types (Vigoreaux, 1994). Thin filaments extend from two adjacent Z lines to interdigitate with the thick filaments. Thick and thin filament arrays form the contractile sy stem and are the repeati ng unit in each myofibril (Bloom and Fawcett, 1968).

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19 Intermediate Filaments (IFs) are one of the th ree major classes of cytoskeletal proteins along with microtubules and microfilaments. The IF proteins and IF-associated proteins such as desmin, vimentin, and nestin, are involved in main taining the structural and functional integrity of all muscle types and align themselves in a head-to-tail dimer along th e central rod domain. For example, in the skeletal muscle injury model, nestin and vimentin IF proteins begin to be expressed 6 hours post-injury in myoblasts a nd their maximal expression was observed around 35 days post injury. Thereafter, vimentin expr ession ceases completely, whereas nestin is found to remain only in the sarcoplasm next to ne uromuscular and myotendi nous junctions. At 6-12 hours post-injury, desmin expression is upregulated and becomes the predominant IF protein in myofibers, coinciding with the appearance of crossstriations. Nestin and vimentin are essential during the early phases of myofiber regeneration and desmin is responsible for maintenance of myofibrils in mature myofiber s (Vaittinen et al., 2001). A protofibril is a complex of eight IF monomers and the network of IFs form a lattice of connections that link the different parts of the muscle fiber. These networks extend from the Zdisk to adjacent myofibrils and from the sa rcolemma to the Z-disc. Additionally, IFs are associated with the neuromuscular junction, nuclear membrane, and mitochondria located between adjacent myofibrils (Agbulut et al., 2001; Re ipert et al., 1999). Skeletal Muscle Function Contraction velocity is based on the efficien cy of the energy metabolism. Velocity is fastest when ATP is generated from an oxidative rather than a glycolytic pathway. In addition, contraction velocity is also de pendent on the type of myosin pr esent in the fiber. The four Myosin Heavy Chain (MyHC) isoforms are the products of a multigene family whose expression during development and in the adult are regul ated by neuronal, hormonal, and mechanical factors. Most single fibers (~86%) express only one MyHC isof orm but there are examples of

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20 co-expression of MyHC isoforms and the MyHC IIA and IIX/D isoforms are predominately expressed among fibers (Rivero et al., 1999). In terms of shortening velocity, the gradient observed for MyHC isoforms is I < IIA < IIX/D < IIB (Bottinelli et al., 1991; Galler et al., 1994). There are also phenotypic differences associ ated with MHC isoform composition in terms of metabolic and size properties of the muscle fi ber types (Br and Pette, 1988; Schiaffino et al., 1989). For example, motor units containing more MyHC I or IIA are more resistant to fatigue than motor units containing MyHC IIX/D or IIB Additionally, MHC IIB fibers are generally larger than MyHC I or II A (Rivero et al., 1999). The globular head on the myosin has ATPase ac tivity. This activity determines how fast the muscles contract, and is determined by how quickly ATP is hydrolyzed. In terms of the force-velocity relationship, this illustrates that the amount of force generated by a muscle is highly proportional to its velocity or velocity is dependent on how much force is resisting the muscle. Maximum contracti on velocity is termed Vmax, which is one of the most commonly used parameters to characterize muscle, and is relative to both fiber type dist ribution and architecture (Lieber, 2002). Function is largely determined by a muscles architecture. While fiber size between muscles does not vary greatly, ther e are major architectural differe nces between different muscle groups and these are the best pred ictors of force generation (Bur kholder et al., 1994). Skeletal muscle architecture is defined as the arrangement of muscle fibers within the muscle relative to the axis of force generation. There are three classifications of muscle arrangements termed parallel, uni-pennate, and multi-pennate. Parallel muscles are composed of fibers that extend parallel to the muscles force-generating axis Uni-pennate muscles have fibers that are orientated at a single angle relative to the force-generating axis typically in an angle of 0 to

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21 30 degrees. Most muscles are multi-pennate, where the fibers are orientated at several angles relative to the axis of force ge neration (Lieber, 2002). Other pa rameters that help characterize the properties of various muscle s or muscle groups are the sarcomere length, which also translates into fiber length, range of movement (ROM), and the physiological cross-sectional area (PCSA). The PCSA of a muscle represents th e sums of the cross-sectional areas of all of the muscle fibers within the muscle and is di rectly proportional to the maximum titanic tension generated by the muscle (Powell et al., 1984). Tying together some of these parameters give a better indication of functional properties of specific muscle groups. For example, the quadr iceps muscles have relatively high pennation angles, large PCSAs, and short fibers. Therefor e, this makes them suited for the generation of large forces. Hamstring muscles like the sart orious, semitendinosus, a nd the gracilis muscles have low PCSAs and extremely long fibers, which allow for large excursions at low forces. Conversely, the soleus muscles have high PCSAs a nd short fiber lengths so that these muscles can generate high forces with small excursi ons making these suitable muscle groups for a postural stabilization role (Edman et al., 1985; Woittiez et al., 1985). There is also different terminol ogy related to different types of contractions. For example, isometric or static contractions are when the muscle tension increases but the body part does not move. Concentric contractions are when the action of the muscle results in muscular shortening as the body part moves and the force is applied to the muscle as it contracts. Eccentric contractions occur when the muscle is activated and force is produced as the muscle lengthens. Therefore, these types of contractions are more likely to result in muscle injury or soreness versus concentric contractions. Additionally, muscle strengthening regimens are greatest when exercises using eccentric contrac tions are utilized (Lieber and Friden, 2002). These factors

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22 demonstrate why muscle architecture is also a consideration in traini ng regimes for human and animal athletes. As more information is gene rated on the basic understanding of muscle form and function, there can be further benefits for athletic training and perf ormance. Skeletal Muscle Development Skeletal muscle development is a highly comple x and orchestrated proc ess, which is still not completely understood, both at the macroscopic and molecular levels. Skeletal muscle cells are derived from the somites, except for the cran iofacial muscles in mammals and birds (Armand et al., 1983). Somites are balls of epithelial cells formed from the paraxial mesoderm in pairs on either side of the neural t ube, and differentiate into two regions the dermomyotome and sclerotome. Due to their interactions with su rrounding tissues, the vent romedial portion of the somite gives rise to the sclerotome, which is the precursor to the ribs a nd axial skeleton, and the dorso-lateral part forms the dermomyotome, whic h is where the myogenic precursors and dermis originate in embryogenesis (Bra un et al., 1992; Rudnicki et al., 1993; Christ and Ordahl, 1995). Within the dermomyotome, the epaxial muscles and subsequent body wall muscles are formed on the dorso-medial lip. The hypaxial muscles, which consist of the limbs, tongue, diaphragm and ventral wall musculature, are formed on the dor so-lateral lip. Endothelial precursors are also derived from the somites (Chevallier et al ., 1977; Beddington and Martin, 1989; Ordahl and LeDouarin, 1992; Wilting et al., 1995; Kardon et al., 2002). Multiple pathways mediate the embryonic proces ses throughout skeletal muscle formation. In terms of limb bud development, the progenitors of this process evolve by delamination and migration distally into the developing limb bud in response to molecular signals from the adjacent lateral plate mesoderm (Chevallier et al ., 1977; Christ et al., 1977; Solursh et al., 1987; Hayashi and Ozawa, 1995). Induction of premyo genic cells in the limb to delaminate is mediated by hepatocyte growth f actor (HGF) or scatter factor and by fibroblast growth factor

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23 (FGF). In experiments where both FGF2 and HG F were applied ectopically in the interlimb flank mesenchyme of chicks, delamination of the lateral dermomyotome was observed (BrandSaberi et la., 1996; Heymann et al., 1996). A dditionally, when c-met, the tyrosine kinase receptor for HGF, is inactivated, delamination in mice is prevented. Both HGF and FGF2 and FGF4 are also found to promote migration by acti ng as a chemotactic sour ce resulting in cell migration towards the distal tip of the limb bud (Itoh et al., 1996; Takayama et al., 1996; Webb et al., 1997; Scaal et al., 1999). Transcription factors are i nvolved in delamination and mi gration, with Pax3 and Lbx1 being initially expressed in the lateral dermom yotome. Pax3 is necessa ry for the epithelialmesenchymal transformation of the latera l dermomyotome (Daston et al., 1996). In Pax3 null mice, which are also called Splotch mice, c-met expression is either si gnificantly reduced or absent in the lateral dermomyotome. This suggests that Pax3 is upstream of c-met since the dermomyotome is disorganized and the limb myogen ic cells do not migrate (Epstein et al., 1996; Yang et al., 1996; Mennerich et al., 1998; Tremblay et al ., 1998). Inactivation of Lbx1 causes premoygenic cells to delaminate properly but have dysfunctional migration. The resulting phenotype exhibits minimal hindlimb musculatur e and the extensors (or dorsal muscles) are missing in the forelimb. These observations are similar to experiments where Gab1 is inactivated, which is also involved in c-met signali ng and is also believed to be a result of cells being unable to respond to limb migratory cu es (Schfer and Bra un, 1999; Brohmann et al., 2000). Therefore, as these premyogenic cells mi grate distally towards the limb bud, they begin to switch on the myogenic determina tion bHLH transcription factors, MyoD and Myf5 and form the dorsal and ventral muscle masses. The myogenic cells then terminally di fferentiate into slow

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24 or fast fibers, which express the relevant form of slow or fast MyHC. The fast fibers are needed for movement with the slow fibers helping maintain posture (Francis-West et al., 2003). Another major player in skeletal muscle formation is Sonic hedgehog (Shh), which is expressed in the notochord and fl oor plate of the neural tube a nd induces the formation of the sclerotome. Along with the Wnt proteins (1, 3a, 7a, 11), Shh also promotes myogenesis by activating expression of Myf5 in the epaxial myotome through its interaction with Wnt1. Wnt1 is expressed in the dorsal neural tube and the dorsal ectoderm. Wnt7a is expressed in the dorsal ectoderm and found to activate the expression of MyoD in hypaxial myotome (Schmidt et al., 2000; Petropoulos and Skerjanc, 2002). The bone morphogenic proteins (B MPs) are expressed in the lateral plate mesoderm and limb bud ectoderm. Noggin an antagonist for BMP, is expressed in the dorsomedial lip of dermomyotome. BMPs and Noggin regulate Pax3 in proliferating muscle precursor cells (MPCs), and Pax3 is critical for the downstream ac tivation for Myogenic Regulatory Factors (MRFs) (Amthor et al., 1998). The levels of BMPs within the somite, in the presence or absence of its antagonists, cont rols the expression of Pax3 and can either promote embryonic muscle growth by expanding Pax3-expressing muscle pr ecursor cells or restricting development by inducing apoptosis (Amthor et al., 2002). In the limb, BMP2 and BMP7 expression is increased by Shh. This causes a stimulation of muscle gr owth and a delay in muscle differentiation by allowing these BMPs to inhibit activation of MyoD and Myf5 by Pax3 (Tajbakhsh et al., 1997). The BMP signals originating from the lateral pl ate delay the activation of myogenic bHLH gene expression (Pourqui et al., 1996) In the presence of Noggin, BMPs are sequestered, Pax3 expression is repressed, and MyoD expression is dramatically e xpanded, inducing formation of a lateral myotome (Duprez et al., 1998; Reshef et al., 1998; Krger et al., 2001). Conversely,

PAGE 25

25 another BMP antagonist, follistatin, promotes Pax3 expression, which transiently delays muscle differentiation and exerts a proliferat ive signal during muscle development (Amthor et al., 2002). Myogenic Regulatory Factors (MRFs) At the molecular level, MRFs mediate the process of myoge nic determination and musclespecific gene expression enabling multipotent, me sodermal cells to give rise to mononucleated myoblasts, withdraw from the cell cycle and diffe rentiate into multinucleated muscle fibers, which are the framework of whol e muscle (Cooper and Konigsber g, 1961; Stockdale and Holtzer 1961; Stockdale et al., 1999). MRFs are a family of skeletal muscle specif ic bHLH transcription factors and contain the genes, Myf5 MyoD MRF4 and myogenin When these factors are overexpressed in non-myogenic cell lines, they caus e them to differentiate into myogenic cells (Davis et al., 1987; Wright et al., 1989; Braun et al., 1989). MyoD and Myf5 are expressed in proliferating, undifferentiated cells and when cells begin to terminally differentiate, myogenin expression is induced (Smith et al ., 1993, 1994; Andres and Walsh, 1996). MRF4 is expressed in both early stages of myogenesis, during muscle development, as well as in adult muscle tissue (Hinterberger et al., 1991). Another family of genes, the MEF2 family augments the myogenic activities of MRFs. (Shen et al., 2003). MyoD and MEF2 interact to control myoblast speci fication, differentiation, and proliferation and MRF4 acts in embryonic ce lls to control myogenic determination (KassarDuchossoy et al., 2004). However, myogenin expression is required for terminal differentiation before birth. In addition, the expression of skeletal muscle -actin is a marker of late differentiation following myogenin expression (Knapp et al., 2006). Further mediation of MRF activities is achie ved through their heter odimer formation with another family of bHLH proteins, the E proteins (Lassar et al., 1991; Shirakata et al., 1993). The

PAGE 26

26 E proteins are class I, bHLH proteins. These clas s I proteins form heterodimers with the class II bHLH proteins such as MyoD. This heterodi mer binds to specific DNA regions (E boxes) that contain the sequence CANNTG and activate transcripti on of muscle target genes (Yutzey and Konieczny, 1992). E47 is an alte rnate splice product of the E2 A gene expressed early in myogenic differentiation (Sun and Baltim ore, 1991; Quong et al., 1993). Emerging data demonstrates that the intracellular signaling pathway, p38 mitogenactivated protein kinase (MAPK), participates in several stages of the myogenesis. The most documented role of p38 is its cooperation with tr anscription factors from the MyoD and MEF2 families in the activation of muscle specific genes. This interaction contributes to the temporal gene expression during differe ntiation (Cuenda and Cohen, 1999; Zetser et al., 1999). Withdrawal of myoblasts at the G1 stage is necessary for differentiation to occur. This is partially mediated by p38 kinase and c-Jun Nterminal protein kinases (JNKs) signaling through its induction of p21 expression in myoblasts (Puri et al., 2000; Mauro et al., 2002). The MyoD/E47 heterodimer is regulated by p38 MAPK and MRF4 is phosphorylated by p38 MAPK on Ser-31 and Ser-42 resulting in a reduced transcriptional activ ity. The modulation of MRF4 activity also results in selective silencing of muscle-specific genes in terminal differentiation (Suelves et al., 2004). During somite development in mice, p38 MAPK plays a crucial role in activating MEF2 transcription factors. In Xenopus laevis p38 signaling is required for early expression of Myf5 and for the expression of several muscle st ructural genes (Keren et al., 2005). Negative myogenic regulators exist such as, Id1 and Twist. Id1 is a dominant negative HLH protein that prevents the interaction of the E proteins with MyoD or Myf5 by preferentially binding to the E proteins (Nort on et al., 1998). Twist complexes with E proteins to repress

PAGE 27

27 myogenic differentiation. Twist also can directly block transc riptional activity of MyoD and MEF2 by binding to E-box sequences (Hebrok et al., 1997; Puri and Sartorelli, 2000). In order for muscle differentiation to occur, myoblast cell cycle arre st is critical. MyoD promotes cell cycle arrest by inducing p21 which is a cyclin-depende nt kinase (cdk) inhibitor (Guo et al., 1995; Halevy et al., 1995). Induction of p21 is subsequent to myogenin expression and it is believed that high levels of p21 are required for cells to remain in a post-mitotic state. MyoD also induces retinoblastoma ( Rb ), which is a negati ve regulator of G1 progression (Martelli et al., 1994). There is an intricately regulated pattern of cell cycle proteins that are temporally expressed based on a variety of molecu lar interactions and furt her discussion of these events is found in a later section. Briefly, G1 progression is controlled by Cdk-mediated phosphorylation of the Rb protein, which allows for the release and activation of E2F1 (Lundberg and Weinberg, 1998). E2F1 mediates S-phase progression by allowing for expression of genes required for DNA replication and mitosi s (Ishida et al., 2001). Withdrawal from the cell cycle is mediated by p21, which directly inhibits Cdk complexe s and interferes with S-phase progression by binding to and inhi biting the activity of prolif erating cell nuclear antigen (PCNA), a subunit of the DNA polymerase (Dotto, 2000). Satellite Cells More than 40 years ago, Alexander Mauro firs t identified muscle progenitor cells in frog skeletal muscle, which were identified as satell ite cells based on their obs erved location adjacent to mature muscle fibers as seen by elect ron microscopy (Katz, 1961; Mauro, 1961). These undifferentiated cells have a degree of plasticity, demonstrating prope rties of stem cells such as, yielding all of the specialized cel l types from which they originate and having the ability to selfrenew. In mature muscle, in response to inju ry, hypertrophy, routine maintenance or disease, satellite cells serve as a reserve of muscle stem cells that are activated from their quiescent or G0

PAGE 28

28 state to re-enter the cell cycl e and provide myonuclei for grow th or repair (Moss and Leblond, 1971). Self-renewal ability in sa tellite cells may occur by either a stochastic event or through asymmetric cell division, and this self-renewa l is required for maintenance of their own population pool (Collins et al., 2005). Satellite cells are derived from a Pax3/Pax7 population of progenitor cells located in fetal muscle (Relaix et al., 2006). In a quiescent state, satellite cells are characterized by a lo w nucleus/cytoplasm ratio, few organelles, and high amount of heterochromatin or condensed chromosomes, resulting in minimal metabolic activity or transcription (Schul tz, 1976). Quiescent satelli te cells can also be distinguished from activated ce lls because there are numerous mo rphological changes that occur upon activation. During activation, cytoplasmic extensions become apparent, along with an increase in cytoplasmic volume of the activated cell, the amount of he terochromatin decreases and organelles such as the Golg i, endoplasmic reticulum, ribosomes, and mitochondria start to appear (Schultz et al., 1978). A dditionally, at the molecular level, MyoD expression is turned on, and a CD34 isoform switch is observed, along with co-expression of Pax7, M-cadherin, and Myf5. Cellular proliferation and division is in dicated by expression of proliferation markers such as PCNA, and induction of myogenin expression designates cells undergoing myogenic differentiation (Fuchtbauer and Westphal, 1992; Grounds et al., 1992; Yabl onka-Reuveni et al., 1994; and Zammit et al., 2004). Satellite Cell Marker Proteins In order to distinguish sate llite cells from other mononucle ated cells within skeletal muscle, protein markers have been identified whic h are specific to satellite cells alone or in combination with other markers that allow for the specific iden tification of a pure population of satellite cells. Some of the most studi ed are CD34, M-cadherin, Pax7, syndecan-3/4, VCAM1, and c-met (Rosen et al., 1992; Beauchamp et al., 2000; Seale et al., 2000; Cornelison et al.,

PAGE 29

29 2001). Pax7 is currently the best marker for iden tifying quiescent satellit e cells (Seale et al., 2000). Markers like CD34 are useful in identifying satellite cells on isolat ed myofibers, but are not specific or unique to satellite cells and a co-staining or a combination of markers is needed to definitively identify these cells (Beauchamp et al., 2000). Although, the truncated form of CD34 (Beauchamp et al., 2000) and the isoform of myocyte nuclear f actor (MNF) are specific to quiescence (Garry et al., 1997; Yang et al., 1997). There is also a level of heterogeneity that exists within the sate llite cell population in te rms of immunohistochemical (IHC) cell staining and in vitro clonal analyses (Schultz a nd Lipton, 1982; Baroffio et al., 19 96; Molnar et al., 1996). For example, most satellite cells are positive for both CD34 and M-cadherin and most of these cells are also positive for Myf5 (Beauchamp et al., 2000), a subpopulation of ~20% are not positive for these markers. Interestingly, functio nal studies suggest that this subpopulation of cells serves as a reserve for satellite cell repl enishment (Rantanen et al., 1995; Schultz, 1996). c-Met c-Met is a transmembrane tyrosine kinase receptor, and represents the activated HGF receptor, which is found in all qui escent satellite cells but not expressed in myofibers (Allen et al., 1995; Cornelison and Wold, 1997; Tatsumi et al ., 1998). However, it is not restricted to satellite cells, and is detected in bot h resting and regenerating muscle. The c-met knockout mouse demonstrates that c-met is necessary fo r proper limb, diaphragm, and skeletal muscle formation. Following a crush injury, the mononu cleated cells surrounding the necrotic fiber express the c-met marker. Additionally, cells negative for another stem cell marker, CD34, but positive for c-met (CD34-/c-met+) are still capab le of giving rise to myotubes in culture (Beauchamp et al., 2000).

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30 Syndecans 3 and 4 Syndecans 3 and 4 have overlapping expression pa tterns in skeletal muscle with c-met and are cell surface trans-membrane heparin sulfate pr oteoglycans. They function in FGF signaling and the expression of Syndecan 3 and 4 is consistent with that of satellite cell position (between basal lamina and sarcolemma) and prolif erating MPCs (Cornelison et al., 2001). Syndecan 3 and 4 knockouts have severe defects in satellite cell activa tion and muscle regeneration (Cornelison et al., 2004). Syndecans play a role in signa ling through the ERK1/2 MAP kinase pathway. Primary mouse satellite cells requ ire heparin sulfate for normal pr oliferation (Cornelison et al., 2001). Activation and initiati on of myogenesis is delayed in vitro when syndecan signaling events are blocked (Cornelison et al., 2004). M-cadherin M-cadherin is a calcium-dependent adhesion mo lecule, which is used as a marker of quiescent satellite cells a nd activated myogenic precu rsors, but is not expressed in differentiated myotubes (Irintchev et al., 1994; Moore and Wa lsh, 1993; Beauchamp et al., 2000; LaFramboise et al., 2003). It is expressed in some, but not all quiescent satelli te cells, demonstrating heterogeneity within satellite cell compartments (Cornelison and Wold, 1997). Those that do express M-cadherin also expre ss CD34 (hematopoietic stem cell marker) (Beauchamp et al., 2000). Studies suggest that M-cad+/CD34+ satel lite cells are also Myf5-positive (Beauchamp et al., 2000). Another observation is that the number of M-cadheri n positive cells increases upon satellite cell activation (Cornelison and Wold, 1997). CD34 CD34 is expressed in cells that express no cardiac, hematopoetic, or skeletal muscle mRNA transcripts, indicating a lack of lineage. This follows with the idea that only a subpopulation of satellite cells e xpresses the CD34 marker and designates a sub-population of

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31 satellite cells that are less committed to the myogenic lineage (Beauchamp et al., 2000). Cells that express CD34 dont express Pax7 and M-cadherin and both CD34 positive and negative progenitor cells can give rise to myotubes in vitro One study examined CD34-positive versus CD34-negative primary myoblasts and found that CD34-positive primary myoblasts were more efficient in participating in regeneration but CD34-negative cells had a higher fusion index in vitro (Beauchamp et al., 2000). Pax7 Pax7 is a member of the paired box family of transcription factors, and is localized to nuclei situated in discreet peripheral locations with in resting adult skeletal muscle (Seale et al., 2000; Relaix et al., 2005). The number of cells positive for Pax7 is believed to correlate well with the expected number of satellite cells. This is because Pax7 expression co-localizes with myostatin, c-met, and m-cadherin in satellite ce lls resting between basal lamina (Seale et al., 2000; LaFramboise et al., 2003; McCroskery et al., 2003; Halevy et al., 2004). In addition, myogenic cells lines, which model quiescent, undi fferentiated myoblasts, appear to be uniquely marked with high levels of Pax7. Furthermore, the expression seen in proliferating primary myoblasts is down-regulated upon myoblast di fferentiation. Although loss of Pax7 does not induce differentiation, indicating that other factors must be presen t or absent for myoblasts to commence terminal differentiation (Seal e et al., 2000; Olguin and Olwin, 2004). Pax7 inhibits myogenic conversion induced by MyoD It is believed that Pax7 is indirectly interfering with its function or competes for proteins ne cessary for MyoD-dependent transcription since the presence of Pax7 cannot inhibit the eff ects of MyoD-E47 heterodimers (Olguin and Olwin, 2004). Yet, ove rexpression of Pax7 in satellite cells does induce cell cycle exit, prevention of BrdU in corporation, a decrease in MyoD expression, and prevention of myogenin induction (Olguin and Olwin, 2004) In addition to its impact on MyoD, it is believed

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32 that myoblasts expressing the Pax7+/MyoDphe notype may return to quiescence to replenish satellite cell pools while myoblasts that acquire MyoD proliferat e and fuse to form myotubes (Zammitt et al., 2004; Relaix et al., 2005). Alte rnatively, one study showed that Pax7 may have anti-apoptotic functions. Myoblasts transduced with dominant negative Pa x7 lead to cell death (Relaix et al., 2006). Myocyte nuclear factor (MNF) MNF is a winged helix transcription factor, also identified as Foxk1, expressed in both cardiac and skeletal muscle, and in quiescent sa tellite cells. The number of MNF positive cells increases when muscle is induced to regenerate and while detected in regenerating myotubes, MNF is down-regulated during late stages of rege neration (Garry et al., 1997). There are two isoforms of MNF, MNFand MNF, and both are detectable in skeletal muscle. Alpha is expressed in prolifera ting MPCs, while beta is limited to quiescent satellite cells and downregulated upon activation (Yang et al., 2000) In quiescent sate llite cells, high MNFexpression allows for the formation of repressi ve complexes with mSin3 family members to repress targeted gene transcrip tion (Garry et al., 2000; Yang et al., 2000). Upon satellite cells activation, isoform switching disrup ts the repression by mSin3, allowing for the targeted genes to become active (Yang et al., 1997). Notch Notch signaling plays a role in cellular homoest asis and cell fate determination (Hing et al., 1994). In mammals, there are four genes, Notch1 Notch2 Notch3 and Notch4 and five ligands, Dll-1, Dll-3, Dll-4, Jagged-1, and Jagged-2 (Chitnis et al., 1995; Li ndsell et al., 1995; Li et al., 1998). The Notch receptors are transmembrane receptors and when bound by the extracellular ligands, Delta, Serrate, or Lag-2, this results in a cleavage of the intracellular domain of

PAGE 33

33 the receptors in the cytoplasm. This cleavage product is the activ e form of Notch, NICD (Notch Intracellular Domain), which translocates to the nucleus where it binds to the family of transcriptional repressors CSL (also known as RBP-J C BF-1, S uppressor of Hairless, and L ag1). This interaction c onverts them into transc riptional activators (Nye et al., 1994; Ahmad et al., 1995). The molecule Numb is an inhibitor of Notc h but little is known about the regulation of Numb at the transcriptional level. Numb localiz es to one pole during ce ll division in crescentshaped patterns so that only one daughter ce ll receives Numb. This as ymmetric expression is believed to play a role in determining which ce ll goes on to differentiate into a myofiber and which cell is going to replenish the satellite ce ll pool. Numb and Pax3 protein expression are mutually exclusive in satellite cells and a Pax3+/N umbcell indicates a sate llite cell that is less committed to a specific phenotype. The daughter cell retaining Numb expression is more committed to progressing along the myogenic lineage (Conboy and Rando, 2002). Constitutively active Notch1 sign aling causes up-regulation of Pax3 and down-regulation of Myf5 MyoD and desmin and a reduction in Pax7 expression. In response to injury, activated Notch expression exhibits a highly localized expression at the site of injury, but not at more distal regions along the fiber. This suggests that it might play a role in targeting or recruiting specific factors to the injury site to pr omote regeneration (Conboy and Rando, 2002). The efficiency of tissue regeneration decreases in response to aging and it was pr oposed that this is due to age-related changes in satellite cell activ ity. Further analyses demonstrated insufficient up-regulation of Delta, resulting in diminished Notch activation in aged, regenerating muscle. When Notch was inhibited in young muscle, impa ired regeneration was observed. Conversely,

PAGE 34

34 when Notch was introduced into old muscle th e regenerative potential was restored (Conboy et al., 2005). To determine if systemic factors play a role in aged progenitor cell s from specific tissues, parabiotic pairings between young and ol d mice were studied (Conboy et al., 2005). Interestingly, the exposure of satellite cells in old mice to young mouse serum resulted in enhanced expression of Delta increased Notch activation, and enhanced proliferation in vitro supporting the notion that there are systemic factors that change w ith age that impact progenitor cell activity (Conboy et al., 2005). Growth Factor Effects on Satellite Cells There are multiple growth factors that regulate satellite cell activation and growth. When myofibers are injured, the re lease of cytokines is stimulate d, and growth factors, such as HGF (Allen et al., 1995; Tatsumi et al., 1998) and FGFs, cause an activation and expansion of satellite cells so that they will re-enter the cell cycle and rapidl y proliferate, although activation of satellite cells has been found to be delayed over time in response to ag ing (Sheehan and Allen, 1999; Clarke et al., 1993; Johnson and A llen, 1995; Cornelison et al., 2001, 2004). Transforming growth factor beta (TGF ) inhibits cell proliferation to some extent, although its more significant effect is in its inhibition of differentiation an d fusion (Florini et al., 1986; Greene and Allen, 1991; Rao and Kohtz, 1995; St ewart et al., 2003; Allegra et al., 2004). MyoD, Myf5, and Pax7 are markers of the myoblasts at this stage (Davis et al., 1987; Wright et al., 1989; Braun et al., 1989; Rhodes and Koniecz ny, 1989; Seale et al., 2004). Factors such as TGF HGF, and FGF inhibit myoblasts from und ergoing differentiation and formation of myofibers (Gospodarowicz et la., 1976; Florini et al., 1986; Olsen et al ., 1986; Massague et al., 1986; Miller et al., 2000).

PAGE 35

35 Skeletal myoblasts serve as a good model to st udy intracellular signaling cascades as they undergo morphological changes characteristic of va rious cellular processes. Previous reports demonstrated the significance of the Raf ki nase signaling axis (Bennett and Tonks, 1997; Coolican et al., 1997; Dorman and Johnson, 1999; Samuel et al., 1999; Dorman and Johnson, 2000; Winter and Arnold, 2000; DeCh ant et al., 2002). Elevated Ra f levels are implicated in causing repression of myoblast differentiation and low-levels enhancing differentiation. These findings highlight the importance of time and dur ation of Raf signal tran smission (DeChant et al., 2002). The molecular basis for inhibition of differentiation may involve direct modification of E-proteins and/or induction of TGF -like gene expression. Additionally, inhibition of myogenic differentiation has been demonstrated when cells were treated with various growth factors such as FGFs (1, 2, 4, 6, and 9), TGF and high concentrations of serum (Allen and Boxhorn, 1989; Sheehan and Allen, 1999). Insulin-like Growth Factor I (IGF-I) IGF-I promotes cell recr uitment to injured muscle by coor dinating a regenerative response to muscle injury. Treatment of isolated satellite cells with IGF-I increases their proliferation rates in response to the JAK/STAT pathway. This growth factor induces both proliferation and subsequent differentiation of sa tellite cells via the type I receptor (Allen and Boxhorn, 1989; Adams and McCue, 1998; Kamanga-Sollo et al., 2004). Activation of myoblast proliferation is mediated through the MAPK pathway and induc tion of differentiation signals through the phosphatidylinositol-3-kinase (PI3K) pathway. In overloaded skeletal muscles, IGF-I peptide levels increase (Adams and Haddad, 1996). In vivo evidence demonstrates that when the tibialis anterior (TA) muscle of a rat is infused with IGF-I, there are measurable increases in muscle protein, muscle DNA content, and absolute weight of the treated muscle (Adams and McCue,

PAGE 36

36 1998). Interestingly, IGF-I is up-regulated in regenerating muscles and in aged rats and application of IGF-I rescued approximately 46% of lost muscle mass and increased the proliferation potential of satelli te cells from atrophied gastrocn emius muscle (Chakravarthy et al., 2000). In mdx mice ( dystrophin null), high levels of muscle-s pecific IGF expression resulted in an increase of approximately 40% in muscle ma ss. A subsequent increase in force generation also was observed along with an elevation of signaling pathwa ys associated with muscle regeneration and protection agai nst apoptosis (Barton et al., 2 002). IGF-I serves as a strong mitogen of satellite cells ev en in the presence of a st rong growth inhibitor, TGF yet the presence of TGF is able to inhibit IGF-I-mediated satellite cell differentiation (Allen and Boxhorn, 1989). Additionally, IGF -I increase the magnitude of the proliferative response elicited by FGF2 and stimulate differentiation wh en treated alone (Greene and Allen, 1991). Fibroblast Growth Factors (FGFs) In muscle tissue, there are several FGFs expr essed and released in response to injury (Anderson et al., 1995). FGF2 and FGF6 are pot ent enhancers of muscle precursor cell (MPC) expansion and satellite cells by increasing pro liferation and promoting muscle regeneration (Johnson and Allen, 1993; Lefauche ur and Sebille, 1995; Floss et al., 1997; Sheehan and Allen, 1999; Yablonka-Reuveni et al., 1999). FGF receptors (FGFR) 1, 2, 3, and 4 are expressed in proliferating rat satellite cells with FGFR1 and 4 being the most prominent (Sheehan and Allen, 1999). FGF2 is a heparin-binding growth factor that increases satellite cell proliferation and PCNA expression (Johnson and Allen, 1993). Treatme nt with FGF2 elicits a greater mitogenic response than IGF-I or TGF l and is as potent as HGF in stim ulating satellite cell proliferation (Sheehan and Allen, 1999). Combinations of FGF2 and HGF are additive with regard to

PAGE 37

37 proliferation, and after injury, disrupted myofibers express FGF2, particularly in regions of hyper-contraction (Anderson et al., 1995). FGF2 is present in newly formed myotubes and injection of FGF2 into the TA muscle of male mdx mice during the first r ound of spontaneous necrosis resu lts in enhanced satellite cell proliferation. This is due to increasing the numbe r of satellite cells that enter the cell cycle but inhibits the differentiation of satellite cel ls (Lefaucheur and Sebille, 1995). The FGF6 knockout in certain genetic backgrounds exhi bits a reduced regenerative ca pacity after crush injury and when they are interbred with mdx mice, a severe dystrophic phenotype is observed. FGF6 is upregulated in response to skeletal muscle injuries and helps completely restore experimentally damaged skeletal muscle (Floss et al., 1997). Hi gher levels of FGF2 and FGFR1 are expressed in the pectoralis major of turkey. Faster prolif erating satellite cells fr om pectoralis major of turkey express higher levels of FGF2 and FGFR1 by comparison to slower proliferating cells. They also show a greater mitogenic response to FGF2, suggesting that FGFs may play a role in proliferative rates of muscle cells (McFarland et al., 2003). Hepatocyte Growth Factor (HGF) HGF, also called Scatter Factor (SF), is a critic al activator of satelli te cells found initially in crushed muscle extract (CME) (Tatsumi and Allen, 2004; Tatsumi et al., 2002). Direct injection of HGF into muscle results in activation of quiescent satellite ce lls, even in the absence of trauma, and when an anti-HGF antibody is inc ubated with CME, the ac tivation capacity of the CME is abolished (Tatsumi et al., 1998; Tatsumi et al., 2002). This growth factor binds to the cMet receptor and signals thr ough the PI3K pathway to prom ote cell survival and MAPK pathways to stimulate the mitogenic effect (Alle n et al., 1995). Placenta, liver, and muscle all express HGF (Brand-Saberi et al., 1996). In additi on, HGF is present in basal lamina of skeletal muscle fibers, which provides a reservoir of HGF within skeletal muscle (Tatsumi and Allen,

PAGE 38

38 2004). The HGF (-/-) mouse is embryonic lethal, and w ithout HGF signaling, skeletal muscle cells cannot migrate from the somite during em bryogenesis (Schmidt et al., 1995; Bladt et al. 2002). Transforming Growth Factor Beta (TGF ) Superfamily The TGF superfamily is involved in cellular pr oliferation, differen tiation, migration, and apoptosis. TGF is one of the most potent negative regu lators of proliferation and differentiation of satellite cells (Florini et al., 1986; Greene and Allen, 1991; Ra o and Koht, 1995; Stewart et al., 2003; Allegra et al., 2004). Inhi bition of myoblast fusion is do se-dependent and reversible (Florini et al., 1986; St ewart et al., 2003). The TGF superfamily can be divide d into three groups: the TGF s, the activins/inhibins, and the bone morphogenic proteins (BMPs). All three groups of growth factors signal through serine/threonine kinase receptors (Massagu et al ., 1994). In the presence of growth factors, ligands bind to a Type II receptor dimer located on the plasma membrane, which causes autophosphorylation of the Type II dimer, recruitment of a Type I receptor dimer, and subsequent phosphorylation of this dimer (Wrana et al., 1992; Attisano et al., 1993; Ebner et al., 1993; Wieser et al., 1993; Wrana et al., 1994). This phosphorylation event re cruits the receptorregulated Smads or R-Smads, which then unde rgo phosphorylation (Aoki et al., 2001). The Rsmads form a complex with the common-part ner Smads (Co-Smads), or Smad4. The RSmad/Co-Smad complex translocates to the nucl eus and binds to the DNA altering transcription of target genes (Derynck et al ., 1996; Liu et al., 1996; Meersse man et al. 1997; Nakao et al., 1997). Additional co-activators and co-repressors lend regulation to the system (Wotton et al., 1999).

PAGE 39

39 Specificity of TGF signaling is mediated by the diffe rent types of Type II and Type I receptors present in the target cell and ligand a ffinities for these receptors (Wrana et al., 1992; Attisano et al., 1993; Ebner et al., 1993; Wieser et al., 1993; Wrana et al., 1994). Additionally, R-Smads (Smads 1, 2, 3, 5, and 8) are ligand-spec ific. Smads are cytoplasmic when inactive and transfer to the nucleus upon phosphorylation (Derynck et al., 1996; Liu et al., 1996; Meersseman et al. 1997; Nakao et al., 1997). I-Smads or i nhibitory Smads (Smad6 and Smad7) bind to the receptor and prevent phosphorylation and signaling activities (Imamura et al., 1997; Nakao et al., 1997; Horiki et al., 2004). Negative regulation of TGF signaling is achieved by follistatin, which binds activin and prevents receptor docking (Nakamura et al., 1990). BMPs (2, 4, and 7) form a trimeric complex between ligand, receptor, and follistatin to inhibit BMP actions (Iemura et al., 1998). Table 1-1 summarizes the receptor and R-Smad specificity for TGF superfamily members (ten Dijke et al., 1994a; ten Dijke et al ., 1994b; Koening et al., 1994; Macas-Silva et al., 1998; Yamashita et al., 1995; Rosenzweig et al ., 1995; Liu et al., 1995; Nohno et al, 1995). Myostatin Myostatin (MSTN), also called growth and differ entiation factor 8 (GDF8) is a member of the TGF superfamily and is responsible for mainta ining muscle size. Mouse devoid of MSTN possesses a larger muscle mass char acterized by hypertrophic fibers. MSTN is expressed in multiple tissues with the greatest mRNA levels found in muscle (Grobet al., 1997; Kambadur et al., 1997; McPherron and Lee, 1997). MSTN circulat es in the blood in an inactive form until the pro-domain is cleaved away by a furin enzyme (McPherron et al., 1997; Lee and McPherron, 2001; Zimmers et al., 2002). Treatment with MSTN inhibits proliferation of muscle precursor cells. Fluorescence Activated Cell Sorting (FACS) analysis determined that MSTN prevents progression of myoblasts from G1 to S-phase transition of th e cell cycle. Subsequently,

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40 upregulation of p21 and a decrease of Cdk2 protein and ac tivity is observed, which results in an accumulation of hypophorylated Rb and arrest in G1 (Thomas et al., 2000). This same cell cycle arrest is observed in MSTN treated satellit e cells. MSTN is thought to be required for maintaining satellite cells in their quiescent stat e, until inhibited by a stim ulus such as injury (McCroskery et al., 2003). In MS TN-deficient satellite cells, a higher number of satellite cells are activated compared to wild type counterparts and addition of MSTN to myofiber explant cultures inhibits satellite cell activation (McCrosk ery et al. 2003). In the MSTN knockouts, hypertrophy and hyperplasi a are observed (McPherron et al., 1997). These animals exhibit an increased num ber of satellite cells versus wild type counterparts, and the satellite cells presen t in the knockout animals have an increased proliferation rate (McCroskery et al., 2003). Gene ablation also resu lts in enlarged hearts of the mutant animals. Myostatin signals through th e Activin Receptor II and transgenic animals carrying a dominant negative receptor have a threefold increase in muscle mass compared with wild type animals (Lee and McPherron, 2001). Bone Morphogenic Proteins (BMPs) BMPs are the largest group of family members in the TGF superfamily (Reddi and Huggins, 1972; Wozney et al., 1988). As the na me implies, BMPs induce bone or cartilage formation ectopically and initia te osteoblast differentiation (Uri st, 1965; Gitelman et al., 1995). BMPs are 30-38kDa homodimers that are synthesized as prepropeptides of approximately 400-525 amino acids. BMPs inhibit the myogenic differentiation of C2C12 cells, and convert their differentiation pathway in to that of osteoblast lineage cells (Katagiri e al., 1994). Additionally, BMPs play a role in somite development by abrogating premature initiation of myogenesis in the presomitic mesoderm (Pourquie et al., 1996). A BMP inhibitory signal is

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41 believed to prevent the premature expression of MyoD before somites are formed (Linker et al., 2003). In pre-myogenic cells, BMP2, 4, and 7 have dose-dependent effects with low concentrations maintaining a Pax3-expre ssing proliferative p opulation and delaying differentiation. Conversely, high concentrations of these BMPs prevent muscle development (Amthor et al., 1998). In vivo BMP2 expression in skeletal-derived cells prevents myogenic differentiation and promotes osteogenic di fferentiation (Musgrave et al. 2001). BMPs mediate non-osteogenic processes. Du ring the developmental and differentiation processes of the embryo, BMPs regulate epithe lial-mesenchymal interactions, cell fate specification, dorsoventral patterning, apoptosis and the secretion of extracellular matrix components (Vainio et al., 1993; Amthor et al., 1998; Weaver et al., 1999; Angerer et al., 2000; Higuchi et al., 2002; Tiso et al ., 2002). More specifi cally, in vertebrates, BMP2, 4, and 7 are found to direct the development of neural crest cells into th eir ultimate phenotypes (Wilson and Hemmati-Brivanlou, 1995; Miya et al., 1997). In terms of signaling, there are Type I and Type II receptors specific for BMPs and Smads1, 5, and 8 are the downstream molecu les phosphorylated by the ligand-receptor complexes (see Table 1-1). At the transcriptiona l level, BMPs regulate target genes such as, Runx2 osteopontin osteonectin and osteocalcin specific for osteogenesis (Ahrens et al., 1993). Smad6 is an I-Smad. It functions by binding to type I BMP receptors thus preventing the activation of BMP Smads 1/5/8 (Imamura et al., 1997). Smad6 overexpr ession in chondrocytes results in delayed differentiation and maturation (Horiki et al., 2004). Noggin and other cystine knot-c ontaining BMP antagonists asso ciate with BMPs to block their signaling. Noggin overexpression in osteobla sts, results in osteoporosis in mice (Devlin et al., 2003; Wu et al., 2003). Noggin is a secreted peptide, which is expressed in condensing

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42 cartilage and immature c hondrocytes. Ablation of Noggin is embryonic lethal at 18.5 days post coitum (dpc) and results in severe hyperplasia of the cartilage with multiple joint fusions, severe defects in somitogenesis, and multiple skelet al defects (Brunet et al., 1998; McMahon et al., 1998). Noggin is found to have different affiniti es for different BMP family members making its regulation of BMP-mediated processes more complex. For example, in the Noggin null transgenic, different bones have varying res ponses to the de-repression of BMP signaling (Zimmerman et al., 1996; Chang and HemmatiBrivanlou, 1999). Depending on location and/or embryonic origin of the bones, one observes i nhibition, delay or acceleration of ossification processes (Tylzanowski et al ., 2006). Another BMP negative regulator is Tob which suppresses the activity of receptorregulated Smads (1/5/8) (Bradbury et al., 1991; Fl etcher et al. 1991; Yo shida et al., 2000). Tob null mice exhibit enhanced BMP signaling and increas ed bone formation (Yoshida et al., 2000). Negative BMP regulation also is achieved by mechan isms that target elements of BMP signaling for degradation. The Hect domain E3 ubiquiti n ligase, Smurf1, targets Smad1 and 5 for degradation, in addition to in teracting with and mediating th e degradation of bone-specific transcription factors such as Runx2 (Zhao et al., 2003). Smurf1 can also target type I BMP receptors for degradation by interacting with Sma d6. They form a complex that can be exported from the nucleus where it inter acts with the BMP receptors to pr omote degradation (Murakami et al., 2003). Furthermore, when Smurf1 is overexpressed in osteoblasts, postnatal bone formation is inhibited (Zhao et al., 2004). While BMPs are capable of redirecting muscle mesenchyme cells to differentiate into bone tissue and stimulate bone formation in vivo (Urist, 1965), previous re ports have demonstrated that mutations of BMP family members results in disruption of skeletal development (Kingsley

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43 et al., 1992; Storm and Kingsley, 1999). Moreov er, BMPs have opposing activities that are concentration-dependent. In the presence of low BMP levels myogenic precursor cells are maintained in a proliferative state in devel oping limb bud, while high BM P levels induce cell death. Thus, BMPs can both stimulate and re strict muscle growth suggesting that a concentration gradient of BMPs is needed for the correct determination and maintenance of the myogenic program (Amthor et al., 1998; Amthor et al., 2002). While BMPs signal through the Smads, a multitude of different transcription factors are recruite d, accounting for the diversity of functional BMP responses. One set of genes i nvolved in skeletal development are the Hox family of transcription factors. In analyses of gainor loss-o f-function and natu rally occurring mutations of Hox genes, BMPs play a central ro le in embryonic skeletal patterning (Manley and Capecchi, 1997; Yueh et al., 1998). Furthermore, several of the Hox genes interact with Smads and there is evidence that Hox gene expressi on may be regulated by BMPs further suggesting that BMPs are a critical mediat or of skeletal myogenesis (Ladhe r et al., 1996; Tang et al. 1998; Liu et al., 2004). BMP6 a member of the BMP subfamily, was origin ally isolated from phage plaques of a g10-based cDNA library derived from 8.5-dpc mu rine embryos. The murine library was hybridized with a 32P-labeled partial Xenopus laevis Vg-1 cDNA under low-stringency conditions (Derynck et al., 1988). Northern blot analyses of mu rine tissues illustrated that BMP6 was present in muscle (Lyons et al., 1989). Th e human and bovine homologs were isolated from bone and designated BMP6 (Celeste et al., 1990). Expre ssion predominates in mature chondrocytes during endochondral ossification and BMP6 treatment stimulates chondrogenic and osteogenic phenotypes in vitro and induction of carti lage and bone formation in vivo (Gitelman et al., 1994; Gitelman et al., 1995; Ya maguchi et al., 1996). Adult muscles express

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44 BMP6 (Lyons et al., 1989) and BMP6 expression increases in Raf-arrested myoblasts (Wang et al., 2004). In keratinocytes, treatment with BMP6 significantly de creases DNA synthesis (DSouza et al., 2001) and triggers differentiation programs (Tennenbaum et al., 1996). In these differentiated keratinocyte cultur es, E2F5 protein levels are si gnificantly increased (DSouza et al., 2001). Treatment of keratinocytes with TGF 1 causes reversible cell cycle arrest without activating the differentiati on program (Pierce et al., 1998; Dicker et al., 2000). BMP Function in vivo : Lessons Learned from Knockouts A compilation of the various BMPs and recepto rs targeted knockouts and their resulting phenotypes are presented in Table 1-2. BMP5 null mice are called short ear mice, because these animals have reduced ear size in comparison to wild type animals, in addition to exhibiting reduced vertebral processes, and a reduced num ber of ribs and sesamoid bones (Green, 1968; Kingsley et al., 1992; King et al ., 1994). Mice with a homozygous BMP7 deletion are found to die at birth due to renal failure because of hypoplastic/dysplastic kidneys (Dudley et al., 1995; Luo et al., 1995; Wawersik et al., 1999). BMP6 null mice are viable and fertile and exhibit no major defects, except for a delay in ossificati on of the developing sternum (Solloway et al., 1998). It is believed that BMP2 may be functionally compensating for BMP6 ablation since BMP2 and BMP6 are required for some overlapping or redundant functions (Solloway et al., 1998). Utilization of BMPs in a clinical setting was demonstrated in various therapeutic interventions such as, bone de fects, non-union fractures, spin al fusion, osteoporosis, and root canal surgery. Multiple studies utilizing BMP2 demonstrate its ability to promote healing of severe long bone defects in rats rabbits, dogs, sheep and non-human primates (Murakami et al., 2002). Adenoviral administration of BMP2 mixed with a bioresorbable polymer and

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45 mesenchymal stems cells was able to repair bone defects (Chang et al., 2003). RhBMP2 administered systemically increased mesenchyma l stem cell activity and reversed age-related bone loss due to ovariectomies in different mous e models (Turgeman et al., 2002) demonstrating a potential application of BMPs in osteoporosis treatment. Interestingly, rhBMP2 has been used as a complete bone graft substitute in spinal fu sion surgery and in some cases BMP2 has been more efficacious in promoting successful bone fusion as compared to autogenous bone grafts. BMP2 was used in other fusion applications such as intervertebral and lumbar posterolaterial fusions (Sandhu, 2003). BMPs can be used in de ntal applications since BMP2 induces bone formation around dental implants used in periodont al reconstruction. It ha s been suggested that BMPs could serve as an alternat ive to root canal surgeries (Sch wartz et al., 1998; Cochran et al., 1999). Current BMP6 Studies Recently, BMP6 was able to induce matrix s ynthesis and induce differentiation of bovine ligaments fibroblasts providing another potentia l source of chondrocytes for tissue repair (Bobacz et al., 2006). Mesenchymal stem cells (MSC s) treated with BMP6 in combination with parathyroid hormone (PTH) and vitamin D(3) incr eased osteocalcin production. Osteocalcin is used as a marker for bone formation and in these MSCs, BMP6 enhanced calcium formation (Sammons et al., 2004). TGF and BMPs may act in an antagonistic manner towards each other. TGF inhibits chondrocyte maturation relatively early in differentiation by down-regulating bmp6 ihh (Indian hedgehog), and colX (collagen type X), which ar e genes specifically expressed by hypertrophic chondrocytes (Vortkamp et al., 1996; Ferguson et al., 2004). BMPs are implicated in the development of can cers of the GI tract, breast, and prostate (Howe et al., 2001; Pouliot and Labrie, 2002; Bruba ker et al., 2004). Currently, much focus is

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46 being placed on TGF superfamily members as targets for ca ncer therapy due to their ability to suppress tumor progression by inhibiting growth of neoplastic tissues. BMP6 in the early stages of carcinogenesis inhibits beni gn and malignant skin tumor fo rmation (Wach et al., 2001). BMP6 exerts an anti-proliferative and pro-apopt otic effects in multiple myeloma (Kawamura et al., 2000; Hjertner et al., 2001; Kersten et al., 2005). Ras/Raf in Skeletal Muscle A key regulator of signal transduction pathways controlling cell proliferation, differentiation, and oncogenesis is Ras, a monomeric guanine nuc leotide-binding protein with intrinsic GTPase activity (Marshall, 1996). Ras is a molecular switch that cycle between a GTPbound active and a GDP-bound inactive state (McCormick, 1993). On cogenic H-Ras inhibits the differentiation of muscle cells i ndependent of their continued pr oliferation (Olson et al., 1987). Ras inhibits myogenesis by disrupt ing MRF function, resulting in th e inhibition of differentiation of muscle cells independent of pr oliferation (Ramocki et al., 1998). Ras proteins are localized at the cytoplasmic face of the plasma membrane a nd activated by a large number of extracellular stimuli, such as growth factors and hormones (McCormick, 1993; Bar-Sagi, 2001). A family of Ras effector molecules specifically binds to and is activated by Ras-GTP, which then carries out the downstream functions of activat ed Ras (Marshall, 1996). Some effectors of Ras are the Ral GTPase signaling pathway involved in the G1 to S progression (Ramoc ki et al., 1998) and the PI3K/AKT pathway involved in cell survival (Murphy et al., 2002). All of the major Ras effectors have been tested for a role in the Ras-mediated inhi bition of skeletal myogenesis but none of them are able to duplicate the effects of oncogenic Ras (Ramocki et al., 1997; Weyman et al., 1997).

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47 The Raf Family The Raf family consists of three serine/threonine specific kinases and are the best characterized effectors of Ras. Members include A-Raf, B-Raf, and cRaf, also called Raf-1 (Marshall, 1996). There are th ree conserved regions (CR1-3) w ithin Rafs consisting of two Nterminal regulatory domains (CR1 and CR2) and a C-terminal catalytic kinase domain (CR3). Rafs lead to the activation of the extracellular signal-related kinase (ERK) pathway, which mediates cellular proliferation and differentiation (Marshall, 1996). Inactive Raf is cytosolic and translocates to the plasma membrane following activation by Ras (Chong and Guan, 2003). The activation of Raf is important in mediating growth factor gene expression and a major function of Raf protein kinase is to phosphorylate MEK1 and MEK2. Subsequently, ERK1 and ERK2 are phosphoryl ated on tyrosine and threonine residues. These activated ERKs then either phosphorylate numerous cytoplas mic targets or migrate to the nucleus to activate transcription factors such as c-fos and Elk1 (H uang et al, 1993). The Raf/MEK/ERK signaling cascade is required for cell cycle progression and overexpression causes cell transformation (Kolch et al., 1991; Cowley et al., 1994; Mansour et al., 1994). The sustained activation of this pathway is imp licated in differentiation with prolonged ERK activated leading to differentiation of PC12 ce lls (Qui and Green, 1992) Raf-activated MEKERK cascades are likely participants in apoptos is because potent ERK activation can protect cells from apoptosis (Le Gall et al., 2000). Signaling through the Raf/MEK/ERK controls several aspects of myogenesis. Overexpression of a constitutively active Raf and MEK results in reduced muscle gene transcription and these myoblasts are unable to form myocytes (Dorman and Johnson, 1999; Dorman and Johnson, 2000; Samuel et al., 1999; Winter and Arnold, 2000). Down-regulation of ERK activity by overexpression of MAPK phospha tase I (MKP-I), or through the use of

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48 synthetic inhibitors, upregulates muscle specif ic gene transcription (Bennett and Tonks, 1997; Dorman and Johnson, 1999). By contrast, other re ports have found no effect of ERK activity on muscle gene expression, myoblast fusion, and myoblast differentiation (Weyman et al., 1997; Jones et al., 2001). Myoblasts expr essing low levels of Raf differe ntiate more efficiently than control cells again suggesting that ERK1/2 promotes myogene sis (Wang et al., 2004). In differentiating C2 myoblasts, ERK1/2 activity in creases MyoD expression and transcriptional activity (Gredinger et al., 1998). Activated ERK1 is associated with myoblast proliferation and activated ERK2 is associated with myoblast differentiation (Sar bassov et al., 1997). ERK1 and ERK2 are both ubiquitously expressed in mo st tissues and similar in structure. ERK1 null mice are viable, fertile, and of normal size (Nekrasora et al., 2005), whereas, ERK2 null mice are embryonic lethal (Yao et al., 2003). E2F Family The major function of the E2F f actors is cell cycle progression, in addition to playing roles in metabolic activities such as proliferation, di fferentiation, and apoptosis (Fujita et al., 2002). Genes expressed at the G1/S transition contain E2F-binding sites within their promoters (DeGregori et al., 1995). E2F me mbers are conserved throughout evolution from invertebrates to mammals and there are currently nine family members, which demonstrate tissue-specific activities (Dynlacht et al., 1994; DSouza et al., 2001). Two major groups, activators and repressors, comprise the E2F family. E2F1, E2F2 and E2F3a are involved in positive cell cycle control and S-phase entry of quiescent cells. These member s preferentially bind pRb with expression peaking in late G1 and association with E2F-regulated promoters during the G1-S transition. Ectopic expression of these members results in S-pha se induction in serum-starved cells (DeGregori et al., 1997; Lukas et al., 1996 ; Leone et al., 1998; Hu mbert et al., 2000). E2F3b, E2F4 and E2F5 are classified as represso r proteins, due to their negative control of cell

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49 cycle progression. E2F4 and E2F5 pre dominately bind p107 and p130 pocket proteins (DeGregori et al., 1997; Leone et al., 2000). E2F6, E2F7, and E2 F8 also are classified as repressors but through a mechanism independent of pocket protein interac tion because they lack the Rb-binding sequence at the C-terminus (Cam panero et al., 2000). Members of the E2F family demonstrate differential mRNA expression throughout the cell cycle. For example, E2F1 and E2F2 mRNA increase in late G1, and peak at the G1/S transition, although E2F2 mRNA expresses at a lower level than E2F1. During quiescence, E2F3 and E2F5 mRNA are expressed, with E2F1 barely present. In early-to-mid G1, E2F3 and E2F5 mRNA levels rise (Sardet et al., 1995; Pierce et al., 1998). Members of the E2F family are grouped based on their homologous DNA binding domain (DBD). All of the E2Fs, except E2F7 and 8, a nd both DP proteins have a conserved DBD and a dimerization domain. E2F4 and E2F5 demonstr ate a 72% amino acid identity to each other and a 35% amino acid identity to E2F1-3 (Vaishna v et al., 1998). E2F4 and E2F5 make up a subclass of the E2F family since their N-termi nus lacks the cyclin A-binding domain found in the other members. The repressive E2Fs c ontain the Rb-binding sequence and have nuclear export sequences instead of nuclear localization sequences in the N-terminus (Helin et al., 1993; Sardet et al., 1995). E2F Signaling Mechanism and Involvement of Pocket Proteins The E2F proteins signal via the formation of active heterodimer complexes with DP proteins (Helin et al., 1993). E2Fs can dimerize with either DP1 or DP2, except for E2F7 which binds DNA in a DP-independent manner (Ormondroyd et al., 1995). These E2F complexes promote gene expression required for G1 progression and DNA replication (Chen et al., 2004). The mammalian Rb family of proteins (pRb, p107, and p130) is also referred to as the pocket proteins. The pocket prot ein motif allows for interactions with cellular proteins that

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50 possess a LXCXE peptide motif (Lee et al., 1998). The LXCXE motif is present in E2Fs, the Dtype cyclins (Gill et al., 1 998), and HDACs. The pocket dom ain is separated into two functionally conserved regions, th e A and B pockets. The spacer region is specific to each pocket protein and contains binding sites for cyclin/CDK complexes in p107 and p130 but not in pRb. p130 and p107 contain approximately 50% am ino-acid identity and 30-35% identity to pRb (Ewen et al., 1991; Hannon et al., 1993). Ov erexpression of cDNAs coding for pocket proteins induces growth arrest at G1 (Classon et al., 2000). Rb was the first tumor suppressor gene cloned an d loss of Rb function serves as a hallmark of oncogenic progression. pRb is a major G1 checkpoint and inhibits S-phase entry. pRb promotes terminal differentiation, cell cycle exit and tissue specific gene expression (Dunaief et al., 1994; DeCaprio et al., 1989; Dick et al., 2000). Cell Cycle Repression Pocket protein dephosphorylation occurs from anaphase to G1 or in response to growth inhibitory signals (Ludlow et al., 1993). Phosphorylation events can also lead to permanent inactivation of the pocket protein and possibly target it for degradation (Ma et al., 2003). The type of functional effect that pocket proteins have on a cell is dependent on what type of coaccessory proteins interact with the target gene (Stevaux et al., 2005). To repress gene transcription needed for the G1 to S-phase transition, the pRb binds directly to the transactivation domain of E2F. pRb also recr uits chromatin remode ling factors such as histone deacetylase I (HDAC1), SUV39H1, hBRM, and BRG1. These factors act on the surrounding nucleosome structure to remodel it, and promote histone acetylation/deacetylation and methylation events (Shao and Robbins, 1995). HDAC1 is recruite d to E2F complexes by pRb to function in repressing cyclin E gene expression (Magnaghi-Jaulin et al., 1998). SUV38H1 is a methyltransferase that methylates K9 of histone H3 and cooperate s with pRb in the repression of

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51 E2F-responding promoters (Rea et al., 2000). hBRM and BRG1 are the mammalian homologs of SNF2/SWI2, which are yeast chromatin remode ling complexes and they associate with pRb (Strober et al., 1996; Kang et al., 2004). E2Fs and Pocket Proteins in Skeletal Muscle pRb is required for muscle differentiation and fo r transcription of myoge nic bHLH factors. Functional Rb is required for the activity of MyoD-mediated transcriptional activation of myogenic genes (Gu et al., 1993). In models of inactive Rb, myoblas t differentiation was inhibited in vitro and terminally differentiat ed myotube nuclei were able to reenter the cell cycle. In the absence of pRb skeletal muscle cells exhibited ec topic DNA synthesis and/or apoptosis, and pRb null mice exhibit severe defect s in skeletal muscle (Schneider et al., 1994; Novitch et al, 1996; Zacksenhaus et al., 1996; Novitc h et al., 1999; de Brui n et al., 2003; Wu et al., 2003). Interestingly, E2Fs in complex with p130 accu mulate in cells such as, myoblasts, and melanocytes undergoing terminal differentiation (Shi n et al., 1995). In rat L6 myoblasts, p107 is normally involved in regulation of E2F proteins during cell cycle progression. Exponentially growing L6 myoblasts demonstrate complexe s of E2F and bound p107 throughout the cell cycle. During the differentiation of L6 cells, p107 leve ls are reduced and p130 levels are greatly increased, suggesting that p130 is a differentiation-specific regulator of E2F activity (Kiess et al., 1995). In summary, skeletal myogenesis is regul ated by a multitude of growth factors and signaling events. Since most studies have focused on the role of BMPs in embryonic development, little is known about its role in postnatal muscle. E2Fs are critical mediators of cell cycle progression. Satel lite cells are requisite for growth and repair of skeletal muscle. It is not well understood how satellite ce lls are activated to enter the cell cycle nor how they exit the cell cycle to return to quiescence. E2F5 appears to have some role in Raf-arrested myoblasts and

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52 BMP6 expression is high in these Raf-arrested cel ls. Therefore, these data will further assess the role of BMP6 and E2F5 in regulati on of skeletal myogenesi s and/or satellite cell bi ology.

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53 Table 1-1. Receptor and R-Smad specificity for TGF superfamily members Ligand Type II Receptor Type I Receptor R-Smad Activin Act-R-II/IIB ALK4 Smad2,3 TGF TGF RII ALK5 ALK1 Smad2,3 Smad1,5,8 BMP BMPRII ActRII/IIB ALK3 ALK6 ALK2 Smad1,5,8 Smad1,5,8 Smad1,5,8

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54 Table 1-2. TGF superfamily transgenic knockouts* Genes Phenotypes References Ligands Bmp2 Delayed primitive streak, small allantois, lack of amnion, heart defects, decreased number of PGCs. Zhang and Bradley, 1996; Ying and Zhao, 2001 Bmp3 Increase bone density Baham onde and Lyons, 2001; Daluiski et al., 2001 Bmp4 Lack of allantois and PGCs, posterior truncation, heart defects, and lack of optic vesicle; heterozygotes-cystic kidney, craniofacial malformations, microphthalmia, Winnier et al., 1995; Dunn et al., 1997; Furuta and Hogan, 1998; Lawson et al., 1999; Ying et al., 2000 Bmp5 Short ear phenotype including defects in skeleton, lung, and kidney Green, 1968; Kingsley et al., 1992; King et al., 1994 Bmp6 Delayed sternum ossification Solloway et al., 1998 Bmp7 Skeletal defects, kidney agenesis, eye defects Dudley et al., 1995; Luo et al., 1995; Wawersik et al., 1999 Bmp8a Defects in spermatogenesis and epididymis Zhao et al., 1998 Bmp8b Defects in PGC formation, testis cord formation, and spermatogenesis Zhao et al., 1996; Ying et al., 2000; Yao et al., 2002 Bmp11 (Gdf11) Defects in A-P patterning of axial skeleton McPherron et al., 1999 Bmp12 (Gdf7) Hydrocephalic abnormalities growth defects in seminal vesicle Lee et al., 1998; Settle et al., 2001 Bmp15 Subfertile due to defects in oogenesis Yan et al., 2001 Gdf1 Defects in left/right asymmetry Rankin et al., 2000 Gdf5 Brachypodism (shorterned skeleton in limbs and reduced number of digit bones) Storm et al., 1994; Storm and Kingsley, 1996 Gdf8 (Myostatin) Skeletal muscle hypertrophy McPherron et al., 1997 Gdf9 Sterile due to defects in oogenesis Dong et al., 1996 Nodal No gastrulation (lack of primitive streak); anterior neural pattern defects; placenta defects (increased number of trophoblast giant cells) Conlon et al., 1994; Varlet et al., 1997; Lowe et al., 2001; Ma et al., 2001; Zhou et al., 1993 Lefty1 Abnormal left-right axis (left isomerism) Meno et al., 1998 Lefty2 Extended streak, excessive mesoderm, left isomerism Meno et al., 1999 Activin a Lack whiskers and low incisors and cleft palate Matzuk et al., 1995b Activin b Defects in eyelid development and female reproduction Vassalli et al., 1994 Inhibin Ovarian cancer Matzuk et al., 1992 MIS Pseudo-hermaphrodites (female reproductive tract present in males) Behringer et al., 1994 Tgf 1 Hyperactive immunity and defects in angiogenesis Shull et al., 1992; Dickson et al., 1995

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55 Table 1-2 continued. Genes Phenotypes References Tgf 2 Perinatal lethality due to multiple defects in heart, lung, limb, spinal column, eye, inner ear, and urogenital system Sanford et al., 1997 Tgf 3 Cleft palate Proetzel et al., 1995 Receptors Alk1 Defects in embryonic angiogenesis Oh et al., 2000 Alk2 (ActrIA) Defects in mesoderm formation as a result of defective visceral endoderm Gu et al., 1999; Mishina et al., 1999 Alk3 (BmprIA) Defects in epiblast proliferation and no mesoderm formation in null mutants, impaired cardiac and limb development in conditional mutants Mishina et al., 1995; Ahn et al., 2001; Gaussin et al., 2002 Alk4 (ActrIB) Defects in epiblast differentiation and lack of mesoderm formation Gu et al., 1998 Alk6 (BmprIB) Defects in seminal vesicle development, female reproduction, and limb skeletal formation Baur et al., 2000; Yi et al., 2000; Yi et al., 2001 BmprII Defects in gastrulation/lack of mesoderm Beppu et al., 2000 TgfbrII Defects in vasculogenesis and hematopoiesis Oshima et al., 1996 ActrIIa Deficiency in reproduction due to suppressed FSH and mild defects in skeletal development Matzuk et al., 1995a ActrIIb Defects in axial patterning and left-right asymmetry (45% right isomerism) Oh and Li, 1997 *Adapted from Zhao, G.H. 2003. Consequences of Knocking out BMP signaling in the mouse. Genesis 35:43-56. Table 1, pgs.45-46.

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56 CHAPTER 2 MATERIALS AND METHODS Myoblast Cell Culture C2C12 skeletal muscle satelli te cells (Blau et al., 1985) were grown in Dulbeccos modified Eagles medium (DMEM) containi ng 10% fetal bovine serum (FBS), 1% v/v penicillin/streptomycin, and 0.1% v/v gentamyci n. 23A2 skeletal muscle cells (Konieczny and Emerson, 1984) were maintained in Basal Medi um Eagle (BME) supplemented with 15% FBS, 1% v/v penicillin/streptomycin, 0.1% v/v gentam ycin reagent solution, 1% v/v L-glutamine. 23A2RafERDD myoblasts were derived from the parent al 23A2 myoblasts and stably express a tamoxifen-inducible chimeric Raf protein. The estrogen receptor fused to the Raf kinase domain is unstable in the absence of the estrogen an alog 4-hydroxytamoxifen (4HT). Addition of 4HT binds to the estrogen receptor a nd allows for a dose-dependent incr ease in Raf protein expression and kinase activity that initiates downstream ERK1/2 activation (Wang et al., 2004). 23A2RafERDD myoblasts were maintained in the same media as 23A2 cells with the addition of 10mM puromycin (Wang et al., 200 4). All culture media, supplements, and sera were purchased from Invitrogen, Carlsbad, CA. For induction of stable Raf expression, cells were washed with phosphate buffered saline (PBS), treated with 10 g/ml protamine sulfate (CalBioChem, San Diego, CA) in serum-free BME for 10 minutes. Ce lls were starved in serum free BME for one hour and treated with 1 M 4-hydroxytamoxifen (4HT; Sigma, St Louis, MO) in 2% FBS BME. Cells for immunofluorescence were cultured on 35 mm glass-bottomed plates (World Precision Institute, Sarasota, FL) coated with 10% v/v BD Matrigel Matrix HC (BD Biosciences, San Jose, CA).

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57 Growth Factor Treatment, and BrdU Pulsing and Fixation C2C12 cells were seeded on gelatin-coated ti ssue culture plates at a concentration of 1x105 cells/well in a 6-well cluster. Myoblasts were treated in the absence and presence of human recombinant BMP6 (R&D Systems, Minneapolis, MN) at final concentration of 25 ng/ml for 48 hours in 2% FBS DMEM. When nece ssary, cells were pulsed with 10 M bromodeoxyuridine (BrdU) during the last thirty minutes of treatme nt and then fixed in 70% Ethanol (EtOH) at 4 C for 30 minutes. Plasmids and Transfections Semi-confluent myoblasts were transfected by calcium phosphate precipitation formation. In brief, each well received DNA precipitate containing 1 g of luciferase reporter, 0.5 g of activator plasmid, 0.5 g of kinase, and 50 ng of pRL-tk, a Renilla luciferase plasmid used as a monitor of transfection efficiency. The lucifera se reporter plasmids used in the study were a multimerized BMP response element (BRE-luc) (Korchynskyi and ten Dijke, 2002), the internal response element of the quail troponin I gene (T nI-luc) (Johnson et al., 1996), a multimerized TGF responsive element (3TP-luc) (Wrana et al., 1992), an Immunoglobulin E box reporter plasmid ( E5-Luc) (Johnson et al, 1996), and E2F-TA -luc (Clontech, Mountain View, CA). Activator plasmids used were CMV-E47 (Page et al., 2004), CMV-E2F5 (OriGene Technologies Inc., Rockville, MD), and pEM-MyoD (Page et al., 2004). Cells were maintained in growth media or differentiation-permissive medium su pplemented with human recombinant BMP6, and TGF (R&D Systems, Minneapolis MN) for 48 hours prior to lysis and measurement of luciferase and -galactosidase or Renilla luciferase activities (Promega, Madison, WI). Final concentrations of BMP6 were 1, 10, 25, or 100 ng/ml. TGF 1 was supplemented at 10 ng/ml.

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58 BrdU Staining and BrdU Incorporation Cells were fixed in 70% ethanol for 10 minutes at room-temperature, followed by incubation in 2N Hydrochloric acid (HCl) for 1 hour at 37 C. Cells were washed well with Phosphate Buffered Saline (PBS) and then incu bated in blocking buffer (PBS containing 5% horse serum (HS), and 0.1% Triton X-100) for 1 hour. Antigen was detected by incubation with Biotynylated Anti-BrdU IgG (H+L) (BA-2000) at 1:100 (Vector Laboratories, Burlingame, CA) for 1 hour, followed by incubation with HRP-St reptavidin at 1:100 (Vector Laboratories, Burlingame, CA) for 1 hour. Antibodies were diluted in blocking buffer (PBS containing 2% Horse Serum, and 0.1% Triton X-100). Visual ization of BrdU staining was accomplished by addition of one part 3,3-Diaminobenzidine tetr ahydrochloride (DAB) and five parts Nickel Chloride (NiCl) in the presence of H2O2. Percentages of BrdU incorporation were calculated by dividing the number of BrdU positive nuclei by the total number of nuclei. The averages of a minimum of six microscopi c fields at 200X per treat ment are shown. Immunofluorescent E2F5 Staining 23A2RafERDD cells were permeabilized for twenty minutes (1X PBS, 0.01% Triton X100), incubated in blocking buffer for one hour, and then incubated overnight at 4 C with E2F5 antibody (sc-999) (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 in blocking buffer (1X PBS, and 2% Horse Serum). Next, cells were incubated with Alexafluor 488 conjugated anti-rabbit diluted 1:100 in bloc king buffer (Molecular Probes, Ca rlsbad, CA) for 45 minutes at room temperature. Secondary antibody contro ls also were include d to demonstrate the specificity of an antibodys expr ession patterns. Hoechst dye (1 g/ml final concentration) was included as a nuclear stain. Re presentative photomicrographs at 630X under oil immersion were captured with a Nikon 1200DMX digital camera.

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59 RNA Isolation and Nylon Arrays Total RNA was isolated from C2C12 myoblasts and myofib ers and 23A2 myofibers with the use of a Stratagene RNA Easy Kit. E qual amounts (3 ug) of pooled RNA were reverse transcribed with Superscript II (B D BioScience, San Jose, CA) in th e presence of biotin-UTP. Biotinylated cDNAs were used to probe a RT2 Gene Expression Assay, a nylon mini-arrays containing 96 genes coding for various members of the TGF superfamily, their receptors, signaling intermediates, and transcriptional regulators (SuperArray Bioscience Corporation, Frederick, MD). Array membranes were pre-hy bridized with 100 g/ml heat-denatured salmon sperm DNA in GEAhyb Hybridiza tion Solution (SuperArray Bioscien ce Corporation, Frederick, MD) for 2 hours at 60 C. Biotinylated probes were adde d to arrays in GEAhyb solution to hybridize overnight (O/N) at 60 C. Following hybridization, th e membranes were washed twice in 2X SSC (saline sodium citrate), 1% SDS (sodium dodecyl sulfate) and twice in 0.1X SSC, 0.5% SDS for 15 minutes each at 60 C. After washing, membranes were blocked with GEAblocking solution Q for 40 minutes at room temperature. Alkaline phosphatase-conjugated streptavidin (AP) was diluted 1:8,000 with Bu ffer F and incubated with the membrane for 10 minutes at room temperature. Membranes were washed four times with 1X Buffer F for 5 minutes each and then rinsed twice with Buffer G and visualized with CDP-Star chemiluminescent substrate for 2-5 minutes at room temperature (SuperArray Bioscience Corporation, Frederick, MD) and expos ure to X-ray film (XAR-5, Kodak). Western Blots C2C12 myoblasts were differentiated in th e presence or absence of 100 ng/ml BMP6. After 48 hours, the cells were lysed in 4X SD S-PAGE sample buffer (250 nM Tris pH 6.8, 8% SDS, 40% glycerol, 0.4% -mercaptoethanol) and protein con centrations were measured (Bio-

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60 Rad; Bradford, 1976). Equal amounts of protei n were electrophorectic ally separated through denaturing gels and transferred to nitrocellulose membrane. The blots were incubated with 5% nonfat dry milk or 5% bovine serum albumin (BSA) in TBST (10mM Tris, pH 8.0, 150nM NaCl, 0.1% Tween 20) to block nonspecifi c binding sites. Primary antibodi es were diluted in blocking buffer, and the blots were incubated overnight at 4 C with shaking. Antibodies and dilutions included the following: anti-myogenin (F5D ascites, Developmental Hybridoma Bank, University of Iowa, 1:5,000), anti-myosin heavy chain (MF20 hybridoma supernatant, Developmental Hybridoma Bank, University of Iowa IA, 1:5), anti-troponin T (R & D Systems, Minneapolis, MN, 1:2,500), anti-desmin (D3, hybridoma supernatant, Developmental Hybridoma Bank, University of Iowa, IA, 1:10), anti-phospho-Smad1/Smad5/Smad8 (Cell Signaling Technology, Beverly, MA, 1:1,000), anti-p38 (1:5,000), and anti-phospho-p38 (1:5,000) (Cell Signaling Technol ogy, Beverly, MA, 1:2,000). After three times of washing with TBST for 15 minutes each, the blots were reacte d with the appropriate peroxidase conjugated secondary antibody diluted 1:5,000 in blocking buffer for 60 minutes at room temperature. Visualization of protein ba nds was accomplished by chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ) and exposu re to X-ray film (XAR-5, Kodak). Alkaline Phosphatase Staining C2C12 myoblasts were treated for 0, 1, 24 or 48 hours with 100 ng/ml BMP6 or vehicle alone (control). Cultures were fixed with 4% paraformaldehyde and alkaline phosphatase (ALP) activity measured by reaction with 165 g/ml 5bromo-4-chloro-3-indolyl -phosphate (BCIP) and 330 g/ml Nitro Blue Tetrazolium (NBT) color de velopment substrates (Promega, Madison, WI) for 18 hours at 37 C. Representa tive photomicrographs at 100X we re captured under bright field conditions with a 1200DMX dig ital camera (Nikon).

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61 p38 Inhibition Assays Myoblasts were treated with 10 M SB202190 (Upstate Biotechnology, Charlottesville, VA), an inhibitor of p38 for 1, 24, and 48 hour s. SB202190 is a cell permeable pyridinyl imidazole that potently inhibits p38 /SAPK2a and p38 /SAPK2b. This specific p38 inhibitor does not effect ERK activity or SAPK/JNK MAP kinase s. After treatment, cells were fixed with 4% paraformaldehyde and assessed for ALP activ ity measured by reaction with NBT + BCIP or lysed and assayed for TnI-Luc activity. Apoptosis Analysis Myoblasts were treated with 25 M staurosporine (Sigma, St. Louis, MO) for 3 hours. Cells were lysed in 4X SDS-PAGE sample buffer and equal amounts of protein were electrophorectically separated th rough denaturing gels and tr ansferred to nitrocellulose membrane. The blots were incubated with 5% n onfat dry milk in TBST and primary antibodies were diluted in blocking buffer. The blots were incubated overnight at 4 C with shaking. Antibodies and dilutions included the following: anti-Poly (ADP-ribose) polymerase (PARP) (1:1000, Cell Signaling Technology, Inc., Danvers, MA) and anti-Bcl -2 (1:500, BD Biosciences, San Jose, CA). After three times washing for 15 minutes each with TBST, the blots were reacted with the appropriate peroxidase conjugated secondary antibody diluted 1:5,000 in blocking buffer for 60 minutes at room temperature. Vi sualization of protein bands was accomplished by chemiluminescence and exposure to X-ray film. Statistics All data presented represents at least thr ee independent experiment s with a minimum of two to three replicates per trea tment group. All numerical data were compared to appropriate controls and each other as indicated for each ex periment and analyzed following the General

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62 Linear Models (GLM) Procedures of the Sta tistical Analysis Syst em (SAS) (SAS, 1988). Differences between treatments were calculated using predicted differenc es between the leastsquares means of treatment divided by standard deviation. Classes were designated as cells, treatment (trt), replicates (rep), and relative luciferase units (rlu). The statistical model included rlu equals treatment and replicates (rlu = trt rep) as main effect s. Data was presented as Means SEM. Treatments were considered significantly different when P 0.05.

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63 CHAPTER 3 DIFFERENTIAL EXPRESSION OF TGF SUPERFAMILY MEMBERS DURING SKELETAL MYOGENESIS Objective Our previous work demonstrated that activ ated Raf, a key regulator of the MEK/ERK pathway inhibits myogenesis based on its overall signaling intensity (R amocki et al., 1997; Dorman and Johnson, 1999; Page et al., 2004; Wa ng et al., 2004). In myoblasts exhibiting strong Raf/Extracellular-Signal Re gulated Kinase (ERK) signaling, TGF1, GDF8 and BMP6 are up-regulated, sugges ting that these TGF family proteins may serve as autocrine inhibitors of differentiation (Wang et al., 2004). Thes e proteins are members of the TGF superfamily, which consist of the BMP, activin, and TGF subfamilies. Skeletal myogene sis is intricately regulated by differential gene expressi on of these ligands. TGF 1 and GDF8 are inhibitors of proliferation and differentiation (Florini et al., 1986; Green e and Allen, 1991; Rao a nd Kohtz, 1995; Stewart et al., 2003; Allegra et al., 2004). Ablation of MSTN in mouse and the naturally occurring mutations found in some Belgium Blue cattle resu lt in greater muscle mass (Grobet al., 1997; Kambadur et al., 1997; McPherron and Lee, 19 97). Much less is known about BMP effects on myogenesis. The objective of thes e experiments was to examine TGF signaling components during myogenesis in embryonic myoblasts and a pos tnatal satellite cell de rivative. Differential Transcriptional Activity in Myoblasts versus Myofibers Autocrine activity during the transition from myoblasts to myofibers was examined in C2C12 cells transfected with a TGF response element promoter re porter (3TP-Lux) (Wrana et al., 1992), a BMP responsive reporter gene (BRE-L uc) (Korchynskyi and ten Dijke, 2002) or a muscle reporter gene (TnI-Luc). Myoblasts were maintained in differentiation-permissive medium for 48 hours prior to lysis, and lucifera se activity measured. As expected, TnI-Luc

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64 levels were barely detectable in myoblasts and increased approximately 12-fold during the conversion to myofibers (Figure 31). Evaluation of BMP and TGF -driven transcription varied in myogenic cells. In myoblasts, 3TP-Lux relati ve activity is decrease d during differentiation. Conversely, BMP-directed transcriptional activ ity doubled in myofibers as compared to myoblasts (Figure 3-1). The di fferent reporter levels observed in myoblasts and myofibers suggests that autocrine loops are pr esent that may contribute to dist inct stages of myogenesis. Differentiation TGF Gene Expression in Myoblasts and Myofibers Numerical changes in reporter activity s uggested differential expression of TGF proteins during myogenesis. RT2 Gene Expression Assays were used to quantify changes in relative TGF superfamily member gene expression profiles in myoblasts and myofibers. Total RNA was isolated from 23A2 (embryonic myoblasts) a nd C2C12 (adult satellite cells) myofibers and evaluated by ethidium bromide impregnated form aldehyde agarose gels. Distinct 18S and 28S bands demonstrate intact RNA (Figure 3-2A). Equal amounts of RNA were reverse transcribed in the presence of biotin-UTP (Figure 3-2B). The TGF miniarray was hybridized with biotynlated cDNA according to manufacturers recommendations (SuperArray). Results demonstrate Stat1 Noggin and Runx1 transcripts are more abundant in 23A2 myofibers than C2C12 myofibers. By contrast, Bmp3 Bmpr1a Itgb5 and Igfbp3 expression is greater in C2C12 myofibers than 23A2 myof ibers (Figure 3-3). Appendix A provides a complete list of genes and spotting locations. Total RNA was isolated from proliferating C2C12 myoblasts or di fferentiated myofibers and relative gene expression was determined by nylon array. Results demonstrate that BMP ligands and receptors, TGF ligands, and Runxs are expressed differentially during the transition from myoblasts to myofibers. Runx1 mRNA levels are low in mononucleated myoblasts and

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65 increase in multinucleated myofibers; Runx2 mR NA levels are high in myoblasts and low in myofibers and TGF 3 is low in myoblasts and increases in myofibers (Figure 3-4). The BMP ligands are inductive and inhibitory in myogene sis (Tzahor et al., 2003). Expression of BMP1 2 3 4 and 6 were detectable in both myobl asts and myofibers. Closer examination of these BMPs shows that BMP3, BMP4 and BMP6 mRNA levels are greater in mononucleated myoblasts than multinucleated myofibers. BMP2 expression does not appear to differ between myoblasts and myofibers. BMP1 mRNA slightly increase s from myoblast to myofibers (Figure 3-5) Relative amounts of BMP1 3 4 and 6 messages differ in myoblasts versus myofibers suggesting these genes may be invol ved in distinct stages of muscle formation. BMP6 expression is greater in myoblasts than myofibers. Autocrine regulation of BMP6 was examined in C2C12 myofibers. Results demonstrated that BMP6 levels, and other BMP ligands, are not affected by ectopic BMP6 treatment (Figure 3-6). In summary, differential gene expression of TGF superfamily members is detected in different myoblast cell lines and during various stages of myogenesis. BMP6 mRNA is abundant in myoblasts but does not up-regulate it own expression. Discussion Assessment of autocrine activ ity during the transition from myoblast to myofiber by evaluation of BMP-responsive, TGF -responsive or muscle specific reporter elements demonstrated the presence of autocrine loops th at may contribute to distinct stages of myogenesis. TnI-Luc activity drastically increased during th e conversion of myoblasts to myofibers, which confirmed successful differentiation. TGF -sensitivity was high in myoblasts and low in myofibers, s upporting the idea that a TGF -like protein is inhi biting differentiation. Conversely, BMP-sensitivity was low in myoblasts and high in myofibers. The different reporter

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66 levels observed in myoblasts and myofibers furthe r suggests that there are differential responses for TGF and BMPs at different stages of skeletal myogenesis or myoblasts versus myofibers. 23A2 cells are embryonic myoblasts that were derived from C3H10T1/2 embryonic fibroblasts treated with 5-Azacytidine (K onieczny and Emerson, 1984). C2C12 are adult satellite cells derived from mu rine limb muscle (Blau et al ., 1985). While both of these immortalized cell lines exhibit myogenic properties, there are al so marked differences between 23A2 and C2C12 cells. Comparison of embryon ic myoblasts and a dult satellite cells demonstrated detectable gene expr ession differences. Transcripts for Stat1 Noggin and Runx1 are greater in embryonic myoblasts than adult satellite cells. Stat1 is involved in modulating anti-proliferative and growth arrest signals by inducing expression of cell cycle inhibitors, p21WAF1/CIP and pro-apoptotic signals (Durbin et al., 1996; Meraz et al., 1996). The Stat1 knockout mouse demonstrates no overt developmental abnormalities although the mice have a significant increase in bone mi neral density and bone mineral content (Xiao et al., 2004). Greater levels of Stat1 in 23A2 myofibers may explain why ALP induction is greater in C2C12 than 23A2 myoblasts since it may act as a ne gative regulator of osteogenic activity. Conversely, Bmp3 Bmpr1a Itgb5 and Igfbp3 transcripts are greater in adult satellite cells than embryonic myoblasts. Bmp3 is the most abundantly expressed member of the BMP subfamily in both mononucleated myoblasts a nd multinucleated myofibers assessed by nylon array. BMP3 is also the most abundant BM P in adult bone and a major component of osteogenin, which does have osteog enic activity (Wozney and Rose n, 1993; Luyten et al., 1989). However, recombinant BMP3 is unable to induce an osteogenic response in multiple cell lines (Bahamonde and Lyons, 2001; Daluiski et al., 2001). Knockout models demonstrate that Bmp3 is a negative determinant of bone density with null mice exhibiting twice as much trabecular

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67 bone as wildtype counterparts. Additiona lly, BMP3 inhibits BMP2-responsiveness in osteoprogenitor cells and acts as an antagonist of osteoge nic BMPs by activating the TGF /activin pathway, which would antagonize BMP signaling (Bahamonde and Lyons, 2001; Daluiski et al., 2001). Therefore, the abundant gene expression of BMP3 would be important for skeletal muscle formation because high levels of Bmp3 play an essential role in modulating osteogenic BMPs. Specifically, Bmp3 would help inhibit osteogenic conversion of myoblasts through competition for signaling co mponents similar to both the TGF /activin and BMP pathways such as Smad4 (Heldin et al., 1997). Gene expression profiles for myoblasts and myofibers demonstrated differential gene expression such as Runx1 and TGF3 are greater in myofibers than myoblasts and Runx2 is greater in myoblasts than myofibers. Results sugg est that these genes play a role in cell origin and regulate distinct stages of skeletal myogenesis. Runx2 (or Cbfa1) is the master regulator for bone development and the Runx2 knockout mouse demonstrates a complete lack of bone formation and chondrocyte hypertrophy in most of the skeleton (Shum and Nuckolls, 2002). Based on nylon array results, Runx2 is greater in myoblasts than myofibers and this may be a function of the ability of myobl asts to undergo transdifferentiation in response to BMP6. Alternatively, myofibers and fibroblasts do not undergo transdifferentia tion, which may be due to lower levels of Runx2 Nylon arrays demonstrated that there are differential gene expression profiles between 23A2 and C2C12 myofibers. Additionally, bot h lines were found to undergo transdifferentiation in response to ectopic BMP6 treatment, but to different magnitudes which also demonstrates differences in the molecu lar signaling of these immortalized lines. In conclusion, these nylon arra ys allow for a glimpse of di fferential gene expression of embryonic myoblasts and postnatal sa tellite cells, and different stages of skeletal myogenesis.

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68 Verification of these results by Real-Time PCR an alyses are needed with specific primers for each uniquely expressed gene. While previous literature demonstrat es a role for these BMPs in embryonic development and somite patterning (W innier et al., 1995; Zh ang and Bradley, 1996; Dunn et al., 1997; Solloway et al., 1998; Daluiski et al., 2001; Yi ng and Zhao, 2001), the role of BMPs in adult myogenic cells has not been high ly researched and this would determine which TGF superfamily members, specifically BMP gene s, are important for mo lecular regulation of myogenic differentiation and fusion.

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69 Figure 3-1. Differential transc riptional activity in myoblasts versus myofibers. C2C12 myoblasts (1 x 105) were transfected with 1 g of 3TP-luc (TGF -specific), BRE-luc (BMP-responsive), or TnI-luc (Muscle-specifi c). Cells were maintained in growth medium (myoblasts) or differentiation-permi ssive medium (myofibers) for 48 hours. Cells were lysed, and luciferase and Renilla luciferase activities were measured. Means and SEM are from three independent ex periments. Different letters indicates a significant difference, P<0.05.

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70 Figure 3-2. Intact RNA and cDNA probe synthesis. Total RNA was isolated from 23A2 and C2C12 myofibers. 15 g was electrophoretically separated through formaldehyde agarose gels. RNA integrity was visualiz ed with ethidium bromide, 18S and 28S ribosomal RNA are noted (A). One g of RNA was reverse transcribed in the presence of biotin-UTP. Serial dilutions of biotin cDNA were spotted to nylon and incubated with streptavidin HRP. Chemiluminescence demonstrates biotin-UTP incorporation (B).

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71 Figure 3-3. Gene expression prof iles in embryonic and satellite cell myofibers. Total RNA was isolated from 23A2 embryonic myofibers (A ) or C2C12 satellite cell myofibers (B) and equal amounts (3 g) of RNA were revers e transcribed in the presence of biotinUTP. Biotinylated cDNAs were used to probe a TGF /BMP Signaling Array (SuperArray). Following hybridization, the blots were incubated with avidinperoxidase and visualized by chemilumines cence. Representative blots are shown. Significant gene expression di fferences are indicated in the column between the two arrays. Abbreviations: Bmp3 Bone Morphogenetic Protein 3, Bmpr1a Bone Morphogenetic Protein Receptor 1a, Col1a1 Procollagen, type I, alpha 1, Itgb5 Integrin beta 5, Igfbp3 Insulin-like growth factor binding protein 3, Runx1 Runt related transcription factor 1, and Stat1 Signal transducer and activator of transcription 1.

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72 Figure 3-4. Differential gene e xpression in myoblasts and myofib ers. Total RNA was isolated from C2C12 myoblasts (A) or myofibers (B ) and equal amounts (3 g) of RNA were reverse transcribed in the presence of bio tin-UTP. Biotinylated cDNAs were used to probe a TGF /BMP Signaling Array (SuperArray) Following hybridization, the blots were incubated with avidin-peroxida se and visualized by chemiluminescence. Representative blots are shown. Gene Classification Groups are indicated on the right of the diagram. Abbreviations : BMP, Bone Morphogenic Protein, TGF Transforming Growth Factor beta, GDFs, Growth and Differentiation Factors, Ids, Inhibitor of DNA binding, SMAD, MAD ho molog, ECMs, Extracellular Matrix Molecules.

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73 Figure 3-5. Relative BMP gene expression in myoblasts and myofibers. Total RNA was isolated from C2C12 myoblasts an d myofibers and equal amounts (3 g) of RNA were reverse transcribed in the presence of biotin-UTP. Biot ynlated cDNAs were used to probe a TGF /BMP signaling array (SuperArra y). Following hybridization, blots were incubated with avidin-peroxidase and visualized by chemiluminescence. Representative subsections of blots for BMP 1, 2, 3, 4, 5, 6, 10, and 15 are shown. (A) Myoblasts; (B) Myofibers; (C) Schematic location of genes.

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74 Figure 3-6. BMP6 does not undergo autocrine gene regulation. C2 C12 myoblasts were differentiated for 48 hours in the absence (A ) or presence (B) of 100 ng/ml BMP6. Total RNA was isolated and equal amounts (3 g) were reverse transcribed in the presence of biotin-UTP. Biotynlated cDNAs were used to probe a TGF /BMP signaling array (SuperArray). Following hybridization, blots were incubated with avidin-peroxidase and visualized by chemilu minescence. Schematic location of BMP genes (C).

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75 CHAPTER 4 IMPACT OF BMP6 ON SKELETAL MYOGENESIS Objective In Raf-arrested myoblasts, removal of TGF 1 biological activity doe s not restore the myogenic program suggesting that a nother factor is mediating this inhibition (Wang et al., 2004). Ectopic GDF8 treatment does not inhibit 23A 2 myoblast differentiation. BMP6 mRNA is present in myoblasts and absent in myofibers suggesting an inhibitory action during myogenesis (Derynck, 1989). The balance of proliferation, cell differentiati on, and apoptosis mediates the pool of myoblasts available fo r skeletal myogenic maintenance. The objective of these experiments was to measure BMP6 effects on myoblast proliferation, differentiation, and apoptosis. Inhibition of Skel etal Myogenic Differe ntiation by BMP6 To further examine the role of BMP6 in embryonic myoblasts and satellite cells, 23A2 myoblasts and C2C12 satellite cells were transien tly transfected with TnI-Luc and treated with 0 or 100 ng/ml BMP6 for 48 hours in differentiati on media prior to lysis and measurement of luciferase activity. Results show that treatment with 100 ng/ml BM P6 resulted in more than an 80-fold inhibition of muscle specific reporte r (TnI-Luc) activity in both myogenic cell types (Figure 4-1, P<0.05). Due to the significant bioc hemical inhibition of differentiation by BMP6, assessment of morphological cha nges was performed. C2C12 myobl asts were differentiated in the presence of 100 ng/ml BMP6 for 48 hours. Subsequently, cells were fixed and immunostained for MyHC. Results show very fe w multinucleated fibers in response to BMP6 treatment (Figure 4-2A). Total cell lysates were isolated from a second set of C2C12 myofibers treated in an analogous manner. Equal amounts of protein (10 g) were analyzed by Western blot for contractile and regulator y protein expression. Inhibition of the muscle contractile protein

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76 MyHC, in addition to, myogenin, and troponin T protein markers wa s observed in response to BMP6 treatment (Figure 4-2B). Thus, BMP6 inhibits the complete differentiation program. Dose-Dependent Effects of Recombinant BMP6 on Myoblasts BMP 2 4 and 7 have opposing activities that ar e concentration-dependent during embryonic muscle growth (Amthor et al., 1998 ; Amthor et al., 2002). Due to the high concentration of BMP6, a retrospective dose-re sponse experiment was performed (1 ng/ml to 50 ng/ml BMP6). Biochemical di fferentiation was measured follo wing transient transfection of TnI-Luc into C2C12 myoblasts. Cells were tr eated for 48 hours in differentiation media with increasing concentrations of BMP6. Analysis of luciferase reporter activity shows an approximate 20% decrease of TnI-Luc activity at 25 ng/ml (P<0.05) and an approximate 50% decrease at 50 ng/ml (P<0.001) (Fig ure 4-3A). Parallel plates we re lysed for protein analyses. Equal amounts of total cell proteins were separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with anti-M yHC and anti-myogenin. Results demonstrate that MyHC protein expression begins to decrease at 50 ng/ml BMP6 (Figure 4-3B). Interestingly, myogenin protein expression begins to decrease at 10 ng/ml BMP6, further at 25 ng/ml and is completely absent in response to 50 ng/ml BMP6 (Figure 4-3B). Induction of ALP Activity in Response to BMP6 BMPs induce bone or cartilage formation ectopi cally (Urist, 1965; Gitelman et al., 1995). BMP2 inhibits the myogenic differentiation of C2C12 cells, by converting their differentiation pathway into that of osteoblast lin eage cells (Katagiri et al., 1994). To determine if the block to differentiation is associated with transdiffere ntiation, assessment of alkaline phosphatase (ALP) activity of myofibers tr eated with BMP6 was measured. C2 C12 myoblasts were treated with vehicle only or increasing amounts of BMP6 in differentiation media for 48 hours. Cells were

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77 fixed and histologically stained for ALP activit y, a marker enzyme of osteogenic cells. As shown in Figure 4-4, as little as 5 ng/ml BMP6 was sufficient to induce ALP activity. To determine if the effect of BMP6 is unique to the committed myoblast, C3H10T1/2 fibroblasts were treated with 0, 1, 10, and 100 ng/ml BMP6 in differentiation media for 48 hours. Cells were fixed and histologically stained for ALP activity. Results indicated that BMP6 does not cause ALP induction in C3H10T 1/2 fibroblasts (Figure 4-5). BMP6 Induces Rapid Transdi fferentiation in Myoblasts C2C12 myoblasts were treated with 100 ng/ml BMP6 for 24 and 48 hours. Subsequently, cells were fixed, and ALP activity was measured. Results show that ALP activity is observed as early as 24 hours and further increases at 48 hours (Figure 4-6). C2C12 cells treated with vehicle alone also demonstrated a slight induction of ALP at 24 and 48 hours indicating endogenous ALP activity. BMP6 Does Not Alter Prolifera tion Rates of Myoblasts Cell cycle withdrawal or inhibition of prolif eration is required for differentiation and fusion of myofibers. To determine if the bloc k to myogenic differentiation by BMP6 is due to altered cell proliferation, C2C 12 myoblasts were treated with 25 ng/ml BMP6 for 48 hours. Cells were pulsed with BrdU during the final thir ty minutes of the treatment interval, fixed with methanol, and immunostained for BrdU (Figure 4-7A ). The numbers of nuclei were not different between control and BMP6 treated cells (Fi gure 4-7B, P<0.05). Ther efore, exposure of myoblasts to BMP6 does not al ter cellular proliferation. BMP6 is not Anti-Apoptotic Previous reports indicate that BMP2 a nd 4 promote cell survival in pluripotent mesenchymal cells by inhibiting TNF-mediated apoptosis (Che n et al., 2001). BMP6 was demonstrated to partially restore survivability in human mesenchymal stem cells (hMSCs)

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78 induced by the BMP antagonist sclerostin (Sutherl and et al., 2004). Bcl-2 is an anti-apoptotic protein expressed abundantly in sa tellite cells (Krajnak et al, 2 006). The protective actions of BMP6 were examined in C2C12 myoblasts treated with staurosporine. C2C12 satellite cells were treated with increasing c oncentrations of BMP6 for 48 hours and Bcl-2 protein content measured by Western blot. Results demonstrat e no changes in Bcl-2 protein concentration following BMP6 treatment (Figure 4-8A). Subs equently, myoblasts were treated for 3 hours with BMP6, 25 M staurosporine or BMP6 and staurosporin e. Cells were lysed and assayed for Poly (ADP-ribose) polymerase (P ARP) protein expression by West ern blot analysis. Results demonstrate that staurosporine s timulates PARP cleavage, a hallmar k of apoptosis (Figure 4-8B). BMP6 does not prevent PARP cleavage in C2C12 myoblasts treated with staurosporine, thus precluding an anti-apoptotic function (Figure 4-8B). In summary, BMP6 significantly inhibits the complete differentiation program as observed by biochemical suppression of muscle specific re porter activity, and morphological disruption of myofiber formation and muscle-specific protein expression. This inhibi tory effect is dose dependent and results in rapid transdiffere ntiation of committed mesodermally-derived myoblasts to an osteogenic lineage. BMP6 doe s not inhibit differentiation by promotion of a proliferative state. Nor does BMP6 serve as an anti-apoptotic factor in myoblasts. Discussion BMP6 is greater in myoblasts than myofibers, and up-regulated in Raf-arrested myoblasts. Therefore, the function of BMP6 on proliferation, differentiati on, and apoptosis in skeletal myogenesis was evaluated. Treatment of 23A 2 and C2C12 myoblasts with 100 ng/ml BMP6 significantly inhibited muscle specific activity (TnI-luc). Muscle fiber fusion and muscle specific protein expression of myosin heavy chain (MyHC), myogenin and Troponin T are also

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79 decreased in response to BMP6 treatment. Resu lts suggest that BMP6 inhibits the complete differentiation program. Other members of the TGF superfamily can also inhibit di fferentiation. One of the most notable examples is GDF8 or myostatin. Myostatin is mutated in double muscled cattle breeds, Belgium Blue and Piedmontese (Grobet et al., 1 997; Kambadur et al., 1997; McPherron and Lee, 1997). It is predominately e xpressed in the muscle and negatively regulates myogenic proliferation. The myostatin knockout mouse also demonstrates two times larger muscle mass as compared to wild type counterparts (Thomas et al., 2000). Therefore, TGF superfamily members are critical mediators of skeletal myogenesis. BMP6 is a morphogen, and morphogens are charact erized by exhibiting different effects at different levels or concentra tions. In pre-myogenic cells, BM P2, 4, and 7 have dose dependent effects with low concentrations maintaining a Pax3-expressing pro liferative population and delaying differentiation. Convers ely, high concentrations of these BMPs prevent muscle development (Amthor et al., 1998). In the pr esence of low BMP levels, myogenic precursor cells are maintained in a pr oliferative state in developi ng limb bud, while high BMP levels induce cell death. Thus, BMPs can both stimulate and restrict muscle growth (Amthor et al., 1998; Amthor et al., 2002). This suggests that a concentration gradient of BMPs is needed for the correct determination and maintenance of the myogenic program (Centrella et al., 1994; Alliston et al., 2001; Reddi, 1994). 23A2 myoblasts treated with increasing concentrations of BMP6 were measured for muscle specific repo rter activity and MyHC and myogenin protein expression. Results demonstrated that muscle repor ter activity is significan tly decreased at both 25 ng/ml and 50 ng/ml. MyHC protein expression began to decrease at 50 ng/ml BMP6 and myogenin decreased at 10 ng/ml BMP6. This suggests that BMP6 exhibits dose dependent

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80 effects on contractile and regulat ory muscle proteins. Smad1 a nd Smad5 binding sites are found within the promoter of myogenin which allow BMP6 to induce downstream Smad signaling. C2C12 cells transiently transfected with Smad1 and Smad5 were able to induced ALP activity and decrease myogenin/chloramphenical acetyl transferase (myogenin-CAT) activity. Although in NIH3T3 fibroblasts, Smad1 and Smad5 decr eased myogenin-CAT but did not induced ALP activity, which demonstrates that Smad1 and Sm ad5 are involved in the BMP signaling that inhibits myogenic differentiation and induces tr ansdifferentiation. Furt hermore, the conversion of these two differentiation path ways is regulated independently at the transcriptional level (Yamamato et al., 1997). Additiona lly, high levels of BMP6 impact or repress stages of skeletal myogenesis prior to te rminal differentiation. Promotion of osteogenic differentiation by BMP2 expression in skeletal muscle-derived C2C12 cells (Musgrave et al. 2001) demonstrated that myoblasts can undergo transdifferentiation from a myoge nic to osteogenic cellular lin eage in response to BMP. ALP staining measures osteogenic activity and ectopic BMP6 treatment of C2C12 myoblasts demonstrated a dose dependent induction of AL P activity. Additionally, when C2C12 myoblasts were treated for 24 and 48 hours, induction of ALP activity in response to BMP6 treatment demonstrated a time dependent transdifferentiation of myofibers into an osteogenic lineage. The substantial induction in the numbers of AL P positive cells in C2C12 cultures maintained in low serum at 24 and 48 hours also suggests a ba sal level of osteogenic activity in C2C12 myofibers. These cells are m ononucleates and not myofibers. Comparison of gene transcripts between myoblasts and myofibers by nylon arra y suggests that greater transcripts of Runx1 could explain why these cells demonstrate a basal level of osteogenic ac tivity. Furthermore, BMP1 3 and 4 transcripts are expressed in both untreated and BMP6 treated myoblasts, but only BMP4

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81 displays osteogenic activity, which could c ontribute to endogenous ALP activity of C2C12 myoblasts. BMPs 2, 4, 6, 7, and 9 are characterized as osteogenic BMPs, with BMPs 2, 7, 6, and 9 displaying the highest osteogenic activity when applied ectopically in vitro and in vivo (Luu et al., 2007). BMP1 is a metalloprotease that regulates de position of fibrous extracellular matrix (ECM) in vertebrates and does not display osteoge nic activity (Bond and Beynon, 1995). It provides procollagen C proteinase (PCP) ac tivity to cleave the C propeptides of procollagens I-III to yield the major fibrous components of ECM (Kessler et al., 1996; Li et al., 19 96; Suzuki et al., 1996; Scott et al., 1999). In bone, BMP1 co-purifies with TGF -like BMPs from osteogenic extracts of bone and is believed to coordinate the depos ition of ECM with the acti vation of certain BMPs in early development and later in the developm ent of bone and other tissues (Wozney et al., 1988; Scott et al., 2000). Furthermore, BMP3 would not contribute to endogenous ALP activity because as mentioned previously, recombinant BMP3 does not display osteogenic activity and acts as an antagonist of osteogenic BM Ps (Bahamonde and Lyons, 2001; Daluiski et al., 2001). Conversely, exposure of C3H10T1/2 fibroblasts to a BMP6 dose-response curve did not induce ALP activity at any dosage for 1, 10, and 100 ng/ml BMP6. The differential responses observed in C2C12 and C3H10T1/ 2 cells suggest that BMP6-med iated transdifferentiation is specific to lineage restricted ce lls. Similar results of a signifi cant ALP induction in C2C12 cells by BMPs (2, 4, 6, 7, and 9) was reported by other groups. C2C12 cells demonstrated the most potent ALP induction (Ebisawa et al ., 1999). Therefore, the origin of the cell line may determine if the cells will respond to BMPs and/or the ove rall magnitude of the ALP response. Yang et al., (2003) did demonstrate induction of ALP activity and stimulation of osteoblast marker genes by 300ng/ml of recombinant BMP6, whereas, our expe riments never used a dosage higher than

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82 100ng/ml BMP6. Another group demonstrated an induction of ALP activity in C3H10T1/2 fibroblasts in response to 100 ng/ml BMP2, s uggesting that other BMP family members or higher dosages of BMP6 may have different impacts on fibroblasts. Additionally, BMP2mediated ALP induction in C3H10T1/2 fibroblasts was increased in the pr esence of HGF (Imai et al., 2005). Therefore, the addition of HGF with BMP6 migh t result in a detectable ALP induction in C3H10T1/ 2 fibroblasts. Comparison of BMP2 and BMP6 finds that both are expressed in skeletal muscle cells but are in different classes of the BMP subfamily (L yon et al, 1989). BMP2 is a member of the DPP class along with BMP4, whereas, BMP6 is a memb er of the 60A class along with BMP5, 7, 8A, and 8B (Gitelman et al., 1997). Structurally, BMP2 contains 3 exons, two of which encode the precursor protein (Feng et al., 1994). The non-coding exon refers to an exon located on the 5 flanking region of the DNA that has been shown to serve as an alternate promoter, which suggests that BMP2 is regulated in both a deve lopmental and tissue-specific manner (Gitelman et al., 1994). BMP6 is composed of 7 coding exon s with the mature protein encoded by 3 full exons and a portion of the fourth exon and is al so developmentally regulated. While BMP2 and BMP6 are members of the same subfamily, there is little similarity in the localization of the intron-exon structures further demo nstrating differences at the stru ctural level (Gitelman et al., 1994). In vivo analyses demonstrates that BMP6 null mice are viable and fertile and exhibit no major defects in known BMP6-expressing tissues, excep t for a delay in ossification restricted to the developing sternum (Solloway et al., 1998). It is believed that BMP2 may be functionally compensating for BMP6 ablation since BMP2 and BMP6 are required for some overlapping or redundant functions (Solloway et al., 1998). Based on nylon arrays, BMP6 is greater in

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83 myoblasts than myofibers, yet BMP2 and BMP4 transcripts dont appear to differ in myoblasts and myofibers. Conversely, when the muscles of athymic nude rats were injected with adenoviral vectors for BMP6 (AdBMP6), a ra pid tissue calcificati on was observed. The induction of bone was produced through mechanis ms similar to both intramembranous and endochondral ossification pathways and AdBMP6 was even more potent than the prototypical adenoviral vector AdBMP2 (Jane et al., 2002). Thes e studies utilized titers of BMP6 that would not mimic physiological conditi ons but do demonstrate the powe rful osteogenic activity of BMP6. BMP2 null mice exhibit multiple developmental defects including a delayed primitive streak, small allantois, lack of amnion, heart defects and a decreased number of primordial germ cells (Zhang and Bradley, 1996; Yin and Zhao, 2001). Therefore, BMP2 and BMP6 appear to have different biologic al functions. It has also been suggested that receptor oligomerization determines BMP2 signaling pathways. Nohe et al., (2002) demonstrated that binding of BMP2 to preformed receptor complexes activates the Smad pathway. Conve rsely, BMP2-induced recruitment of receptors activates a Smad-independent pathway, which re sults in the induction of ALP activity via p38 MAPK (Nohe et al., 2002). These different re ceptor complexes may also recruit different adaptor proteins such as, XIAP (Yamaguchi et al., 1999), BRAM-1 (Kurozumi et al., 1998), and FKBP12 (Wang et al., 1996). BMP2 treatment of C3H10T1/2 cells stimulates ERK1 and ERK2 during osteoblastic differentiation (Lou e al., 2000), and ERK activati on can inhibit nuclear translocation of Smad1, which w ould block the Smad pathway (Kretzschmar et al., 1997). Our experiments demonstrate a strong Smad1/5/ 8 activation by BMP6 and only a slight p38 activation suggesting that BMP6 induces ALP ac tivity through a different signaling mechanism than BMP2.

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84 Characterization of BMP6 and BMP2 in C2C 12 cells demonstrates that BMP6 strongly binds to activin receptor-like kinase (ALK)-2 or ActR-I (Ebisa wa et al., 1999). ALK2 forms complexes with receptors like, BMPR-II or ActR-II. BMP6 can also weakly bind to ALK3, which also can bind BMP2 (Ebisawa et al., 1999 ) but BMP2 preferentially binds to BMPRIA and IB. C2C12 cells only express mRNA for BMPRIA (Akiyama et al., 1997). C3H10T1/2 fibroblasts express both BMPR IA and BMPRIB but BMPRIA expression levels are endogenously higher. Transfection experiment s with BMPRIA and BMPRIB in C3H10T1/2 cells also demonstrate that the dominant role in BMP2 mediated osteogenic development was mediated by BMPRIA, with BMPRIB only partially influencing osteogen ic development (Kaps et al., 2004). Additionally, type IB and IA BMP receptors appear to transmit different signals during the specification and diffe rentiation of mesenchymal lin eages (Kaps et al., 2004). Truncation and overexpression of BMP receptors BMPRIA and BMPRIB, have demonstrated that overall receptor levels e xpressed in cells play a critical role in specification and differentiation of osteoblasts by BMP2 (Chen et al., 1998). Therefore, while BMP2 and BMP6 can interact with the same BMP receptor, they demonstrate the strongest affinity for different type I receptors. Therefore, differences in signaling could be based on like receptor oligomerization, cross-talk with other signaling pathways, competition for signaling components, interaction with additional prot eins, and activation of differe nt downstream transcriptional targets, which would result in different biological responses of BMP2 and BMP6. Transdifferentiation of myogeni c cells to an osteogenic lina ge results in induction of specific markers at the transcri ptional level. Therefore, m easurement of genes such as, Runx1 Runx2 osteopontin osteonectin and osteocalcin (Ahrens et al., 1993) would determine if the cells were exhibiting more bone-like characteris tics and what genes are up-regulated in BMP6-

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85 treated myoblasts. Interestingly, based on the ny lon array assessment of myoblasts versus fibers, Runx1 appeared to be greater in myofibers, yet Runx2 appeared to be greater in myoblasts. Runx2 is required for later stages of chondr ocyte and osteoblast differentiation, while Runx1 mediates early events of endochondral and intram embranous bone formation (Smith et al., 2004). These results would need to be confirmed by Real-Time PCR and may have implications in transcriptional regulation of the myoblast to m yofiber transition and/or transdifferentiation of myoblasts to osteoblasts. Since myogenic precursor cells (MPCs) also have the ability to form skeletal muscle, bone, or cartilage, the differentia l expression of these tr anscription factors may demonstrate how these precursor cells de termine their ultimate cell fate. Inhibition of embryonic skeletal muscle differentiation through promotion of a proliferative state was monitored. Others have suggested that Raf inhib its skeletal myogenesis by keeping myoblasts in a proliferative state (S amuel et al., 1999). Sinc e BMP6 expression is high in Raf-arrested myoblasts, it was proposed that BMP6 may also be promoting a proliferative state of cells. When myoblasts were treated w ith BMP6, the number of BrdU positive nuclei or cells in S-phase was not significantly different from untreated myoblasts. The percent BrdU incorporation was calculated by dividing the number of BrdU positive nuclei by the total number of cells and taking the average of six fields. There also was no significant difference in BMP6 treated percent BrdU incorp oration versus control myoblasts. Therefore, exposure of myoblasts to BMP6 does not inhib it proliferation suggesting that BMP6-mediated inhibition of differentiation is not mediated through modulation of pr oliferative events. Conversely, another group did demonstrate an inhibition of proliferation by [3H] thymidine incorporation assays in a dose-dependent manne r (Ebisawa et al., 1999). Comparatively, this study did a dose response up to 1000 ng/ml BM P6 and only observed approximately a 20%

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86 decrease at 300ng/ml, whereas our analyses asse ssed BrdU incorporation at 25 ng/ml, which is considered a more physiological dose. Apoptosis is a mechanism of programmed ce ll death that promotes tissue turnover, embryonic development, and immunological defe nse mechanisms (Kerr et al., 1972; Adams, 2000; Siu et al., 2005). The intrinsic and extrin sic signaling pathways are the two principal pathways involved in apoptosis. The intrinsi c route uses cell signaling pathways to alter mitochondrial function. Permeabiliza tion of the outer mitochondrial membrane results in release of cytochrome c, thereby forming an apoptos ome. This macromolecular complex activates caspase-9, mediated by apoptotic protease activign factor (APA F)-1 (Roy and Nicholson, 2000). Conversely, the extrinsic pathway sends death liga nd signals of the the Tumor Necrosis Factor (TNF) or Fas families through appropriate receptors that activate caspase-8 in conjunction with the adaptor molecule, Fas-associated death domain (FADD). At this point, intrinsic and extrinsic cascades converge to activate effector caspases ( caspase-3 and -7), whic h cause the proteolytic degradation of cellular materi al (Roy and Nicholson, 2000). BMP2, BMP4, and BMP6 promote cell survival in mesenchymal cells (Chen et al., 2001; Sutherland et al., 2004). Yet when myoblasts were treated with ectopi c BMP6, Bcl-2 protein expression, an anti-apoptotic marker did not change in response to increasing amounts of BMP6. Additionally, BMP6 did not prev ent PARP cleavage in response to staurosporine, a common inducer of apoptosis. This suggests that BMP6 does not have an anti-apoptotic function in skeletal myoblasts. BMP6 was able to partia lly protect hMSC cells from sclerostin-induced apoptosis by decreasing caspase activity (Sutherland et al., 2004). Sclerostin is a BMP antagonist that binds to BMPs and blocks downstream signaling pathways. Furthermore, the protective effect of BMP6 obser ved was only a partial block of sclerostin-mediated apoptosis

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87 suggesting that additional factor s are involved (Winkler et al., 2003). Therefore, the protective effect of BMP6 observed in this scenario may be a result of additiona l signaling components not present in myoblasts or is due to an alternative apoptot ic signaling cascade sp ecific to Sclerostin or different from staurosporine-induced apoptosis. In conclusion, BMP6 inhibits the complete differentiation program in myoblasts. BMP6 treatment results in a rapid transdifferentiation of myoblasts that is specific to a committed mesodermal derived myogenic cell. BMP6 does not appear to involve modulation of proliferation rates of myoblasts. Nor does BMP6 appear to demonstrate a survival role in myoblasts.

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88 Figure 4-1. Biochemical inhibi tion of skeletal myogenesis by BMP6. Myoblasts were transiently transfected with 2 g of TnI-Luc reporter construct and 50 ng pRL-tk (Renilla) and treated 0 or 100ng/ml BMP6 in differentiation media for 48 hours. Cells were lysed and assessed for TnI-L uc activity in both 23A2 and C2C12 myofibers. Reporter luciferase activ ity was normalized to the amount of Renilla luciferase activity and vehicle only was set to 100%. Data represents the mean and standard error of the mean (SEM) of three independent experiments. Different letters indicates a significant difference, P<0.05.

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89 Figure 4-2. Inhibition of skel etal myogenesis by BMP6. C2C12 myoblasts were treated with 100 ng/ml BMP6 for 48 hours. Myoblasts were fixed and immunostained for MyHC expression. Representative microscopic im ages (200X) are shown (A). Parallel plates were lysed and analyzed for muscle specific proteins by Western blot (B). Tubulin expression was used as a loading control.

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90 Figure 4-3. BMP6 dose response curve. C2C12 myoblasts (1x105) were transiently transfected with 2 g of TnI-Luc reporter construct and 50 ng pRL-tk ( Renilla ). Cells were maintained in differentiation media supplemented with BMP6 for 48 hours. Cells were lysed and luciferase activities were m easured (A). Reporter luciferase activity was normalized to the amount of Renilla luciferase activity and the control (vehicle only) was set to 100%. Data represents th e mean and standard error of the mean (SEM) of three independent experiments. Di fferent letters indicates significance at p<0.05. Total cell lysates were analyzed by Western blot for MyHC and myogenin protein expression (B). Tubulin expressi on was used as a loading control.

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91 Figure 4-4. BMP6 induction of alkaline phosphatase activity. C2C12 myoblasts were treated with 0, 5, 10, 25, 50, and 100 ng/ml BMP6 for 48 hours. Cells were fixed and ALP activity detected colo rimetrically. Representative photomicrographs at 200X are shown.

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92 Figure 4-5. BMP6 does not i nduce alkaline phosphatase (ALP ) activity in fibroblasts. C3H10T1/2 fibroblasts were treated with 0, 1, 10, and 100 ng/ml BMP6 for 48 hours. Cells were fixed, and stained for ALP activ ity. Representative photomicrographs at 200X are shown.

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93 Figure 4-6. BMP6 induces rapi d transdifferentiation in myobl asts. C2C12 myoblasts were treated for 1, 24 or 48 hours with vehicl e alone (control) or 100 ng/ml BMP-6. Cultures were fixed with 4% paraformaldehyde and ALP activity measured colorimetrically. Representative photo-mic rographs at 200X demonstrate intense ALP staining as early as 24 hours of treatment.

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94 Figure 4-7. BMP6 treatment does not alter m yoblast proliferation. C2C12 myoblasts were treated with 25 ng/ml BMP6 in differentiation medium for 48 hours, pulsed with 10 M BrdU for 30 minutes, fixed with 70% ethanol at 4 C for 30 minutes and immunostained for BrdU expression (A). Representative photomicrographs at 100X shown. % BrdU incorporation was calcul ated by dividing the number of BrdU positive nuclei by the total number of nuclei (B ). The averages of a minimum of six fields per treatment are shown.

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95 Figure 4-8. BMP6 is not anti-apoptotic. C2C 12 myoblasts were placed in differentiation permissive media with increasing concentr ations of BMP6 for 48 hours. Whole cell lysates were prepared and analyzed for e xpression of Bcl-2 by Western blot (A). C2C12 myoblasts were treated with 100ng/ml BMP6 in the absence or presence of 25 M staurosporine (STS). Whole cell lysa tes were assessed for PARP cleavage and tubulin protein expression (B).

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96 CHAPTER 5 BMP6 SIGNALING DURI NG SKELETAL MYOGENESIS Objective During skeletal myogenesis, BMP6 actions can be mediated by multiple signaling mechanisms (Figure 5-1). The sp ecificity of the BMP6 and TGF 1 signaling responses are achieved by different types of Type I and Type II receptors and R-Smads (Wrana et al., 1992; Attisano et al., 1993; Ebner et al., 1993; Wieser et al., 1993; Wrana et al., 1994). Traditionally, BMPs signal through Smads 1/5/8, while TGF 1 signals through Smads 2/3. BMPs also initiate p38 signaling and within muscle, this regulation is specific to the p38 isoform. A third, more novel regulation, is through crosstalk with Notch signaling. The objective of these experiments was to validate the presence and activation of three putative intracellu lar signaling cascades induced by BMP6. Analysis of BMP Signaling Systems in Myoblasts The ability of BMP6 and TGF 1 to induce Smad phosphorylati on and nuclear translocation was evaluated. In brief, C2C12 myob lasts were treated with 10 ng/ml TGF 1 and 25 ng/ml BMP6 for 48 hours. Total cell lysates were analyzed by Western blot for total and phosphoSmad1/5/8 expression. Myoblasts treated with vehicle alone demonstrated lows levels of phospho-Smad1/5/8, which were increased in response to both 10 ng/ml TGF 1 and BMP6 (Figure 5-2A). No differences in total Smad1/5/ 8 or tubulin were eviden t. Parallel plates of C2C12 myoblasts treated as described were fixed and immunostained for phospho-Smad1/5/8. In both instances, the signaling molecules are located in the nucleus (Figure 5-2B). Therefore, the archeotypical Smad signaling system is intact and functional in myoblasts. These results also support previous observations that demonstrated expression of Smad1/5/8 in C2C12 cells. Subsequent phosphorylation of Smad5 and weak phosphorylation of Smad1 was also observed

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97 by BMP6. Smad8 was found to be constitutively phosphorylated in C2C12 cel ls (Ebisawa et al., 1999). In addition to signaling through the Smad prot eins, BMPs also may utilize components of the MAPK and Janus kinase (JAK)/signal transd ucers and activators of transcription (STAT) family of signaling proteins, culminating in activation of JNK or p38 (von Bubnoff and Cho, 2001). p38 signaling is a requirement for muscle fo rmation (Zetser et al., 1999; Lee et al.,2002). The interplay of BMP6 signaling and p38 kinase activity during myogenesis was examined. C2C12 myoblasts were treated with increasing amounts of BMP6 and assessed for total and phospho-p38 protein expression by West ern blot. Results demonstrate that while levels of total p38 dont change in response to BMP6, phospho-p38 is slightly induced in myoblasts treated with 25 and 50 ng/ml BMP6 (Figure 5-3). C2C12 myoblasts were transiently transfected with TnI-Luc activ ity and treated with 100 ng/ml BMP6, 10 M SB202190 or a combination of both for 48 hours. Results show 100 ng/ml BMP6 significantly inhibited TnI-Luc ac tivity, as observed previously (Figure 5-4A, P<0.05). Treatment with 10 M SB202190 inhibited TnI-Luc activity (Figure 5-4A, P<0.05). Importantly, the combination of BMP6 and SB2 02190 demonstrated an additive effect indicating independent pathways (Figure 5-4A, P<0.0001). Mo rphological observati ons showed myofibers in control cells, few myofibers in SB202190 treated, and no myofibers in either BMP6 treated or the combination of BMP6 and SB202190 (Figure 5-4B). C2C12 myoblasts were treated with BM P6, SB202910, and BMP6 with SB202910 for 48 hours in differentiation media. Cells were ly sed and assayed for My HC, myogenin, Troponin T, and desmin protein expression by We stern blot. Results demonstrate that muscle specific protein markers (MyHC, myogenin, and Troponin T) are re duced in the presence of BMP6, p38 inhibitor

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98 (SB202910), and the combination (Figure 5-4). C2C12 myoblasts treated with both BMP6 and SB202910 demonstrated a more severe inhi bition of myogenesis. The importance of p38 signaling during BMP6-mediated transdifferentiation was examined. Parallel plates of C2C12 m yoblasts treated with BMP6, SB202190, and the combination of BMP6 and SB202190. ALP activity was detected colorimetrically. Results show that inhibition of p38 func tion does not alter ALP induction in response to BMP6 treatment (Figure 5-6). This further demonstrates that BMP6 is signaling through an independent pathway to cause inhibition of different iation and transdifferentiation. Impact of Notch Inhibitor on BMP6-Med iated Inhibition of Differentiation A third signaling mechanism used by BMP ligands involves Notch. Notch is a transmembrane receptor that is cleaved on th e intracellular surface to release a proteolytic fragment that translocates to the nucleus and a ffects gene transcription (Nye et al., 1994; Ahmad et al., 1995). Notch inhibits myogenic differe ntiation similar to BMP6 (Kopan et al., 1994; Takahashi et al., 1994). BMP4 repression of musc le gene expression invo lves Notch activation (Dahlqvist et al., 2003). The involvement of BMP6 in Notch controlled signaling was examined in C2C12 myoblasts. In brief, m yoblasts were induced to differentia te in the presence or absence of 25 ng/ml BMP6 and 10 M L685,458, a Notch inhibitor, for 72 hours. Cells were fixed and immunostained for MyHC. Results show that MyHC was expressed in control cells and significantly inhibited in BMP6 treated (Figure 5-7A and 5-7B, P<0.05). Treatment of C2C12 myoblasts with L685,458 alone does not alte r MyHC expression a nd interestingly, the combination of BMP6 and L685,458 partially restor es MyHC expression (Figure 5-7A and 5-7B, P<0.05). This suggests that the BMP6-mediate d inhibition of differe ntiation is partially controlled by the Notch signaling pathway.

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99 In summary, differential BMPand TGF -responsiveness and gene expression exists in myoblasts versus myofibers. This suggests that TGF superfamily members play different regulatory roles at various stag es of myogenesis as myoblasts undergo terminal differentiation. Treatment of myoblasts with BMP6 results in a dramatic increase of ALP activity in a dosedependent and time-dependent manner. Exogen ous BMP6 treatment of skeletal myoblasts results in inhibition of myoblast differentiation as observed by signi ficant inhibition of muscle reporter activity, muscle specific protein synthe sis, and myoblast fusion. BMP6 treatment does not alter proliferation rate s of myoblasts or promote a proliferat ive state to inhibit differentiation. Inhibition of p38 activity combined with BMP6 treatment caused inhibition of TnI-Luc activity that was greater than either treatment alone suggesting an additive effect between BMP6 and inhibition of p38 activity. Interestingly, the co mbination of BMP6 and Notch inhibition by L685,458 partially restores MyHC expression in fibers. This suggests that the myogenic inhibitory effect observed in the presence of BMP6 is partially medi ated by functional Notch signaling. Discussion BMPs can be mediated by multiple signaling pathways and these experiments validated the presence and activation of these cascades in skeletal myoblasts. BMPs signal through serine/threonine kinase receptors (Massagu et al ., 1994). In the presence of growth factors, ligands bind to a Type II receptor dimer located on the plasma membrane, which causes autophosphorylation of the Type II dimer and recrui tment and transphosphorylation of a Type I receptor dimer (Wrana et al., 1992; Attisano et al ., 1993; Ebner et al., 1993; Wieser et al., 1993; Wrana et al., 1994). This phosphorylation event recruits the receptor -regulated Smads (RSmads), which then undergo phosphorylation (Aoki et al., 2001). The R-Smads form a complex

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100 with the common-partner Smad (Co-Smad), Sma d4. The R-Smad/Smad4 complex translocates into the nucleus and binds to DNA causing activati on of target genes (Derynck et al., 1996; Liu et al., 1996; Meersseman et al. 1997; Nakao et al., 1997). Co-ac tivators and co-repressors lend additional regulation to the system (Wotton et al., 1999). BMPs signal through Smads1/5/8 and the TGF s typically signal through Smads2/3. BMPactivated Smads, most importantly Smad5, are necessary for inhibition of myogenic diffe rentiation and osteoblas tic induction in C2C12 cells (Lee et al., 2000). Assessment of functiona l Smad 1/5/8 activation was observed in the absence and presence of TGF 1 or BMP6 treatment by immu nostaining for phospho-Smad1/5/8 expression. Untreated C2C12 myofibers demonstr ated low levels of phospho-Smad1/5/8 protein expression, which were increased in response to both 10 ng/ml TGF 1 and 25 ng/ml BMP6 observed by Western analyses. Additionally, nuclear phospho-S mad1/5/8 protein expression was induced in response to 100 ng/ml BMP6 and 25 ng/ml TGF 1 by immunoflourescence. Therefore, induction of phospho-Sm ad1/5/8 protein expression in response to BMP6 or TGF 1 treatment demonstrates functional Smad1/5/8 ac tivation in C2C12 myofib ers and this induction of phospho-Smad1/5/8 is not due to an increase in total Smad1/5/8 protein expression. Since both BMP6 and TGF 1 induce functional Smad1/5/8 activ ation in myofibers, it may be argued that similar downstream effects coul d result from either ligand treatment. While BMP6 induces dramactic transdifferentiation of m yoblasts, this same effect is not observed in response to TG 1 treatment. Previous reports have demonstrated that TGF 1 and BMP6 ligand bind to different Type II receptors and induce diffe rent target genes. For example, transfection of Smad1 and Smad5 into C2C12 myoblasts and NIH3T3 fibroblasts re sulted in decreased myogenin promoter activity in both lines but ALP activity induction was only observed in C2C12 myoblasts and not in NIH3T3 fibroblasts These results demonstrated that these

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101 differentiation pathways are regulated independen tly at the transcriptional level (Yamamoto et al., 1997). Additionally, treatment of developi ng stage 24-25 chick limb buds with TGF 1 soaked beads resulted in dowregulation of Indian hedgehog (ihh) collagen type X (col X) expression, which are markers of chondrocyte maturation, and further explain why BMP6 induces ALP activity but TGF 1 does not (Ferguson et al., 2004) Previous studies in the laboratory have also dem onstrated that when TGF 1 biological activity was removed with an antibody that binds up endogenous TGF 1 ligand, restoration of the myogenic program is not restored, which suggests that TGF 1 is not targeting the myogenic differentiation program in the same manner as BMP6 (Wang et al., 2004). BMP6 also induces signaling ALP activity, which is not observed in response to TGF 1. TGF 1 does result in transdiffere ntiation of hepatic stellate cells to myofibroblasts and this process has ac tually been demonstrated to not be based on different regulation of Smad expression in these cells (Dooley et al., 2001). Therefore, even though BMP6 and TGF 1 induced the same Smads in m yoblasts, the further downstream induction of gene targets is the critical mediator of the ultimate response and helps support why BMP6 causes transdifferentiation, but not TGF 1, in myoblasts. BMPs can also initiate p38 si gnaling cascade and p38 is known to play a role in myogenic regulation. Most studies have shown that p38 si gnaling is a positive effector of skeletal myogenic differentiation and is required for myocyte formation and muscle-specific gene transcription. p38 MAPK can trigger cell cycle a rrest during muscle diffe rentiation (Lee et al., 2002). Other studies have demonstrated that inte rference of p38 activity with synthetic inhibitors abolish muscle cell fusion and expression of muscle -specific proteins (Zetse r et al., 1999). Total p38 levels were unchanged in response to BMP6 treatment but phospho-p38 was slightly induced suggesting that low levels of p38 signali ng may be active in myoblasts in response to

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102 BMP6. Yet when myofibers were treated wi th a combination of 100 ng/ml BMP6 and a p38 inhibitor (SB202190), inhibition of p38 activity did not appear to alter ALP induction or inhibition of myofiber fusion in response to BMP6 treatment. Treatment of myofibers with BMP6 or SB202190 alone significantly inhibited TnI-Luc activity. The combination of 100 ng/ml BMP6 and 1 M SB202190 further inhibited TnI-Luc activity as compared to either treatment alone. This demonstrates an additive effect of BMP6 and p38 inhibition on inhibition of TnI-luc reporter activity sugge sting BMP6 and p38 are mediating inhibitory effects on skeletal myogenesis through independent pathways. Furt her support of independe nt signaling is the Western results demonstrating inhibition of both contractile and myogenic regulatory proteins by BMP6 and p38 independently and to different degrees. For ex ample, BMP6 inhibits both contractile and regulatory proteins whereas, p38 inhibition appears to target contractile proteins (MyHC). Furthermore, the complete ablation of muscle specific markers by BMP6 plus SB202901supports the additive effect demonstrated observed in the transient transfections on muscle specific reporter activ ity. The reason the phospho-p38 was slightly induced in response to BMP6 may be an antibody specificity issue. The antibody is supposed to be specific for only phospho-p38 but appears to be either picking up hypo-phosphorylated p38 or nonphosphorylated p38 at high levels of BMP6 treat ment, which are not physiologically revelent dosages. Therefore, the activ ation of p38 signaling by BMP6 may not be relevant in an in vivo environment. A third signaling pathway BMPs are regul ated through is Notch signaling. Notch signaling plays a role in cellular homoestasis a nd cell fate determination and is important for proper cellular differentiation in many tissues (H ing et al., 1994). In mammals, there are four genes, Notch-1, Notch-2, Notch-3, and Notch-4 (Ch itnis et al., 1995; Lindse ll et al., 1995; Li et

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103 al., 1998). The Notch receptors are transmem brane receptors and when bound by the extracellular ligands, Delta, Serrate, or Lag-2, this results in a cleavage of the intracellular domain of the receptors in the cytoplasm. This cleavage product is the activ e form of Notch, NICD (Notch Intracellular Domain), which translocates to the nucleus where it binds the family of transcriptional repressors CSL (also known as RBP-J C BF-1, S uppressor of Hairless, and L ag1), and converts them into transcriptional ac tivators, which target genes involved in the inhibition of neurogenesis and myogenesis (Nye et al., 1994; Ahmad et al., 1995). Functional Notch signaling is required for BMP4 -mediated inhibition of differentiation of myogenic cells (Dahlqvist et al., 2003). Both Notch and BM P signaling can block differentiation of myogenic cells with ligand induction of Notch causing a dramatic block in myotube formation (Kopan et al., 1994; Takahashi et al., 1994; Kuroda et al., 1999). Therefore, to determine the involvement of Notch signaling in BMP6-mediate d inhibition, Notch signaling was inhibited with L685,458 in the absence and presence of BMP6 in 23A2 myoblasts. Notch inhibition alone did not alter MyHC expression, although, BMP6 plus Notch inhi bition did partially restore MyHC expression in fibers. This suggests that BMP6-mediated inhibition of differentiation is regulated or partially controll ed by the Notch signaling pathway. Crosstalk could be further confirmed through utilization of a dominant negative version of the CSL, R218H. The cleavage product, NICD (Notch Intracellula r Domain), is the activ e form of Notch, which translocates to the nucleus and binds the transcri ptional repressors, CSL, convert ing them into transcriptional activators, which then target genes involved in the inhibition of myogenesi s (Nye et al., 1994; Ahmad et al., 1995). CSL can also be thought of as the Notch signal mediator and R218H is thought to block activation of genes downstream of Notch by forming a complex with Notch ICD that cannot bind to the promoter (Chung et al., 1994; Wettstein et al., 1997). Transfection

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104 of R218H alone and in combination with BMP6 in C2C12 and satellite cells would allow further confirmation of the Notch inhibitory effect of BMP6-mediated actions by determining MyHC, MyoD, myogenin, and Troponin T expression in response to the dominant negative CSL, R218H. Analyses of Notch responsive genes and re porter activity would also demonstrate the importance of Notch signaling in BMP6-mediated myogenic inhibition. Hes1 and Hey1 are two immediate Notch responsive genes and could determ ine if BMP6 treatment of both satellite cells and C2C12 cells increased Hes1 and Hey1 expression by quantitative PCR. Hey1 has been suggested to be important for i nhibition of muscle development (S un et al., 2001). Hey1-luc is a promoter construct that contai ns both CSL-binding sites and GC-ri ch domains (Kusanagi et al., 2000). Transfection of Hey1-luc into C2C12 and satellite cells and assessment of Hey1-luc activity in response to BMP6 and L685,458 alone and in combina tion would further demonstrate the role of Notch signaling in BMP6-mediate myogenic effects. The Notch and BMP signaling pathways are evolutionarily conserved and in fluence cellular differentiation in many tissues. Determination of how Notch and BMP6 signaling interact or crosstalk would contribute to further understanding of how cells respond to complex extracellula r cues. In summary, the functional role of BMP6 during myogenesis still remains unclear but these data provide further insigh t into the complex re gulation of myogenesis mediated by BMP6. Functional Smad1/5/8 cascades are present in myoblasts, and BMP6-mediated myogenic inhibition is independent of p38 signaling but does partially requi re functional Notch sigaling. The requirement of additional f actors required for this BMP6-mediated effect requires further evaluation. Activated Notch expression is highly localized expression to the site of injury suggesting that it might play a role in targeting or recruiting specifi c factors to the injury site to

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105 promote regeneration. Interestingly, BMP6 is upregulated in response to wounding in keratinocytes (Wach et al., 2001). Therefore, du e to the involvement of Notch in response to injury and BMPs utilization in many clinical stud ies for the generation of artificial tissues and various therapeutic interventions during bone in jury repair, a more thorough understanding of their mechanism of action will assist in the deve lopment of therapeutics targeting muscle disease and repair.

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106 Figure 5-1. Potential BMP6 signaling pathways affecting skeletal myogenesis. Three intracellular signaling pathways highli ght BMP6 signaling. Pathway#1 is the predominate Smad1/5/8 cascade; Pathway #2 is p38 signaling; Pathway #3 involves Notch signaling.

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107 Figure 5-2. Verificat ion of BMP and TGF signaling axis. C2C12 myoblasts were treated with 25 ng/ml BMP6 and 10 ng/ml TGF 1. Parallel plates were immunostained for phospho-smad1/5/8 and Hoescht 33245 was used to visualize nuclei. Representative immunoflorescent images at 200X are shown (B ). Total cell lysates were analyzed by Western blot for total Smad1/5/8 a nd phospho-Smad1/5/8 protein expression. Tubulin expression was used as a loading control (A).

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108 Figure 5-3. BMP6 treatment increases p38 phosphoryl ation. Cells were tr eated with increasing concentrations of BMP6 and to tal cell lysates were analyzed by Western blot for total and phospho-p38 protein expres sion. Desmin expression was used as a loading control.

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109 Figure 5-4. p38 inhibition and BMP6 treatment resu lt in additive inhibition of muscle specific reporter activity. C2C12 myoblasts treated with 100 ng/ml BMP6 +/10 M SB 202190 (p38 inhibitor) in differentiation medi a for 48 hours. Cells were lysed, and measured for TnI-Luc Activity (A). Repor ter luciferase activity was normalized to the amount of Renilla luciferase activity and the control was set to 100% (vehicle only). Means and SEM are from three inde pendent experiments. Different letters indicates significance at P< 0.05. Representative phase c ontrast photomicrographs at 200X (B).

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110 Figure 5-5. BMP6 and p38 signal through indepe ndent pathways to influence myogenic differentiation. C2C12 myoblasts were trea ted with 50 ng/ml BMP6 in the absence and presence of 10 M SB202190 (p38 inhibitor) for 48 hours in differentiation permissive media. Cells were lysed a nd assessed for MyHC, myogenin, troponin T, and desmin protein expre ssion by Western blot.

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111 Figure 5-6. p38 signaling does not play a significant role in transdifferentiation. C2C12 myoblasts were treated with 100 ng/ml BMP6 +/10 M SB 202190 (p38 inhibitor) for 48 hours. ALP activity was measured by reaction with NBT + BCIP colorimetrically. Representative pho to-micrographs at 100X are shown.

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112 Figure 5-7. BMP6 inhibition of differentiation is mediated in part by Notch. C2C12 myoblasts were cultured with 25 ng/ml BMP6 +/1 M L685,458 (Notch Inhibitor) for 72 hours. Cells were fixed and immunostain ed for myosin heavy chain (MyHC). Hoescht dye was used to visualize nuclei. Representative photomicrographs at 200X are shown (A). Quantification of the percentage of MyHC positive fibers in response to treatments (B). Different letters indicates a significant difference, P<0.05.

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113 CHAPTER 6 IMPACT OF E2F5 ON SKELETAL MYOGENESIS Objective BMP6 expression is increased in Raf-arrested myocytes (Wang et al., 2004) and E2F5 is present in the nucleus of these Ra f-arrested myoblasts (Reed et al., 2007). E2F5 is classified as a transcriptional repressor due in part to its negative regulation of cell cycle progression (DeGregori et al., 1997). In keratinocytes, BMP6 stimulates keratinocyte differentiation, which is associated with E2F5 -upregulation and nuclear accumulati on of E2F5 (DSouza et al., 2001). The objective of these experiments was to examine the effect of E2F5 on skeletal myogenesis. BMP6 Treatment does not cause E2F5 Nuclear Accumulation in Myoblasts In keratinocytes, treatment with BMP6 signi ficantly decreases DNA synthesis (DSouza et al., 2001) and triggers differentiation programs (T ennenbaum et al., 1996). In these cultures, E2F5 protein levels are significantly increase d (DSouza et al., 2001). C2C12 myoblasts were treated with 25 ng/ml BMP6 for 48 hours, fixe d and immunostained for E2F5 expression. Results demonstrate cytoplasmic retention of E2 F5 in both control and BMP6 treated myoblasts (Figure 6-1). This indicates that BMP6 does not alter E2F5 translocati on further supporting our contention that autocrine BMP6 does not particip ate in Raf-induced growth arrest. Presence of E2F5 in Satellite Cell Position E2F5 expression is found in the nucleus of quiescent cells in vitro and there also is a change in E2F expression with the onset of myoblast differentiation. Complexes of E2F5-p130 are present in G0 phase but not throughout the remainder of the cell cycle (Olson et al., 1995). In rat cardiomyocytes, hypertrophic stimulus promotes E2F1 / 3 / 4 and DP1 expression while down-

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114 regulating E2F5 These observations suggest that E2F5 plays a role in maintenance of growth arrest or quiescence (Hibi et al., 1996). C onversely, E2F5 expression is primarily in the cytoplasm of differentiated L6 myotubes while pRb, p130, and p107 are present in the myonuclei suggesting cell type spec ificity (Epstein et al., 1995) To monitor the expression patterns of E2F5 in skeletal muscle, cryosections we re collected form the tibialis anterior (TA) of adult male mice. The tissue was immunostained for E2F5 and dystrophin. As shown in Figure 6-2, E2F5 protein localizes to cells lying adjacent to the muscle fibers or putative satellite cells. Few E2F5 immunopositive nuclei were localized within the dystrophin boundary (Figure 6-2). Therefore, E2F5 is present in non-di viding, non-differentiating muscle cells in vivo E2F5 Does Not Inhibit Myofiber Differentiation The presence of E2F5 in satellite cells suggest s a role in cell cycle a rrest and/or inhibition of differentiation. To determine if E2F5 wa s impacting subsequent myoblast fusion and differentiation, C3H10T1/2 fibrobl asts were transiently co-transfected with TnI-Luc, pEMMyoD, and CMV-E2F5. Cells were maintained in differentiation-permissive media for 48 hours prior to lysis and luciferase measurement. Mu scle reporter gene activity was normalized to Renilla luciferase activity to account for transf ection efficiency. C3 H10T1/2 fibroblasts transfected with MyoD induced TnI-luc activ ity. Co-transfection of CMV-E2F5 did not significantly inhibit muscle specific reporter activ ity (Figure 6-3A). Therefore, E2F5 is not repressing myogenic gene transcription. MyoD and E47 form heterodimers to enhance muscle gene transcription (Lassar et al., 1991). E47 plays a crucial role during skelet al myogenesis by serving as the preferred heterodimer binding partner of myogenin (Becker et al., 2001). C3H10T1/2 fibroblasts were transfected with an immunogl obulin E box reporter plasmid, E5-luc, CMV-E47 and CMV-

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115 E2F5. After 48 hours, cells were lysed and a ssayed for luciferase activity. Assessment of E5luc activity demonstrated that E47 function was not significantly affected by the addition of E2F5 (Figure 6-3B). pRb does not interact with E2F5 to Exert Inhi bitory Effects on Muscle Specific Activity E2F gene activity is mediated through inter action with the pocket proteins, pRb, p107, and p130. E2F5 preferentially interacts with p130 and may require interaction with a pocket protein for a functional transcriptiona l effect. Previous work has demonstrated that only pRb translocates to the nucleus in response to Ra f-induced quiescence (Reed, 2007). To determine if E2F5 requires pRb, myoblasts were transiently transfected with TnI-luc, CMV-E2F5, CMV-pRb, CMV-p107, and CMV-p130. Luciferase activity was m easured after 48 hours. A reduction of TnI-Luc activity was observed in cells ectopically expressing p130 (Figure 6-4). Repression of reporter gene activity was not affected by E2F5. Therefore, the inability of E2F5 to inhibit myogenic differentiation is not a product of insufficient p130. E2F5 is Transcriptionally Active Since E2F5 did not cause an effect on myof iber differentiation, functional assessment of E2F5 activity was determined. C3H10T1/2 fibrobl asts were transiently transfected with E2FTA-luc, pRL-tk, and CMV-E2F5. Cells were lysed and assayed for luciferase activity. Both the complex thymidine kinase promoter reporter and the simple E2F cis element reporter were activated by E2F5 (Figure 6-5). Th ese results imply that E2F5 is functional in myoblasts. In summary, BMP6 treatment does not induce E2F5 nuclear accumulation. E2F5 does not inhibit myoblast differentiation. E2F5 does not appear to interact with pRb to mediate a transcriptional effect, although p130 does cause an inhibition of muscle reporter activity. Furthermore, the absence of a significant effect on myofiber differentiation was not due to an

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116 inactive plasmid because the CMV-E2F5 was able to significantly induce E2F response reporters. Discussion Previous studies have demonstr ated that E2F5 is necessary for pocket protein-mediated G1 arrest in cycling cells (Gaubatz et al., 2000). E2 F5 was believed to be involved in inhibition of the cell cycle in myoblasts, but in BMP6-treat ed myoblasts there was no induction of nuclear E2F5 accumulation in C2C12 myoblasts. This suggests that Raf signaling is not working through autocrine BMP6 nor is E2F5 ac tivity directly mediated by BMP6. Members of the E2F family are important in cell cycle progression and found to play roles in cellular processes like proliferation, differentiation, and ap optosis (Fujita et al., 2002). The E2F family regulates cell cycle progr ession and genes expressed at the G1/S transition contain E2F binding sites within their promoters (Nev ins, 1992; DeGregori et al., 1995). Two major groups, activators and repressors, comprise the E2F family. E2F 1, E2F2, and E2F3 are involved in activating the cell cycle control and S-phase entry of quiescent cells. E2F4 and E2F5 are classified as repressor proteins, due to their i nhibitory actions of cell cycle progression. E2F6, E2F7, and E2F8 are also classified as represso rs, but the mechanism is independent of pocket protein interaction, demonstrati ng different regulatory mechanisms in comparison to the other E2F proteins (Campanero et al., 2000). Since in vitro studies have demonstrated E2F5 expression to be associated with quiescent cells and believed to play a role in maintenance of growth arrest or quiescence (Hibi et al., 1996), ap plicability of these obs ervations need to be assessed in an ex vivo setting. Murine fibers isolated from retired breeders did demonstrate E2F5 expression in non-dividing muscle cells, which co-localized with Hoescht staining outside of the dystrophin border, demonstrating that active nuc lear E2F5 expression is associated with quiescent muscle cells in the sate llite cellular location. This further supports or mimics nuclear

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117 E2F5 expression observed in Raf-arrested myoblasts and demonstrates that E2F5 is expressed in putative satellite cells in vivo E2F5 is expressed in the murine brain, hear t, lung, liver, and kidney, and low levels in dermis and epidermis (DSouza et al., 2001). Th e E2F5 knockout mouse is viable because it is believed that E2F4 and E2F5 have overlappi ng functions. The E2F5 knockout does develop hydrocephalus due to overproduction of cerebral spinal fluid resu lting from a choroid plexus defect (Lindeman et al., 1998). The E2F4/5 doubl e knockout is embryonic leth al (Gaubatz et al., 2000). Several labs have found E2F5 expresse d in the nucleus, along with p130 in quiescent cells. E2F5 lacks a cyclin A bi nding domain, resulting in a shorte r N-terminal as compared to other E2Fs and contains a C-terminal transa ctivation domain (Sardet et al., 1995). Skeletal myoblasts undergo cell cycle withdraw al prior to subsequent differentiation and fusion. Since E2F5 is localized in putative sate llite cells outside of the dystrophin border, it was believed that E2F5 may contribute to inhi bition of differentiation. When C3H101T1/2 fibroblasts were transiently transfected with My oD and assayed for muscle specific activity, TnIluc activity was induced demonstrating proper m yofiber differentiation. Co-transfection of MyoD and E2F5 did not alter TnI-luc induction suggesting that fibroblas ts differentiation was not altered in the pres ence of E2F5 plasmid. Therefore, CH310T1/2 myofiber differentiation is not impacted by E2F5 expression assessed by Tn I-luc activity and may be more crucial to maintaining growth arrest or quiescence of myoblasts. Activation of muscle gene expression can be achieved through binding of Myogenic Regulatory Factors (MRF)-containing protein complexes to E-box el ements within the regulatory regions of muscle specific genes (Johnson et al., 1996). In vivo MRFs form heterodimers with members of the E protein family (E47, E2-2 or HEB), which are ubiquitously expressed bHLH

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118 proteins and the molecular basis for inhibi tion of differentiation could involve direct modification of E-proteins (Shira kata et al., 1993). E47 is ubiqu itously expressing in many cells including skeletal muscle cells and is an alternate splice product of the E2A gene (Murre et al., 1989; Sun and Baltimore, 1991). E2A knockout mice leads to subtle deficiencies in skeletal muscle function and null animals appear hunchba ck and are physically weak, suggesting that modest muscle defects exist (Bain et al., 1994; Yan et al., 1997). Luci ferase analysis of fibroblasts demonstrated that E-box reporter activity is significantly stimulated by E47 yet the co-transfection of E47 with E2F5 is similar to E-box reporter activity observed in the presence of E47 alone demonstrating that E2F5 does not alte r E-box reporter stimulation by E47. Therefore, the addition of E2F5 does not effect E47 stimul ation of E-box reporter activity. These results demonstrate that E2F5 does not effect or inhi bit myogenic differentiation. Previous reports demonstrate that Smad3 mediates myogenic differention in MyoD expressing C3H10T1/2 fibroblasts and C2C12 myoblasts by interacting with the HLH domain of MyoD/E protein heterodimerization. Smad3 interferes with the binding of MyoD complexes to E-box sites and results in inhibition of differen tiation (Liu et al., 2001). Theref ore, since BMP6 does not induced E2F5 nuclear accumulation and E2F5 does not im pact E47 or E-box activity, BMP6-mediated inhibition of differentiation does not involve E2F5. E2F5 is known to form a complex with pocket proteins, and interacts preferentially with p130 under physiological conditions to translocate to the nucleus for it to exert its transcriptional effect on target genes (Hijmans et al., 1995). C2 C12 myoblasts were transiently transfected with E2F5 and pocket proteins, pRb, p107, and p130 along with TnI-luc reporter. Results demonstrate that the inability of E2F5 to inhib it differentiation is not du e to insufficient pocket protein levels. Results also s uggest that p130 alone is mediating an inhibitory effect on muscle

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119 specific reporter activity in myoblasts. E2F5 stimulated transcription was found by other groups to be inhibited by cotransfection with p130, p107, and pRb in U2-OS osteosarcoma cells although this effect was DP-1 dependent (Hijma ns et al., 1995). Previous studies in the laboratory have demonstrated that translocation of E2F5 to the nucleus in response to Raf is observed without subsequent p130 nuclear tran slocation. Although another report that overexpressed E2Fs in cardiomyocytes, which are also striated muscles, also demonstrated pocket proteins were not required (Ebelt et al., 2 005). Alternatively, additional co-factors, such as HDAC1, may be necessary for nucl ear translocation and repression of transcriptional events to occur. E2F5 has also been demonstrated to require dimerization with DP-1 to bind DNA suggesting that addition of DP-1 may have been needed for E2F5 to exert transcri ptional activity (Hijmans et al,. 1995). Although, a l ack of transcriptional effect by E2F5 on reporter activity is not due to an inactive plasmid because CMV-E2F5 was able to significantly induce a complete thymidine kinase promoter reporter, which is E2F responsive. E2F5 also significantly induced a simple E2F cis element reporter. This suggests that E2F5 is f unctional in myoblasts. Since E2F5 is expressed in the satellite cell position but does not inhibit differentiation, creation of a stable myoblast line expressing a knockdown of E2F5 by siRNA or a dominant negative E2F5 (DN-E2F5) would further determine th e role of E2F5 in skeletal myogenesis. If the cellular quiescence observed in Raf-arrested myoblasts were being mediated through E2F5, then the stable myoblasts with little to no E2F5 expression w ould not undergo this cell cycle arrest observed in the 23A2RafERDD myoblasts. This would also demonstrate that E2F5 is responsible for maintenance of cellular quiescence of myoblasts. Conversely, assessment of a constitutively active E2F5 could be achieved by construction of a plas mid where the repressor domain was removed and replaced with a viral activator such as VP16. E2F5-VP16 could then

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120 be utilized to create a stable myoblastic line that express constitutiv ely active E2F5. In this line, one would expect that these lines would be in a state of continued cell cycle arrest and might demonstrate a phenotype similar to induced 23A2RafERDD or Raf-arrested myoblasts and could serve as another model of quiescent skeletal myoblasts. Assessment of signaling mechanisms and re gulation events invol ved in skeletal myogenesis and further understandi ng of satellite cell activation w ill allow for better utilization of these muscle precursor cells in repairing a nd regenerating skeletal muscle. In addition, further insight into how myoblastic entrance a nd exit from the cell cycle is regulated and the specific gene expression patterns exhibited by quiescence myoblasts that are critical to the maintenance and regulation of the G0 state. In conclusion, BMP6 does not induce E2F5 nuclear accumulation. E2F5 expresssion is locali zed in the putative satellite cell position in vivo in nondividing muscle. E2F5 was not f ound to inhibit differentiation and this inability is not due to insufficient p130 or a non-functional plasmid. Ther efore, due to the mult ipotency of satellite cells, applications in repair and regeneration of cartilage a nd bone might be further added by these data.

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121 Figure 6-1. BMP6 treatment does not cause E2 F5 nuclear accumulation in myoblasts. C2C12 myoblasts were treated with vehicle only () or 25 ng/ml BMP6 (+) for 48 hours and immunostained for E2F5. Representati ve photomicrographs at 200X are shown.

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122 Figure 6-2. Presence of E2F5 in sa tellite cell position. Cryosections (10 M) from murine TA muscle were immunostained for E2F5 and dystrophin. Hoechst 33245 was used as a nuclear counterstain. Representative photomicrographs at 200X are shown.

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123 Figure 6-3. E2F5 does not inhibit myogenic di fferentiation. C3H10T1/ 2 fibroblasts were transiently transfected with pEM-MyoD CMV-E2F5, and the muscle specific reporter (TnI-Luc) (A). Cells were tr ansiently transfected with CMV-E47, CMVE2F5, and the Immunoglobulin E box reporter plasmid E5-Luc (B). Cells were lysed and luciferase activities measured. Reporter luciferase activity was normalized to the amount of Renilla luciferase activity. Data repr esents the mean and standard error of the mean (SEM) of thr ee independent experiments.

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124 Figure 6-4. pRb does not interact with E2F5 to exert inhibitory effects on muscle specific activity. C2C12 myoblasts were transiently transfec ted with CMV-E2F5, pocket proteins (pRb, p107, and p130), and a TnI-Luc reporter. pRL-tk was included as a transfection efficiency monito r. Cells were lysed and luciferase measured. Reporter luciferase activity was normalized to the am ount of Renilla luciferase activity. Data represents the mean and standard error of the mean (SEM) of three independent experiments. Different letters indi cates a significant difference, P<0.05.

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125 Figure 6-5. E2F5 is transcripti onally active. C3H10T1/2 fibroblas ts were transiently transfected with pRL-tk, CMV-E2F5, and E2F-TA-luc repo rter. Cells were lysed and luciferase activity measured. Data represents the mean and standard error of the mean (SEM) of three independent experiments. Different letters indicates a si gnificant difference, P<0.0001.

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126 CHAPTER 7 SUMMARY AND CONCLUSIONS There appears to be differential gene expression and BMPand TGF -responsiveness in embryonic myoblasts and adult satelli te cells, as well as, different stages of skeletal myogenesis suggesting that different TGF superfamily members play differe nt regulatory roles at various stages of muscle formation. BMP6 was also found to be greater in myoblasts than myofibers but does not autocrinely regulate its own or other BMP ligands. The regulatory role of TGF superfamily member BMP6 on skeletal muscle differentiation was determined. Exogenous BMP6 results in inhibition of myoblastic differe ntiation as observed by significant inhibition of muscle reporter activity, musc le specific protein markers, and myoblast fusion. BMP6 also results in rapid transdifferentiation of myoblasts to an osteogenic ce ll lineage specific to mesodermally derived committed cells. Treatment of myoblasts with BMP6 does not alter the percentage of myoblasts in S-phase or promote a proliferative state to inhibit differentiation. Induction of functional active phospho-Smad1/5/8 in response to BMP6 and TGF 1 was demonstrated by both immunofluorescence and We stern blot analyses. Besides signaling through the Smad proteins, BMPs can also ut ilize components of the MAPK and JAK/STAT family, resulting in p38 activation (von Bubnoff and Cho, 2001). Inhibition of p38 activity and BMP6 treatment caused inhibition of TnI-Luc activ ity that was greater than either treatment alone suggesting an additive effect between BMP6 and p38 inhibition upon inhibition of myogenic differentiation. Results suggest that BMP6-mediated inhibition of differentiation is independent of p38 signaling. BMPs can also interact with Notch signaling a nd previous studies have demonstrated that functional Notch signaling is required for BMP4-m ediated inhibition of di fferentiation of muscle stem cells and C2C12 myogenic cell s (Dahlqvist et al., 2003). In terestingly, the combination of

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127 BMP6 and Notch inhibition by L685,458 partiall y restores MyHC expression in fibers. Therefore, BMP6-mediated inhibition of differentiation is regulated or partially controlled by the Notch signaling pathway similar to BMP4-mediated inhibition. Although, since MyHC expression is only partially restored there must also be additional f actors mediating this inhibition of myogenic differentiation. In summary, the functional role of BMP6 during myogenesis still remains unclear but these data provide further insigh t into the complex re gulation of myogenesis mediated by BMP6. There does appear to be differences in expressi on of BMPs in myoblasts versus myofibers and BMPand TGF -responsiveness. BMP6 inhibits the complete differentiation program in myoblasts. BMP6 does not appear to involve modulation of proliferation. BMP6 treatment results in a rapid transdifferentiation of myoblas ts that is specific to a committed mesodermal derived myogenic cell. BMP6-mediated myogenic inhibition does partially require functional Notch sigaling. The requirement of additional fa ctors required for this BMP6-mediated effect requires further evaluation. BMP6 is known to induce phospho-Smad1/5/8 and could potentially be forming a complex with NCID to bind to the promoter of muscle sp ecific genes such as, MyHC to induced gene transcription required fo r myogenic differentiation (Figure 7-1). Additionally, BMP6 expression is found to be increased in Raf-arrested myoblasts (Wang, 2004) and BMP6 is found to be expressed in ma ture muscle fibers (Dernyk, 1989). E2F5 is localized primarily in cytoplasm in proliferating myoblasts and is translo cated primarily to the nucleus in response to elevated Raf expression. BMP6 stimulates kera tinocyte differentiation associated with E2F5-upregulation (DSouza et al., 2001). E2F5 is known to inhibit the cell cycle and is uniquely expressed in Raf-arrested myoblasts. The exit and entrance of skeletal myoblasts from the cell cycle and maintenance of cellular quiescence is an area of research not

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128 well understood. In order to determine what role E2F5 plays in cellular quiescence of Rafarrested myoblasts, BMP6 treatment was found to not induce E2F5 nuclear accumulation. E2F5 was found present in non-dividing cells in vivo in the satellite cellular position yet did not impact myogenic differentiation or E-box reporter activity. These results demonstrate that while E2F5 is localized in putative satel lite cells outside the dystrophin border, E2F5 does not inhibit differentiation. Additionally, the inability of E2F5 to inhibit differentiation is not due to insufficient pocket proteins, although p130 appears to be inhibiting differen tiation independently. Further understanding of the regulatory mechan isms involved in skeletal myogenesis and satellite cell activation will allow for determining better in vitro conditions of either injury or disease muscle models. Transcriptional modulat ion of processes such as differentiation and fusion could also be translated into ex vivo conditions for the preventi on and treatment of skeletal muscle myopathies. Additionally, better utilizati on of muscle precursor cells and BMPs would prove applicable in the repair and regene ration of skeletal mu scle, bone, and cartilage.

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129 Figure 7-1. Proposed model of BMP6 on myogenic differentiation and interaction with Notch.

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130 APPENDIX A GENE ARRAY LAYOUT AND TABLE Array Layout Acvr1 1 Acvr1b 2 Acvr2a 3 Acvr2b 4 Acvrl1 5 Amh 6 Bambi 7 Bglap1 8 Bmp1 9 Bmp10 10 Bmp15 11 Bmp2 12 Bmp3 13 Bmp4 14 Bmp5 15 Bmp6 16 Bmp7 17 Bmp10 18 Bmp8b 19 Bmpr1a 20 Bmpr1b 21 Bmpr2 22 Cdc25a 23 Cdkn1a 24 Cdkn2b 17 Cer1 26 Chrd 27 Grem1 28 Col1a1 29 Col1a2 30 Col3a1 31 Dlx2 32 Lefty 33 Eng 34 Evi1 35 Fkbp1b 36 Fos 37 Fst 38 Gdf1 39 Gdf11 40 Gdf2 41 Gdf3 42 Gdf5 43 Gdf6 44 Gdf8 45 Gdf9 46 Id1 47 Id2 48 Id3 49 Id4 50 Cd79a 51 Igf1 52 Il6 53 Inha 54 Inhba 55 Inhbb 56 Inhbc 57 Inhbe 58 Itgb5 59 Itgb7 60 Ivl 61 Jun 62 Junb 63 Lap3 64 Lefty2 65 Igfbp3 66 Smad1 67 Smad2 68 Smad3 69 Smad5 70 Smad6 71 Smad7 72 Smad9 73 Nbl1 74 Nodal 75 Nog 76 Pdgfb 77 Plat 78 Plau 79 Runx1 80 Runx2 81 Serpine1 82 Sox4 83 Stat1 84 Tgfb1 85 Tgfb1i1 86 Tsc22d1 87 Tgfb2 88 Tgfb3 89 Tgfbi 90 Tgfbr1 91 Tgfbr2 92 Tgfbr3 93 Tgif 94 Timp1 95 Zfhx1a 96 PUC18 97 PUC18 98 PUC18 99 Blank 100 Blank 101 Blank 102 Gapdh 103 Gapdh 104 Ppia 105 Ppia 106 Ppia 107 Ppia 108 Rpl13a 109 Rpl13a 110 Actb 111 Actb 112 Gene Table Position Unigene GeneBank Symbol Description Gene Name 1 Mm.689 NM_007394Acvr1 Activin A receptor, type 1 ALK2/ActR-I 2 Mm.308467 NM_007395Acvr1b Activin A receptor, type 1B 6820432J04/ActR-IB 3 Mm.314338 NM_007396Acvr2a Activin receptor IIA ActRIIa/Acvr2 4 Mm.8940 NM_007397Acvr2b Activin receptor IIB ActRIIB 5 Mm.279542 NM_009612Acvrl1 Activin A receptor, type IIlike 1 AI115505/AI427544

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131 Position Unigene GeneBank Symbol Description Gene Name 6 Mm.376094 NM_007445Amh Anti-Mullerian hormone MIS 7 Mm.284863 NM_026505Bambi BMP and activin membrane-bound inhibitor, homolog (Xenopus laevis) 2610003H06Rik 8 Mm.87858 NM_007541Bglap1 Bone gamma carboxyglutamate protein 1 Bglap/OC 9 Mm.27757 NM_009755Bmp1 Bone morphogenetic protein 1 TLD 10 Mm.57171 NM_009756Bmp10 Bone morphogenetic protein 10 BMP10 11 Mm.42160 NM_009757Bmp15 Bone morphogenetic protein 15 AU015375/AU018861 12 Mm.103205 NM_007553Bmp2 Bone morphogenetic protein 2 AI467020/Bmp2a 13 Mm.209571 NM_173404Bmp3 Bone morphogenetic protein 3 9130206H07/9530029I04Rik 14 Mm.6813 NM_007554Bmp4 Bone morphogenetic protein 4 Bmp2b/Bmp2b-1 15 Mm.118034 NM_007555Bmp5 Bone morphogenetic protein 5 AU023399/se 16 Mm.374781 NM_007556Bmp6 Bone morphogenetic protein 6 D13Wsu115e/Vgr-1 17 Mm.595 NM_007557Bmp7 Bone morphogenetic protein 7 OP1 18 Mm.318417 NM_007558Bmp8a Bone morphogenetic protein 8a Bmp7r1/OP-2 19 Mm.30413 NM_007559Bmp8b Bone morphogenetic protein 8b Op3

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132 Position Unigene GeneBank Symbol Description Gene Name 20 Mm.237825 NM_009758Bmpr1a Bone morphogenetic protein receptor, type 1A 1110037I22Rik/ALK3 21 Mm.39089 NM_007560Bmpr1b Bone morphogenetic protein receptor, type 1B AI385617/ALK-6 22 Mm.7106 NM_007561Bmpr2 Bone morphogenic protein receptor, type II (serine/threonine kinase) 2610024H22Rik/AL117858 23 Mm.307103 NM_007658Cdc25a Cell division cycle 25 homolog A (S. cerevisiae) D9Ertd393e 24 Mm.195663 NM_007669Cdkn1a Cyclin-dependent kinase inhibitor 1A (P21) CAP20/CDKI 25 Mm.269426 NM_007670Cdkn2b Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) AV083695/INK4b 26 Mm.6780 NM_009887Cer1 Cerberus 1 homolog (Xenopus laevis) Cerr1/cer-1 27 Mm.20457 NM_009893Chrd Chordin Chd 28 Mm.166318 NM_011824Grem1 Gremlin 1 Cktsf1b1/Drm 29 Mm.277735 NM_007742Col1a1 Procollagen, type I, alpha 1 Col1a-1/Cola-1 30 Mm.277792 NM_007743Col1a2 Procollagen, type I, alpha 2 AA960264/AI325291 31 Mm.249555 NM_009930Col3a1 Procollagen, type III, alpha 1 AW550625/Col3a-1 32 Mm.3896 NM_010054Dlx2 Distal-less homeobox 2 AW121999/Dlx-2 33 Mm.378911 NM_010094Lefty1 Left right determination factor 1 AI450052/Leftb 34 Mm.225297 NM_007932Eng Endoglin AI528660/CD105 35 Mm.56965 NM_007963Evi1 Ecotropic viral integration site 1 D630039M04Rik/Evi-1

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133 Position Unigene GeneBank Symbol Description Gene Name 36 Mm.20453 NM_016863Fkbp1b FK506 binding protein 1b AW494148 37 Mm.246513 NM_010234Fos FBJ osteosarcoma oncogene D12Rfj1/c-fos 38 Mm.4913 NM_008046Fst Follistatin FST 39 Mm.258280 NM_008107Gdf1 Growth differentiation factor 1 AI385651/Gdf-1 40 Mm.299218 XM_125935Gdf11 Growth differentiation factor 11 Bmp11 41 Mm.343728 NM_019506Gdf2 Growth differentiation factor 2 Bmp9 42 Mm.299742 NM_008108Gdf3 Growth differentiation factor 3 C78318/Gdf-3 43 Mm.4744 NM_008109Gdf5 Growth differentiation factor 5 CDMP-1/bp 44 Mm.302555 NM_013526Gdf6 Growth differentiation factor 6 BMP13/GDF16 45 Mm.3514 NM_010834Gdf8 Growth differentiation factor 8 Cmpt/Mstn 46 Mm.9714 NM_008110Gdf9 Growth differentiation factor 9 Gdf-9 47 Mm.444 NM_010495Id1 Inhibitor of DNA binding 1 AI323524/D2Wsu140e 48 Mm.34871 NM_010496Id2 Inhibitor of DNA binding 2 AI255428/C78922 49 Mm.110 NM_008321Id3 Inhibitor of DNA binding 3 HLH462/Idb3 50 Mm.283273 NM_031166Id4 Inhibitor of DNA binding 4 Idb4 51 Mm.1355 NM_007655Cd79a CD79A antigen (immunoglobulinassociated alpha) Ig-alpha/Iga 52 Mm.268521 NM_010512Igf1 Insulin-like growth factor 1 C730016P09Rik/Igf-1 53 Mm.1019 NM_031168Il6 Interleukin 6 Il-6 54 Mm.1100 NM_010564Inha Inhibin alpha AW555078

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134 Position Unigene GeneBank Symbol Description Gene Name 55 Mm.8042 NM_008380Inhba Inhibin beta-A INHBA 56 Mm.3092 NM_008381Inhbb Inhibin beta-B INHBB 57 Mm.2594 NM_010565Inhbc Inhibin beta-C INHBC 58 Mm.3510 NM_008382Inhbe Inhibin beta E INHBE 59 Mm.6424 NM_010580Itgb5 Integrin beta 5 AA475909/AI874634 60 Mm.58 NM_013566Itgb7 Integrin beta 7 Ly69 61 Mm.207365 NM_008412Ivl Involucrin 1110019C06RIK 62 Mm.275071 NM_010591Jun Jun oncogene AP-1/Junc 63 Mm.1167 NM_008416Junb Jun-B oncogene JUNB 64 Mm.286830 NM_024434Lap3 Leucine aminopeptidase 3 2410015L10Rik/AA410100 65 Mm.87078 NM_177099Lefty2 Left-right determination factor 2 6030463A22Rik/AV214969 66 Mm.29254 NM_008343Igfbp3 Insulin-like growth factor binding protein 3 AI649005/IGFBP-3 67 Mm.223717 NM_008539Smad1 MAD homolog 1 (Drosophila) AI528653/Madh1 68 Mm.152699 NM_010754Smad2 MAD homolog 2 (Drosophila) Madh2/Madr2 69 Mm.7320 NM_016769Smad3 MAD homolog 3 (Drosophila) AU022421/Madh3 70 Mm.272920 NM_008541Smad5 MAD homolog 5 (Drosophila) 1110051M15Rik/AI451355 71 Mm.325757 NM_008542Smad6 MAD homolog 6 (Drosophila) Madh6 72 Mm.34407 NM_008543Smad7 MAD homolog 7 (Drosophila) Madh7 73 Mm.244353 NM_019483Smad9 MAD homolog 9 (Drosophila) MADH6/Madh9 74 Mm.9404 NM_008675Nbl1 Neuroblastoma, suppression of tumorigenicity 1 D4H1S1733E/DAN 75 Mm.57195 NM_013611Nodal Nodal Tg.413d 76 Mm.135266 NM_008711Nog Noggin NOG 77 Mm.144089 NM_011057Pdgfb Platelet derived growth factor, B polypeptide PDGF-B/Sis 78 Mm.154660 NM_008872Plat Plasminogen activator, tissue AU020998/AW212668 79 Mm.4183 NM_008873Plau Plasminogen activator, urokinase u-PA/uPA

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135 Position Unigene GeneBank Symbol Description Gene Name 80 Mm.4081 NM_009821Runx1 Runt related transcription factor 1 AI462102/AML1 81 Mm.263975 NM_009820Runx2 Runt related transcription factor 2 AML3/Cbf 82 Mm.250422 NM_008871Serpine1Serine (or cysteine) peptidase inhibitor, clade E, member 1 PAI-1/PAI1 83 Mm.240627 NM_009238Sox4 SRY-box containing gene 4 AA682046/Sox-4 84 Mm.277406 NM_009283Stat1 Signal transducer and activator of transcription 1 2010005J02Rik/AA408197 85 Mm.248380 NM_011577Tgfb1 Transforming growth factor, beta 1 TGF-beta1/Tgfb 86 Mm.3248 NM_009365Tgfb1i1 Transforming growth factor beta 1 induced transcript 1 ARA55/Hic5 87 Mm.153272 NM_009366Tsc22d1 TSC22 domain family, member 1 AA589566/AW105905 88 Mm.18213 NM_009367Tgfb2 Transforming growth factor, beta 2 BB105277/Tgf-beta2 89 Mm.3992 NM_009368Tgfb3 Transforming growth factor, beta 3 Tgfb-3 90 Mm.14455 NM_009369Tgfbi Transforming growth factor, beta induced 68kDa/AI181842 91 Mm.197552 NM_009370Tgfbr1 Transforming growth factor, beta receptor I ALK5/AU017191 92 Mm.172346 NM_009371Tgfbr2 Transforming growth factor, beta receptor II 1110020H15Rik/AU042018 93 Mm.200775 NM_011578Tgfbr3 Transforming growth factor, beta receptor III 1110036H20Rik/AU015626 94 Mm.101034 NM_009372Tgif TG interacting factor AA959811/AI462167

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136 Position Unigene GeneBank Symbol Description Gene Name 95 Mm.8245 NM_011593Timp1 Tissue inhibitor of metalloproteinase 1 Clgi/TIMP-1 96 Mm.3929 NM_011546Zfhx1a Zinc finger homeobox 1a 3110032K11Rik/AREB6 97 N/A L08752 PUC18 PUC18 Plasmid DNA pUC18 98 N/A L08752 PUC18 PUC18 Plasmid DNA pUC18 99 N/A L08752 PUC18 PUC18 Plasmid DNA pUC18 100 101 102 103 Mm.343110 NM_008084Gapdh Glyceraldehyde3-phosphate dehydrogenase Gapd 104 Mm.343110 NM_008084Gapdh Glyceraldehyde3-phosphate dehydrogenase Gapd 105 Mm.5246 NM_008907Ppia Peptidylprolyl isomerase A 2700098C05/Cphn 106 Mm.5246 NM_008907Ppia Peptidylprolyl isomerase A 2700098C05/Cphn 107 Mm.5246 NM_008907Ppia Peptidylprolyl isomerase A 2700098C05/Cphn 108 Mm.5246 NM_008907Ppia Peptidylprolyl isomerase A 2700098C05/Cphn 109 Mm.180458 NM_009438Rpl13a Ribosomal protein L13a 1810026N22Rik/Tstap198-7 110 Mm.180458 NM_009438Rpl13a Ribosomal protein L13a 1810026N22Rik/Tstap198-7 111 Mm.297 NM_007393Actb Actin, beta, cytoplasmic Actx/E430023M04Rik 112 Mm.297 NM_007393Actb Actin, beta, cytoplasmic Actx/E430023M04Rik

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137 APPENDIX B SUMMARY OF ABBREVIATIONS BMPs: Bone Morphogenetic Proteins SR: sarcoplasmic reticulum T system: Transverse Tubule system IF: Intermediate Filaments RyR: ryanodine receptors DHP: dihydropyridine receptors MyHC: Myosin Heavy Chain MPCs: Muscle Precursor Cells MRFs: Myogenic Regulatory Factors NMJ: neuromuscular junction MNF: Myocyte Nuclear Factor HGF: Hepatocyte Growth Factor TGF : Transforming Growth Factor Beta FGF: Fibroblast Growth Factor IGF: Insulin Growth Factor PI3K: Phosphatidylinositol-3-kinase TA: Tibialis anterior MSTN: Myostatin ATP: adenosine tri-phosphate ADP: adenosine di-phosphate VO2 max: maximal oxygen uptake dpc: days post coitum

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138 Tn: Troponin Id: inhibitor of differentiation/DNA binding GDF: growth and differentiation factor MAPK: mitogen-activated protein kinase JNK: Jun N-terminal kinase bHLH: basic helix-loop-helix STAT: signal transducers and activators of transcription MEK: mitogen-activated prot ein kinase/extracellular signa l regulated kinase kinase MEKK: MEK kinase ERK: extracellular regu lated signal kinase G0: quiescence or cell cycle arrest MEF: myocyte enhancer-binding factor cdk: cyclin-dependent kinase S-phase: synthesis phase MSCs: Mesenchymal stem cells PTH: parathyroid hormone PCNA: proliferating cell nuclear antigen NICD: Notch Intracellular Domain CSL: RBP-J or C BF-1, S uppressor of Hairless, and L ag-1 Mdx: dystrophin null transgenic mice CME: crushed muscle extract Rb: retinoblastoma Shh: Sonic hedgehog

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139 Ihh: Indian hedgehod ColX: collagen type X ROM: range of motion PCSA: physiological cr oss-sectional area PCR: polymerase chain reaction RT: reverse transcription IHC: immunohistochemical FACS: Fluorescence Activated Cell Sorting DAPI: 4,6-diamidino-2-phenylinodale 4HT: 4-hydroxytamoxifen FBS: fetal bovine serum PBS: phosphate buffered saline TnI-luc: Troponin I luciferase GAPDH: glyceraldehydes-3 -phosphate dehydrogenase tk: thymidine kinase ER: estrogen receptor DBD: DNA binding domain EtOH: ethanol HS: horse serum HCl: Hydrochloric acid DAB: 3,3-Diaminobenzidine tetrahydrochloride NiCl: Nickel Chloride H2O2: hydrogen peroxide

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140 BSA: bovine serum albumin BCIP: 5-bromo-4-chloro-3-indolyl-phosphate NBT: Nitro Blue Tetrazolium PARP: anti-Poly (ADP-ribose) polymerase AP: Alkaline phosphatase-c onjugated streptavidin ALP: alkaline phosphate SDS: sodium dodecyl sulfate SSC: saline sodium citrate O/N: overnight Bmpr: Bone Morphogenetic Protein Receptor Col1a1: Procollagen, type I, alpha 1 Itgb5: Integrin beta 5 Igfbp3: Insulin-like growth factor binding protein 3 Runx: Runt related transcription factor myogenin-CAT: myogenin/chlora mphenical acetyltransferase ECM: extracellular matrix PCP: procollagen C proteinase ALK: activin receptor-like kinase APAF-1: apoptotic protease activign factor 1 TNF: Tumor Necrosis Factor FADD: Fas-associated death domain STS: staurosporine JAK: Janus kinase

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180 BIOGRAPHICAL SKETCH Jennelle Robin McQuown was born in Oswego, NY to Dan and Linda McQuown. She is the middle child of three, having one older brot her, Dan and a younger sister, Michelle. After graduating valedictorian of her high school class in Hannibal, NY, she moved to Melbourne, FL to pursue her bachelors degree. She received a Bachelor of Scienc e (B.S.) degree in both molecular biology and marine biology at the Flor ida Institute of Technology in Melbourne, FL, in May, 2000. Under the mentorship of Dr. Rosa lia Simmen, she received a Master of Science (M.S.) in animal sciences in August, 2002. Af ter spending 2 years at the Moffitt Cancer and Research Institute at the University of South Fl orida, Jennelle returned to the University of Florida to complete her Ph.D. in animal sciences under the advi sement of Dr. Sally Johnson. She is also the proud aunt and godmother of her ne phew Brennen and has three cats, Merlin, Cali, and Barley.


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INVOLVEMENT OF BMP6 AND E2F5 INT SKELETAL MYOGENESIS


By

JENNELLE ROBINT MCQUOWN















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007


































@ 2007 Jennelle Robin McQuown



































To Bill and my family for all of their love and support. I would be lost without you guys! All of
my love, J.









ACKNOWLEDGMENTS

I would like to thank my maj or professor, Dr. Sally Johnson, for serving as my supervisory

committee chair. I would also like to thank my committee members (Dr S. Paul Oh, Dr. Lori

Warren, and Dr. Lokenga Badinga) for their support and advice. I could not have asked for a

more supportive and encouraging group. I would also like to thank the members of Dr. Sally

Johnson' s lab for their assistance with my dissertation proj ect. I also count myself lucky to have

a great group of friends and family who served as my sounding board, my cheering squad, and

my source of inspiration.

The completion of my Ph.D. has definitely been a family goal. Without the love and

support of my family, especially my mom and dad, Linda and Dan McQuown, I might have not

achieved this accomplishment. Another huge source of encouragement and support was William

Pittsley. I feel that I share this degree with him since he has gone through every triumph, every

failure, and every fear and hope along with me, and I am eternally grateful. I also send prayers

up to my Grandpa Cossentine and Grandpa McQuown whom weren't able to see me finish this

degree but I know that they're both smiling down and sending me their love. For my Grandma

McQuown and Grandma Cossentine, I am so thankful that they are here to encourage me, send

me their love, and call me the first Dr. McQuown of the family!i Finally, I would like to thank all

of my wonderful friends. I feel lucky to have such a great cheering squad!












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............8............ ....


LIST OF FIGURES .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 1 1..


CHAPTER


1 LITERATURE REVIEW ................. ...............13...............


Introducti on ................. ...............13......__ ......

Skeletal Muscle Biology .................. .... ....._._ ..... .............. ...... .........1
The SR and Transverse Tubule Systems within Skeletal Muscle ..........._...._ ................14

Maj or Fiber Type Classification ................. ...............15........... ...
Muscle Architecture .............. ...............16....
Skeletal Muscle Function .............. ...............19....
Skeletal Muscle Development ................... .......... ...............22......

Myogenic Regulatory Factors (MRF s) ................. ...............25........... ...
Satellite Cells............... ....... ............2
Satellite Cell Marker Proteins .............. ...............28....
c-M et .................. ........... ...............29.......

Syndecans 3 and 4 .............. ...............30....
M -cadherin .............. ...............30....
CD3 4............... ...............3 0.
Pax7 ................. .... ..... .... .......... ............3

Myocyte nuclear factor (MNF) .............. ...............32....
N otch .............. .. ... ............. .... .........3
Growth Factor Effects on Satellite Cells .............. ...............34....
Insulin-like Growth Factor I (IGF-I) .............. ...............35....
Fibroblast Growth Factors (FGFs) .............. ...............36....
Hepatocyte Growth Factor (HGF) .................. ........... .... ...............37....
Transforming Growth Factor Beta (TGFP) Superfamily .............. .....................3
M yostatin ................... ... ... ....... ............ .............3
Bone Morphogenic Proteins (BMPs) .............. ... .... ... .............4
BMP Function in vivo: Lessons Learned from Knockouts .............. .....................4
Current BMP6 Studies............... ...............45
Ras/Raf in Skeletal Muscle............... ...............46.
The Raf Family ............. ..... __ ...............47..












E2F Fam ily .................. ... ............ ... ....................4
E2F Signaling Mechanism and Involvement of Pocket Proteins .............. ........_......49
Cell Cycle Repression ...................... ..... .............5
E2Fs and Pocket Proteins in Skeletal Muscle .............. ...............51....


2 MATERIALS AND METHODS .............. ...............56....


M yoblast Cell Culture................ ...... ..... ................5
Growth Factor Treatment, and BrdU Pulsing and Fixation............... ...............57
Plasmids and Transfections .............. ...............57....
BrdU Staining and BrdU Incorporation............... .............5
Immunofluorescent E2F5 Staining ................. ...............58._._. ......
RNA Isolation and Nylon Arrays .............. ...............59....
Western Blots................ .. ...............59
Alkaline Phosphatase Staining .............. ...............60....
p38 Inhibition Assays .............. ...............61....
Apoptosi s Analysi s ................ ...............61................
Stati sti cs ................. ...............61........... ....


3 DIFFERENTIAL EXPRESSION OF TGFP SUPERFAMILY MEMBERS DURING
SKELETAL MYOGENESIS .............. ...............63....


O bj ective ............... .. ........ .... .... ...._........ ... ..........6
Differential Transcriptional Activity in Myoblasts versus Myofibers ................. ...............63
Differentiation TGFP Gene Expression in Myoblasts and Myofibers ................. ...............64
Discussion ................. ...............65.................


4 IMPACT OF BMP6 ON SKELETAL MYOGENESIS ................ ............................75


Obj ective ................... ............ ........ ........ .. .................7
Inhibition of Skeletal Myogenic Differentiation by BMP6 ....._____ .........__ ..............75
Dose-Dependent Effects of Recombinant BMP6 on Myoblasts ................. .....................76
Induction of ALP Activity in Response to BMP6 ....___......_____ ..... ....___........7
BMP6 Induces Rapid Transdifferentiation in Myoblasts ................ ......... ................77
BMP6 Does Not Alter Proliferation Rates of Myoblasts ................. ......... ................77
BMP6 is not Anti-Apoptotic............... .............7
Discussion ................. ...............78.................


5 BMP6 SIGNALING DURING SKELETAL MYOGENESIS............... ...............9


Obj ective .................. .. ........... ......... .... .... ..........9
Analysis of BMP Signaling Systems in Myoblasts ........... ................... ......... .......... .....9
Impact of Notch Inhibitor on BMP6-Mediated Inhibition of Differentiation .........._...........98
Discussion ........._..... ...._... ...............99.....











6 IMPACT OF E2F5 ON SKELETAL MYOGENESIS ......____ ........_ ...............113


O bj ective ............... ...... .........__ ...... ... ... .......... ......... 1
BMP6 Treatment does not cause E2F5 Nuclear Accumulation in Myoblasts ..................113
Presence of E2F5 in Satellite Cell Position ....._ .....___ ........__ ...........1
E2F5 Does Not Inhibit Myofiber Differentiation.............___ ...... ... .. .. .........__ ......11
pRb does not interact with E2F5 to Exert Inhibitory Effects on Muscle Specific Activity .115
E2F5 is Transcriptionally Active ................. ...............115...............
Discussion ................. ...............116................


7 SUMMARY AND CONCLUSIONS ................ ...............126...............

APPENDIX


A GENE ARRAY LAYOUT AND TABLE ................. ...............130..............


B SUMMARY OF ABBREVIATIONS .............. ...............137....


LITERATURE CITED ................. ...............141.............

BIOGRAPHICAL SKETCH ....___ ................ ......._. ..........18










LIST OF TABLES


Table page

1-1. Receptor and R-Smad specificity for TGFP superfamily members ................ ................. 53

1-2. TGFP superfamily transgenic knockouts .............. ...............54....










LIST OF FIGURES


Figure page

3-1. Differential transcriptional activity in myoblasts versus myofibers ............... ...............69

3-2. Intact RNA and cDNA probe synthesis............... ...............7

3-3. Gene expression profies in embryonic and satellite cell myofibers ...........__.................71

3-4. Differential gene expression in myoblasts and myofibers............... ...............7

3-5. Relative BMP gene expression in myoblasts and myofibers .............. ....................7

3-6. BMP6 does not undergo autocrine gene regulation ........._._.._......_.. .................7

4-1. Biochemical inhibition of skeletal myogenesis by BMP6............... ...............88..

4-2. Inhibition of skeletal myogenesis by BMP6............... ...............89..

4-3. BMP6 dose response curve............... ...............90.

4-4. BMP6 induction of alkaline phosphatase activity .............. ...............91....

4-5. BMP6 does not induce alkaline phosphatase (ALP) activity in fibroblasts. .......................92

4-6. BMP6 induces rapid transdifferentiation in myoblasts .............. ...............93....

4-7. BMP6 treatment does not alter my oblast proliferation .............. ...............94....

4-8. BMP6 is not anti-apoptotic............... .............9

5-1. Potential BMP6 signaling pathways affecting skeletal myogenesis .............. ..................106

5-2. Verifieation of BMP and TGFP signaling axis. ............. ...............107....

5-3. BMP6 treatment increases p38 phosphorylation ......___ .... ... ..__ .. ....__ .......10

5-4. p38 inhibition and BMP6 treatment result in additive inhibition of muscle specific
reporter activity. ........... ..... .._ ...............109...

5-5. BMP6 and p38 signal through independent pathways to influence myogenic
different ati on ................. ...............110._._._ .....

5-6. p38 signaling does not play a significant role in transdifferentiation. ........._.._... ...............111

5-7. BMP6 inhibition of differentiation is mediated in part by Notch. .............. .............. .1 12

6-1. BMP6 treatment does not cause E2F5 nuclear accumulation in myoblasts ......................121










6-2. Presence of E2F5 in satellite cell position............... ...............12

6-3. E2F5 does not inhibit myogenic differentiation ........................... ........._. ......12

6-4. pRb does not interact with E2F5 to exert inhibitory effects on muscle specific activity ....124

6-5. E2F 5 i s transcriptionally active ................ ...............125......_.__..

7-1. Proposed model of BMP6 on myogenic differentiation and interaction with Notch..........129










Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INVOLVEMENT OF BMP6 AND E2F5 INT SKELETAL MYOGENESIS

By

Jennelle Robin McQuown

May 2007

Chair: Sally E. Johnson
Major: Animal Sciences

An amazing dynamic exists within skeletal muscle that is required for the development and

maintenance of the musculature system in response to stimuli. A differential BMP- and TGFP-

responsiveness and gene expression profile in myoblasts versus myofibers, suggests that TGFP

superfamily members play distinct regulatory roles at specific stages of myogenesis. Treatment

of myoblasts with BMP6 results in an increase in alkaline phosphatase activity in a dose-

dependent and time-dependent manner. Exogenous BMP6 treatment results in inhibition of

myoblast differentiation as observed by significant inhibition of muscle reporter activity, muscle

specific protein synthesis, and myoblast fusion. BMP6 treatment does not alter proliferation

rates or play an anti-apoptotic role in myoblasts. Inhibition of p3 8 activity and BMP6 treatment

caused significant inhibition of TnI-Luc activity and muscle specific protein expression markers

suggesting an additive effect through independent signaling cascades. Interestingly, the

combination of BMP6 and Notch inhibition partially restores MyHC expression in fibers. This

suggests that the myogenic inhibitory effect observed in the presence of BMP6 is not a cell cycle

effect and not a direct target of the RaflMEK/ERK signaling axis, but is partially mediated by

functional Notch signaling. Therefore, BMP6-mediated inhibition of differentiation is regulated

or partially controlled by the Notch signaling pathway. Ectopic BMP6 treatment did not induce









E2F5 nuclear accumulation in myoblasts further suggesting BMP6 is not causing a cell cycle

effect. E2F5 is present in non-dividing cells in vivo outside the dystrophin border in the putative

satellite cell position, although E2F5 did not impact myogenic differentiation. Additionally, the

inability of E2F5 to inhibit differentiation is not due to insufficient pocket protein function.

Multiple signaling axes are key factors during Raf-imposed block to myogenic differentiation.

Further understanding of the regenerative ability in skeletal muscle in response to stimuli would

be useful for patients or animals that experience severe muscle trauma, and for individuals with

skeletal muscle disorders, and would provide additional information for human therapeutic and

agricultural applications that would benefit from enhanced muscle mass.









CHAPTER 1
LITERATURE REVIEW

Introduction

Within an organism, skeletal muscle functions in locomotor activity, postural behavior,

and breathing. Skeletal muscle will undergo injury in response to direct trauma or as an indirect

result of neurological dysfunction or genetic defects, and if not repaired, muscle mass and

locomotion abilities may be lost, and death can possibly occur (Charge and Rudnicki, 2004). An

amazing dynamic exists within skeletal muscle that is required for the maintenance of the

musculature in regards to the states of atrophy, injury, and subsequent repair. Understanding the

regenerative ability of skeletal muscle in response to these stimuli are important areas of

research. For instance, patients who experience severe muscle trauma, individuals with genetic

skeletal muscle disorders like muscular dystrophy, and improving the recovery of astronauts

experiencing a weightless environment would benefit from such studies. Many changes to

muscle physiology occur in an adaptive response to decreased or increased usage of skeletal

muscle (Stein and Wade, 2005). The adaptive responses of decreased usage includes a shift in

myosin isoforms from slow to fast fiber type, replacement of protein with fat within the muscle,

shifts in energy source for metabolism away from lipids to glucose, a loss of bone mineral

density, and an increase in bone resorption (Shackelford et al., 2004; Stein and Wade, 2005).

Skeletal Muscle Biology

The mechanics of skeletal muscle contraction begin with a neural stimulus for contraction.

This results in a generation of an action potential in a motor neuron causing the release of

acetylcholine into the synaptic cleft of the neuromuscular junction (NMJ). This released

acetylcholine binds with the receptors on the motor end-plate, producing an end plate potential,

which leads to the depolarization that is conducted down the transverse tubules deep into the









muscle fiber. Depolarization results in calcium being released from the sarcoplasmic reticulum

(SR) (Crouch, 1985).

In the resting state, myosin cross-bridges remain connected to actin in a weak binding

state. When depolarization signal reaches the SR, calcium is released into the sarcoplasm, which

binds to troponin, causing a shift in troponin position to uncover the "active" sites on actin. The

"energized" or "cocked" myosin cross-bridge then forms a strong bond at the active sites on

actin. Inorganic phosphate released from the myosin cross-bridge energizes the cross-bridge to

allow it the ability to pull the actin molecules. Cross-bridge movement is completed by the

release of ADP from the myosin cross bridge. At this point, the myosin cross-bridges are in

what is considered the "strong binding state" with actin. Attachment of ATP to myosin allows

the cross-bridge to break the strong binding state and form a "weak binding state". ATP is then

broken down to ADP + Pi + energy. The energy is released and used to "energize" the myosin

cross-bridges. This cycle can repeat as long as calcium and ATP are present. The cycle is

stopped when the SR actively removes calcium from the sarcoplasm (Lieber, 2002).

The SR and Transverse Tubule Systems within Skeletal Muscle

Within the muscle, two membrane systems are present to activate the filaments. These two

systems are called the transverse tubule (T) system and the SR system. The T-system consists of

invaginations in the surface membrane physically contiguous with the sarcolemma, or holes that

extend into the fiber and transversely crosses the long axis of the fiber. The T-system conveys

activation signals received from the motor neuron into the myofibrils. Since motor neurons are

not in direct contact, the T-system helps increase activation time versus a simple diffusion

system. The release of calcium switches on muscle activation and the removal of calcium from

the myofilament cause muscle relaxation. Embedded in the SR membrane are specific calcium

channels and pumps that control this calcium release and uptake. The SR envelops each









myofibril to provide contact between the activation and force generation systems. The SR also

contacts the T-system, thus acting as a middle man in skeletal muscle activation and relaxation.

The T and SR systems are arranged in a structure called a "triad", which is a single T tubule

surrounded by two SR tubules located at each Z disk and the triad functions as the interface

between the extracellular and intracellular surfaces of the fiber (Lieber, 2002).

Additionally, the SR and T systems are linked by junctional feet structures. Junctional feet

are composed primarily of two components: ryanodine receptors (RyR) and dihydropyridine

receptors (DHP). RyRs are voltage sensing proteins embedded in the SR that sense action

potential traveling across the T system. DHPs are calcium release channels through which the

myoHilament receives activating calcium (Lieber, 2002).

Major Fiber Type Classification

Skeletal muscles can be classified as either red or white based on the maj or proportion of

red or white fibers they contain. Few muscles are composed solely of one fiber type; most are of

a heterogeneous composition. The red coloration of red fibers is due to higher myoglobin

content as compared to white fibers and the myoglobin allows for oxygen storage required for

oxidative metabolism. Conversely, white fibers have a high content of glycolytic enzymes and

low levels of oxidative enzyme activity (Smith, 1972).

Another method of skeletal muscle fiber designation is based upon contraction rates: slow-

twitch vs. fast-twitch fibers or type I vs. type II. There are four different adult myosin isoforms

in skeletal muscle: type I, IIA, IIX(D), and IIB with type I and IIA designated as red muscle

fibers and type IIX(D) and IIB designated as white muscle fibers (Barany, 1967; Roy et al.,

1984). Slow-twitch fibers, which are also called Type I fibers, are characterized by higher

number of mitochondria, larger mitochondria, and the mitochondria are located in two general

areas: the subsarcolemma, and the intermyofibrillar. The mitochondria within Type I fibers are









more efficient than in Type II fibers. For example, in the subsarcolemma, the mitochondria are

nine times more efficient in Type I and in the myofibrillar, they are five times more efficient in I.

These fibers are surrounded by numerous capillaries and have a higher concentration of

myoglobin, which gives the red muscle fiber types their visual color. Type I fibers have a large

capacity for aerobic metabolism and can use ATP more efficiently than Type II without lactic

acid buildup. The lipid content is greater in red muscle fibers, which serves as a metabolic fuel

source and while these fibers contract slower, they can contract for a longer time. These fibers

play a role in posture because they are less easily fatigued, as long as oxygen is available and due

to their higher resistance to fatigue and allowance for a higher maximal oxygen uptake (VO2

max) they are selected for in endurance type training (Evans et al., 1994; Van Swearingen and

Lance-Jones, 1995; Lieber, 2002).

Fast twitch or Type II fibers are glycolytic and can produce force at a higher rate. This is

called a phasic mode of action since contraction occurs in short bursts and these fibers are more

easily fatigued. White fibers have a more extensively developed SR and T-tubule system along

with more narrow Z disks versus white fibers. They also have fewer and smaller mitochondria

than Type I and the glycolytic metabolism that predominates in these fibers can occur both

aerobically or anaerobically. White fibers have a lower capillary density than red fibers.

Functional implications demonstrate that Type II fibers are increased during strength training due

to their ability to produce force at a higher rate (Zhang and McLennan, 1998; Lieber, 2002).

Muscle Architecture

The anatomical and biomechanical properties of skeletal muscle determine the efficiency

and overall ability of the muscle to generate force and movement. The largest functional unit of

contractile filaments is the myofibril, which is a string of sarcomeres. The muscle fiber is










composed of myofibrils arranged side-by-side and the groups of muscle fibers are sheathed in

connective tissue termed the perimysium (Lieber, 2002).

The number of sarcomeres within a fiber depends on the muscle fiber length and diameter

of the fiber. They are the most important determinant of muscle fiber function. The total

distance of myofibrillar shortening is equal to the sum of the shortening distance of each

sarcomere. This allows for whole muscles to be able to shorten up to several centimeters even

though each sarcomere can only shorten ~1 Clm giving the muscle a tremendous ability to adapt.

Within the sarcomere are two maj or types of contractile filaments, the thick filaments and the

thin filaments. These filaments are large polymers of myosin and actin proteins. In thick

filaments, which are the myosin-containing filament, a "feathered" appearance is observed with

proj sections coming out at either end of the filament because one molecule rotates ~600 relative to

molecules on either side. These filaments generate the tension during muscle contraction and the

thin filaments or actin-containing filament regulates the tension generated (Gordon et al., 1966a;

Gordon et al., 1996b; Lieber, 2002). Tension generation is also a function of the magnitude of

the overlap between the actin and myosin filaments. Passive tension plays a role in providing

resistance in the absence of muscle activation. The source of passive tension is due to the protein

titin, which connects the thick myosin filaments end to end (Magid and Reedy, 1980; Labeit and

Kolmerer, 1995).

Striated skeletal muscle is composed of bundles of enormous, multi-nucleated cells and the

striations arise from a repeating pattern in the myofibrils. Active interdigitations of these

filaments produce muscle shortening and give muscle its striated appearance. Some

characteristics of striated muscle are its ability to contract and generate tension when activated

and its ability to return to its original length and form after contraction or stretching ceases.









These properties are primarily the result of separate sets of filament systems: contractile actin

and myosin filaments and viscoelastic titin and intermediate filaments (Vigoreaux, 1994; Knupp

et al., 2002).

Within the thin filaments, approximately 5% of the myofibrillar protein is composed of

troponin molecules, which are located approximately every seven or eight actin molecules along

the actin filament. Troponins are the regulatory proteins responsible for turning on contraction.

Troponin is composed of three subunits: Tn-I, Tn-C, and Tn-T. Tn-I exerts an inhibitory

influence on tropomyosin when calcium is not present. Tn-C: binds calcium during contraction.

Tn-T: binds troponin to tropomyosin. Tropomyosin is a long, rigid, and insoluble rod-shaped

molecule that stretches along in close contact with each strand of the thin filament. Within each

groove of the actin super-helix lays a strand of tropomyosin and a single molecule extends the

length of seven G-actin molecules (Lieber, 2002).

The dark striations observed in skeletal muscle are a lattice of thick filaments termed the A

band because it appears dark under a microscope. The light striations are termed I bands, which

are regions of the myofibril containing only thin filaments and Z disks. The Z disks are the

structure that attach to the thin filaments to act as actin-anchoring structures and function as a

common link to mechanically integrate contractile and elastic elements (Schroeter et al., 1996).

They are also involved in transmission of active and passive forces and differences in Z band

structure have been described for distinct classes of muscle and fiber types (Vigoreaux, 1994).

Thin filaments extend from two adj acent Z lines to interdigitate with the thick filaments. Thick

and thin filament arrays form the contractile system and are the repeating unit in each myofibril

(Bloom and Fawcett, 1968).









Intermediate Filaments (IFs) are one of the three maj or classes of cytoskeletal proteins

along with microtubules and microfilaments. The IF proteins and IF-associated proteins such as

desmin, vimentin, and nestin, are involved in maintaining the structural and functional integrity

of all muscle types and align themselves in a head-to-tail dimer along the central rod domain.

For example, in the skeletal muscle injury model, nestin and vimentin IF proteins begin to be

expressed 6 hours post-injury in myoblasts and their maximal expression was observed around 3-

5 days post injury. Thereafter, vimentin expression ceases completely, whereas nestin is found

to remain only in the sarcoplasm next to neuromuscular and myotendinous junctions. At 6-12

hours post-injury, desmin expression is upregulated and becomes the predominant IF protein in

myofibers, coinciding with the appearance of cross-striations. Nestin and vimentin are essential

during the early phases of myofiber regeneration and desmin is responsible for maintenance of

myofibrils in mature myofibers (Vaittinen et al., 2001).

A protofibril is a complex of eight IF monomers and the network of IFs form a lattice of

connections that link the different parts of the muscle fiber. These networks extend from the Z-

disk to adjacent myofibrils and from the sarcolemma to the Z-disc. Additionally, IFs are

associated with the neuromuscular junction, nuclear membrane, and mitochondria located

between adj acent myofibrils (Agbulut et al., 2001; Reipert et al., 1999).

Skeletal Muscle Function

Contraction velocity is based on the efficiency of the energy metabolism. Velocity is

fastest when ATP is generated from an oxidative rather than a glycolytic pathway. In addition,

contraction velocity is also dependent on the type of myosin present in the fiber. The four

Myosin Heavy Chain (MyHC) isoforms are the products of a multigene family whose expression

during development and in the adult are regulated by neuronal, hormonal, and mechanical

factors. Most single fibers (~86%) express only one MyHC isoform but there are examples of









co-expression of MyHC isoforms and the MyHC IIA and IIX/D isoforms are predominately

expressed among fibers (Rivero et al., 1999). In terms of shortening velocity, the gradient

observed for MyHC isoforms is I < IIA < IIX/D < IIB (Bottinelli et al., 1991; Galler et al., 1994).

There are also phenotypic differences associated with MHC isoform composition in terms

of metabolic and size properties of the muscle fiber types (Bar and Pette, 1988; Schiaffino et al.,

1989). For example, motor units containing more MyHC I or IIA are more resistant to fatigue

than motor units containing MyHC IIX/D or IIB. Additionally, MHC IIB fibers are generally

larger than MyHC I or IIA (Rivero et al., 1999).

The globular head on the myosin has ATPase activity. This activity determines how fast

the muscles contract, and is determined by how quickly ATP is hydrolyzed. In terms of the

force-velocity relationship, this illustrates that the amount of force generated by a muscle is

highly proportional to its velocity or velocity is dependent on how much force is resisting the

muscle. Maximum contraction velocity is termed Vmax, which is one of the most commonly used

parameters to characterize muscle, and is relative to both fiber type distribution and architecture

(Lieber, 2002).

Function is largely determined by a muscle's architecture. While fiber size between

muscles does not vary greatly, there are maj or architectural differences between different muscle

groups and these are the best predictors of force generation (Burkholder et al., 1994). Skeletal

muscle architecture is defined as the arrangement of muscle fibers within the muscle relative to

the axis of force generation. There are three classifications of muscle arrangements termed

parallel, uni-pennate, and multi-pennate. Parallel muscles are composed of fibers that extend

parallel to the muscle's force-generating axis. Uni-pennate muscles have fibers that are

orientated at a single angle relative to the force-generating axis typically in an angle of 0 to









30 degrees. Most muscles are multi-pennate, where the fibers are orientated at several angles

relative to the axis of force generation (Lieber, 2002). Other parameters that help characterize

the properties of various muscles or muscle groups are the sarcomere length, which also

translates into fiber length, range of movement (ROM), and the physiological cross-sectional

area (PCSA). The PCSA of a muscle represents the sums of the cross-sectional areas of all of

the muscle fibers within the muscle and is directly proportional to the maximum titanic tension

generated by the muscle (Powell et al., 1984).

Tying together some of these parameters give a better indication of functional properties of

specific muscle groups. For example, the quadriceps muscles have relatively high pennation

angles, large PCSAs, and short fibers. Therefore, this makes them suited for the generation of

large forces. Hamstring muscles like the sartorious, semitendinosus, and the gracilis muscles

have low PCSAs and extremely long fibers, which allow for large excursions at low forces.

Conversely, the soleus muscles have high PCSAs and short fiber lengths so that these muscles

can generate high forces with small excursions making these suitable muscle groups for a

postural stabilization role (Edman et al., 1985; Woittiez et al., 1985).

There is also different terminology related to different types of contractions. For example,

isometric or static contractions are when the muscle tension increases but the body part does not

move. Concentric contractions are when the action of the muscle results in muscular shortening

as the body part moves and the force is applied to the muscle as it contracts. Eccentric

contractions occur when the muscle is activated and force is produced as the muscle lengthens.

Therefore, these types of contractions are more likely to result in muscle injury or soreness

versus concentric contractions. Additionally, muscle strengthening regimens are greatest when

exercises using eccentric contractions are utilized (Lieber and Friden, 2002). These factors









demonstrate why muscle architecture is also a consideration in training regimes for human and

animal athletes. As more information is generated on the basic understanding of muscle form

and function, there can be further benefits for athletic training and performance.

Skeletal Muscle Development

Skeletal muscle development is a highly complex and orchestrated process, which is still

not completely understood, both at the macroscopic and molecular levels. Skeletal muscle cells

are derived from the somites, except for the craniofacial muscles in mammals and birds (Armand

et al., 1983). Somites are balls of epithelial cells formed from the paraxial mesoderm in pairs on

either side of the neural tube, and differentiate into two regions the dermomyotome and

sclerotome. Due to their interactions with surrounding tissues, the ventromedial portion of the

somite gives rise to the sclerotome, which is the precursor to the ribs and axial skeleton, and the

dorso-lateral part forms the dermomyotome, which is where the myogenic precursors and dermis

originate in embryogenesis (Braun et al., 1992; Rudnicki et al., 1993; Christ and Ordahl, 1995).

Within the dermomyotome, the epaxial muscles and subsequent body wall muscles are formed

on the dorso-medial lip. The hypaxial muscles, which consist of the limbs, tongue, diaphragm

and ventral wall musculature, are formed on the dorso-lateral lip. Endothelial precursors are also

derived from the somites (Chevallier et al., 1977; Beddington and Martin, 1989; Ordahl and

LeDouarin, 1992; Wilting et al., 1995; Kardon et al., 2002).

Multiple pathways mediate the embryonic processes throughout skeletal muscle formation.

In terms of limb bud development, the progenitors of this process evolve by delamination and

migration distally into the developing limb bud in response to molecular signals from the

adjacent lateral plate mesoderm (Chevallier et al., 1977; Christ et al., 1977; Solursh et al., 1987;

Hayashi and Ozawa, 1995). Induction of premyogenic cells in the limb to delaminate is

mediated by hepatocyte growth factor (HGF) or scatter factor and by fibroblast growth factor










(FGF). In experiments where both FGF2 and HGF were applied ectopically in the interlimb

flank mesenchyme of chicks, delamination of the lateral dermomyotome was observed (Brand-

Saberi et la., 1996; Heymann et al., 1996). Additionally, when c-met, the tyrosine kinase

receptor for HGF, is inactivated, delamination in mice is prevented. Both HGF and FGF2 and

FGF4 are also found to promote migration by acting as a chemotactic source resulting in cell

migration towards the distal tip of the limb bud (Itoh et al., 1996; Takayama et al., 1996; Webb

et al., 1997; Scaal et al., 1999).

Transcription factors are involved in delamination and migration, with Pax3 and Lbx1

being initially expressed in the lateral dermomyotome. Pax3 is necessary for the epithelial-

mesenchymal transformation of the lateral dermomyotome (Daston et al., 1996). In Pa-x3 null

mice, which are also called Splotch mice, c-met expression is either significantly reduced or

absent in the lateral dermomyotome. This suggests that Pax3 is upstream of c-met, since the

dermomyotome is disorganized and the limb myogenic cells do not migrate (Epstein et al., 1996;

Yang et al., 1996; Mennerich et al., 1998; Tremblay et al., 1998). Inactivation of Lbx1 causes

premoygenic cells to delaminate properly but have dysfunctional migration. The resulting

phenotype exhibits minimal hindlimb musculature and the extensors (or dorsal muscles) are

missing in the forelimb. These observations are similar to experiments where Gab1 is

inactivated, which is also involved in c-met signaling and is also believed to be a result of cells

being unable to respond to limb migratory cues (Schafer and Braun, 1999; Brohmann et al.,

2000). Therefore, as these premyogenic cells migrate distally towards the limb bud, they begin

to switch on the myogenic determination bHLH transcription factors, M~yoD and M~yf5, and form

the dorsal and ventral muscle masses. The myogenic cells then terminally differentiate into slow









or fast fibers, which express the relevant form of slow or fast MyHC. The fast fibers are needed

for movement with the slow fibers helping maintain posture (Francis-West et al., 2003).

Another maj or player in skeletal muscle formation is Sonic hedgehog (Shh), which is

expressed in the notochord and floor plate of the neural tube and induces the formation of the

sclerotome. Along with the Wnt proteins (1, 3a, 7a, 11), Shh also promotes myogenesis by

activating expression of Myf5 in the epaxial myotome through its interaction with Wntl. Wnt1 is

expressed in the dorsal neural tube and the dorsal ectoderm. Wnt7a is expressed in the dorsal

ectoderm and found to activate the expression ofhyoD in hypaxial myotome (Schmidt et al.,

2000; Petropoulos and Skerjanc, 2002).

The bone morphogenic proteins (BMPs) are expressed in the lateral plate mesoderm and

limb bud ectoderm. Noggin, an antagonist for BMP, is expressed in the dorsomedial lip of

dermomyotome. BMPs and Noggin regulate Pa-x3 in proliferating muscle precursor cells

(MPCs), and Pax3 is critical for the downstream activation for Myogenic Regulatory Factors

(MRFs) (Amthor et al., 1998). The levels of BMPs within the somite, in the presence or absence

of its antagonists, controls the expression of Pax3 and can either promote embryonic muscle

growth by expanding Pax3-expressing muscle precursor cells or restricting development by

inducing apoptosis (Amthor et al., 2002). In the limb, BM~P2 and BM~P7 expression is increased

by Shh. This causes a stimulation of muscle growth and a delay in muscle differentiation by

allowing these BMPs to inhibit activation of MyoD and Myf5 by Pax3 (Tajbakhsh et al., 1997).

The BMP signals originating from the lateral plate delay the activation of myogenic bHLH gene

expression (Pourquie et al., 1996). In the presence of Noggin, BMPs are sequestered, Pax3

expression is repressed, and MyoD expression is dramatically expanded, inducing formation of a

lateral myotome (Duprez et al., 1998; Reshef et al., 1998; Kriiger et al., 2001). Conversely,









another BMP antagonist, follistatin, promotes Pax3 expression, which transiently delays muscle

differentiation and exerts a proliferative signal during muscle development (Amthor et al., 2002).

Myogenic Regulatory Factors (MRFs)

At the molecular level, MRFs mediate the process of myogenic determination and muscle-

specific gene expression enabling multipotent, mesodermal cells to give rise to mononucleated

myoblasts, withdraw from the cell cycle and differentiate into multinucleated muscle fibers,

which are the framework of whole muscle (Cooper and Konigsberg, 1961; Stockdale and Holtzer

1961; Stockdale et al., 1999). MRFs are a family of skeletal muscle specific bHLH transcription

factors and contain the genes, Myf5, M~yoD, M~RF4, and myogenin. When these factors are

overexpressed in non-myogenic cell lines, they cause them to differentiate into myogenic cells

(Davis et al., 1987; Wright et al., 1989; Braun et al., 1989). M~yoD and M~yf5 are expressed in

proliferating, undifferentiated cells and when cells begin to terminally differentiate, myogenin

expression is induced (Smith et al., 1993, 1994; Andres and Walsh, 1996). M~RF4 is expressed in

both early stages of myogenesis, during muscle development, as well as in adult muscle tissue

(Hinterberger et al., 1991).

Another family of genes, the MEF2 family augments the myogenic activities of MRFs.

(Shen et al., 2003). MyoD and MEF2 interact to control myoblast specification, differentiation,

and proliferation and MRF4 acts in embryonic cells to control myogenic determination (Kassar-

Duchossoy et al., 2004). However, myogenin expression is required for terminal differentiation

before birth. In addition, the expression of skeletal muscle a-actin is a marker of late

differentiation following myogenin expression (Knapp et al., 2006).

Further mediation of MRF activities is achieved through their heterodimer formation with

another family of bHLH proteins, the E proteins (Lassar et al., 1991; Shirakata et al., 1993). The









E proteins are class I, bHLH proteins. These class I proteins form heterodimers with the class II

bHLH proteins such as MyoD. This heterodimer binds to specific DNA regions (E boxes) that

contain the sequence CANNTG and activate transcription of muscle target genes (Yutzey and

Konieczny, 1992). E47 is an alternate splice product of the E2A gene expressed early in

myogenic differentiation (Sun and Baltimore, 1991; Quong et al., 1993).

Emerging data demonstrates that the intracellular signaling pathway, p38 mitogen-

activated protein kinase (MAPK), participates in several stages of the myogenesis. The most

documented role of p3 8 is its cooperation with transcription factors from the MyoD and MEF2

families in the activation of muscle specific genes. This interaction contributes to the temporal

gene expression during differentiation (Cuenda and Cohen, 1999; Zetser et al., 1999).

Withdrawal of myoblasts at the G1 stage is necessary for differentiation to occur. This is

partially mediated by p38 kinase and c-Jun N-terminal protein kinases (JNKs) signaling through

its induction of p21 expression in myoblasts (Puri et al., 2000; Mauro et al., 2002). The

MyoD/E47 heterodimer is regulated by p38 MAPK and MRF4 is phosphorylated by p38 MAPK

on Ser-3 1 and Ser-42 resulting in a reduced transcriptional activity. The modulation of MRF4

activity also results in selective silencing of muscle-specific genes in terminal differentiation

(Suelves et al., 2004). During somite development in mice, p38 MAPK plays a crucial role in

activating MEF2 transcription factors. In Xenopus laevis, p38 signaling is required for early

expression of2~yf5 and for the expression of several muscle structural genes (Keren et al., 2005).

Negative myogenic regulators exist such as, Id1 and Twist. Id1 is a dominant negative

HLH protein that prevents the interaction of the E proteins with MyoD or Myf5 by preferentially

binding to the E proteins (Norton et al., 1998). Twist complexes with E proteins to repress









myogenic differentiation. Twist also can directly block transcriptional activity of MyoD and

MEF2 by binding to E-box sequences (Hebrok et al., 1997; Puri and Sartorelli, 2000).

In order for muscle differentiation to occur, myoblast cell cycle arrest is critical. MyoD

promotes cell cycle arrest by inducing p21, which is a cyclin-dependent kinase (cdk) inhibitor

(Guo et al., 1995; Halevy et al., 1995). Induction of p21 is subsequent to myogenin expression

and it is believed that high levels of p21 are required for cells to remain in a post-mitotic state.

MyoD also induces retinoblastoma (Rb), which is a negative regulator of G1 progression

(Martelli et al., 1994). There is an intricately regulated pattern of cell cycle proteins that are

temporally expressed based on a variety of molecular interactions and further discussion of these

events is found in a later section. Briefly, G1 progression is controlled by Cdk-mediated

phosphorylation of the Rb protein, which allows for the release and activation of E2F 1

(Lundberg and Weinberg, 1998). E2F 1 mediates S-phase progression by allowing for expression

of genes required for DNA replication and mitosis (Ishida et al., 2001). Withdrawal from the

cell cycle is mediated by p21, which directly inhibits Cdk complexes and interferes with S-phase

progression by binding to and inhibiting the activity of proliferating cell nuclear antigen

(PCNA), a subunit of the DNA polymerase (Dotto, 2000).

Satellite Cells

More than 40 years ago, Alexander Mauro first identified muscle progenitor cells in frog

skeletal muscle, which were identified as satellite cells based on their observed location adj acent

to mature muscle fibers as seen by electron microscopy (Katz, 1961; Mauro, 1961). These

undifferentiated cells have a degree of plasticity, demonstrating properties of stem cells such as,

yielding all of the specialized cell types from which they originate and having the ability to self-

renew. In mature muscle, in response to injury, hypertrophy, routine maintenance or disease,

satellite cells serve as a reserve of muscle stem cells that are activated from their quiescent or Go









state to re-enter the cell cycle and provide myonuclei for growth or repair (Moss and Leblond,

1971). Self-renewal ability in satellite cells may occur by either a stochastic event or through

asymmetric cell division, and this self-renewal is required for maintenance of their own

population pool (Collins et al., 2005). Satellite cells are derived from a Pax3/Pax7 population of

progenitor cells located in fetal muscle (Relaix et al., 2006).

In a quiescent state, satellite cells are characterized by a low nucleus/cytoplasm ratio, few

organelles, and high amount of heterochromatin or condensed chromosomes, resulting in

minimal metabolic activity or transcription (Schultz, 1976). Quiescent satellite cells can also be

distinguished from activated cells because there are numerous morphological changes that occur

upon activation. During activation, cytoplasmic extensions become apparent, along with an

increase in cytoplasmic volume of the activated cell, the amount of heterochromatin decreases

and organelles such as the Golgi, endoplasmic reticulum, ribosomes, and mitochondria start to

appear (Schultz et al., 1978). Additionally, at the molecular level, MyoD expression is turned on,

and a CD34 isoform switch is observed, along with co-expression of Pax7, M-cadherin, and

Myf5. Cellular proliferation and division is indicated by expression of proliferation markers

such as PCNA, and induction of myogenin expression designates cells undergoing myogenic

differentiation (Fuchtbauer and Westphal, 1992; Grounds et al., 1992; Yablonka-Reuveni et al.,

1994; and Zammit et al., 2004).

Satellite Cell Marker Proteins

In order to distinguish satellite cells from other mononucleated cells within skeletal

muscle, protein markers have been identified which are specific to satellite cells alone or in

combination with other markers that allow for the specific identification of a pure population of

satellite cells. Some of the most studied are CD34, M-cadherin, Pax7, syndecan-3/4, VCAM1,

and c-met (Rosen et al., 1992; Beauchamp et al., 2000; Seale et al., 2000; Cornelison et al.,










2001). Pax7 is currently the best marker for identifying quiescent satellite cells (Seale et al.,

2000). Markers like CD34 are useful in identifying satellite cells on isolated myofibers, but are

not specific or unique to satellite cells and a co-staining or a combination of markers is needed to

definitively identify these cells (Beauchamp et al., 2000). Although, the truncated form of CD34

(Beauchamp et al., 2000) and the P isoform of myocyte nuclear factor (MNF) are specific to

quiescence (Garry et al., 1997; Yang et al., 1997). There is also a level of heterogeneity that

exists within the satellite cell population in terms of immunohi stochemical (IHC) cell staining

and in vitro clonal analyses (Schultz and Lipton, 1982; Baroffio et al., 1996; Molnar et al., 1996).

For example, most satellite cells are positive for both CD34 and M-cadherin and most of these

cells are also positive for Myf5 (Beauchamp et al., 2000), a subpopulation of ~20% are not

positive for these markers. Interestingly, functional studies suggest that this subpopulation of

cells serves as a reserve for satellite cell replenishment (Rantanen et al., 1995; Schultz, 1996).

c-Met

c-Met is a transmembrane tyrosine kinase receptor, and represents the activated HGF

receptor, which is found in all quiescent satellite cells but not expressed in myofibers (Allen et

al., 1995; Cornelison and Wold, 1997; Tatsumi et al., 1998). However, it is not restricted to

satellite cells, and is detected in both resting and regenerating muscle. The c-met knockout

mouse demonstrates that c-met is necessary for proper limb, diaphragm, and skeletal muscle

formation. Following a crush injury, the mononucleated cells surrounding the necrotic fiber

express the c-met marker. Additionally, cells negative for another stem cell marker, CD34, but

positive for c-met (CD34-/c-met+) are still capable of giving rise to myotubes in culture

(Beauchamp et al., 2000).










Syndecans 3 and 4

Syndecans 3 and 4 have overlapping expression patterns in skeletal muscle with c-met and

are cell surface trans-membrane heparin sulfate proteoglycans. They function in FGF signaling

and the expression of Syndecan 3 and 4 is consistent with that of satellite cell position (between

basal lamina and sarcolemma) and proliferating MPCs (Cornelison et al., 2001). Syndecan 3 and

4 knockouts have severe defects in satellite cell activation and muscle regeneration (Comelison

et al., 2004). Syndecans play a role in signaling through the ERKl/2 MAP kinase pathway.

Primary mouse satellite cells require heparin sulfate for normal proliferation (Comelison et al.,

2001). Activation and initiation of myogenesis is delayed in vitro when syndecan signaling

events are blocked (Comelison et al., 2004).

M-cadherin

M-cadherin is a calcium-dependent adhesion molecule, which is used as a marker of

quiescent satellite cells and activated myogenic precursors, but is not expressed in differentiated

myotubes (Irintchey et al., 1994; Moore and Walsh, 1993; Beauchamp et al., 2000; LaFramboise

et al., 2003). It is expressed in some, but not all quiescent satellite cells, demonstrating

heterogeneity within satellite cell compartments (Cornelison and Wold, 1997). Those that do

express M-cadherin also express CD34 (hematopoietic stem cell marker) (Beauchamp et al.,

2000). Studies suggest that M-cad+/CD34+ satellite cells are also Myf5-positive (Beauchamp et

al., 2000). Another observation is that the number of M-cadherin positive cells increases upon

satellite cell activation (Comelison and Wold, 1997).

CD34

CD34 is expressed in cells that express no cardiac, hematopoetic, or skeletal muscle

mRNA transcripts, indicating a lack of lineage. This follows with the idea that only a sub-

population of satellite cells expresses the CD34 marker and designates a sub-population of









satellite cells that are less committed to the myogenic lineage (Beauchamp et al., 2000). Cells

that express CD34, don't express Pax7 and M~-cadherin, and both CD34 positive and negative

progenitor cells can give rise to myotubes in vitro. One study examined CD34-positive versus

CD34-negative primary myoblasts and found that CD34-positive primary myoblasts were more

efficient in participating in regeneration but CD34-negative cells had a higher fusion index in

vitro (Beauchamp et al., 2000).

Pax7

Pax7 is a member of the paired box family of transcription factors, and is localized to

nuclei situated in discreet peripheral locations within resting adult skeletal muscle (Seale et al.,

2000; Relaix et al., 2005). The number of cells positive for Pax7 is believed to correlate well

with the expected number of satellite cells. This is because Pax7 expression co-localizes with

myostatin, c-met, and m-cadherin in satellite cells resting between basal lamina (Seale et al.,

2000; LaFramboise et al., 2003; McCroskery et al., 2003; Halevy et al., 2004). In addition,

myogenic cells lines, which model quiescent, undifferentiated myoblasts, appear to be uniquely

marked with high levels of Pax7. Furthermore, the expression seen in proliferating primary

myoblasts is down-regulated upon myoblast differentiation. Although loss of Pax7 does not

induce differentiation, indicating that other factors must be present or absent for myoblasts to

commence terminal differentiation (Seale et al., 2000; Olguin and Olwin, 2004).

Pax7 inhibits myogenic conversion induced by MyoD. It is believed that Pax7 is indirectly

interfering with its function or competes for proteins necessary for MyoD-dependent

transcription since the presence of Pax7 cannot inhibit the effects of MyoD-E47 heterodimers

(Olguin and Olwin, 2004). Yet, overexpression of Pax7 in satellite cells does induce cell cycle

exit, prevention of BrdU incorporation, a decrease in M~yoD expression, and prevention of

myogenin induction (Olguin and Olwin, 2004). In addition to its impact on MyoD, it is believed









that myoblasts expressing the Pax7+/MyoD- phenotype may return to quiescence to replenish

satellite cell pools while myoblasts that acquire MyoD proliferate and fuse to form myotubes

(Zammitt et al., 2004; Relaix et al., 2005). Alternatively, one study showed that Pax7 may have

anti-apoptotic functions. Myoblasts transduced with dominant negative Pax7 lead to cell death

(Relaix et al., 2006).

Myocyte nuclear factor (MNF)

MNF is a winged helix transcription factor, also identified as Foxkl, expressed in both

cardiac and skeletal muscle, and in quiescent satellite cells. The number of MNF positive cells

increases when muscle is induced to regenerate, and while detected in regenerating myotubes,

M~NF is down-regulated during late stages of regeneration (Garry et al., 1997). There are two

isoforms of MNF, MNF-oc and MNF-P, and both are detectable in skeletal muscle. Alpha is

expressed in proliferating MPCs, while beta is limited to quiescent satellite cells and down-

regulated upon activation (Yang et al., 2000). In quiescent satellite cells, high MNF-P

expression allows for the formation of repressive complexes with mSin3 family members to

repress targeted gene transcription (Garry et al., 2000; Yang et al., 2000). Upon satellite cells

activation, isoform switching disrupts the repression by mSin3, allowing for the targeted genes to

become active (Yang et al., 1997).

Notch

Notch signaling plays a role in cellular homoestasis and cell fate determination (Hing et

al., 1994). In mammals, there are four genes, Notchl, Notch2, Notch3, and Notch4 and five

ligands, Dll-, Dll-3, Dll-4, Jagged-1, and Jagged-2 (Chitnis et al., 1995; Lindsell et al., 1995; Li

et al., 1998). The Notch receptors are transmembrane receptors and when bound by the extra-

cellular ligands, Delta, Serrate, or Lag-2, this results in a cleavage of the intracellular domain of









the receptors in the cytoplasm. This cleavage product is the active form of Notch, NICD (Notch

Intracellular Domain), which translocates to the nucleus where it binds to the family of

transcriptional repressors, CSL (also known as RBP-JK, CBF-1, Suppressor of Hairless, and Lag-

1). This interaction converts them into transcriptional activators (Nye et al., 1994; Ahmad et al.,

1995).

The molecule Numb is an inhibitor of Notch but little is known about the regulation of

Numb at the transcriptional level. Numb localizes to one pole during cell division in crescent-

shaped patterns so that only one daughter cell receives Numb. This asymmetric expression is

believed to play a role in determining which cell goes on to differentiate into a myofiber and

which cell is going to replenish the satellite cell pool. Numb and Pax3 protein expression are

mutually exclusive in satellite cells and a Pax3+/Numb- cell indicates a satellite cell that is less

committed to a specific phenotype. The daughter cell retaining Numb expression is more

committed to progressing along the myogenic lineage (Conboy and Rando, 2002).

Constitutively active Notchl signaling causes up-regulation of Pax3 and down-regulation

of Myf5, M~yoD, and desmin and a reduction in Pax7 expression. In response to injury, activated

Notch expression exhibits a highly localized expression at the site of injury, but not at more

distal regions along the fiber. This suggests that it might play a role in targeting or recruiting

specific factors to the injury site to promote regeneration (Conboy and Rando, 2002). The

efficiency of tissue regeneration decreases in response to aging and it was proposed that this is

due to age-related changes in satellite cell activity. Further analyses demonstrated insufficient

up-regulation of Delta, resulting in diminished Notch activation in aged, regenerating muscle.

When Notch was inhibited in young muscle, impaired regeneration was observed. Conversely,









when Notch was introduced into old muscle the regenerative potential was restored (Conboy et

al., 2005).

To determine if systemic factors play a role in aged progenitor cells from specific tissues,

parabiotic pairings between young and old mice were studied (Conboy et al., 2005).

Interestingly, the exposure of satellite cells in old mice to young mouse serum resulted in

enhanced expression of Delta, increased Notch activation, and enhanced proliferation in vitro,

supporting the notion that there are systemic factors that change with age that impact progenitor

cell activity (Conboy et al., 2005).

Growth Factor Effects on Satellite Cells

There are multiple growth factors that regulate satellite cell activation and growth.

When myofibers are injured, the release of cytokines is stimulated, and growth factors, such as

HGF (Allen et al., 1995; Tatsumi et al., 1998) and FGFs, cause an activation and expansion of

satellite cells so that they will re-enter the cell cycle and rapidly proliferate, although activation

of satellite cells has been found to be delayed over time in response to aging (Sheehan and Allen,

1999; Clarke et al., 1993; Johnson and Allen, 1995; Cornelison et al., 2001, 2004).

Transforming growth factor beta (TGFP) inhibits cell proliferation to some extent, although its

more significant effect is in its inhibition of differentiation and fusion (Florini et al., 1986;

Greene and Allen, 1991; Rao and Kohtz, 1995; Stewart et al., 2003; Allegra et al., 2004).

MyoD, Myf5, and Pax7 are markers of the myoblasts at this stage (Davis et al., 1987; Wright et

al., 1989; Braun et al., 1989; Rhodes and Konieczny, 1989; Seale et al., 2004). Factors such as

TGFP, HGF, and FGF inhibit myoblasts from undergoing differentiation and formation of

myofibers (Gospodarowicz et la., 1976; Florini et al., 1986; Olsen et al., 1986; Massague et al.,

1986; Miller et al., 2000).









Skeletal myoblasts serve as a good model to study intracellular signaling cascades as they

undergo morphological changes characteristic of various cellular processes. Previous reports

demonstrated the significance of the Raf kinase signaling axis (Bennett and Tonks, 1997;

Coolican et al., 1997; Dorman and Johnson, 1999; Samuel et al., 1999; Dorman and Johnson,

2000; Winter and Arnold, 2000; DeChant et al., 2002). Elevated Raf levels are implicated in

causing repression of myoblast differentiation and low-levels enhancing differentiation. These

findings highlight the importance of time and duration of Raf signal transmission (DeChant et

al., 2002). The molecular basis for inhibition of differentiation may involve direct modification

of E-proteins and/or induction of TGFP-like gene expression. Additionally, inhibition of

myogenic differentiation has been demonstrated when cells were treated with various growth

factors such as FGFs (1, 2, 4, 6, and 9), TGFP, and high concentrations of serum (Allen and

Boxhorn, 1989; Sheehan and Allen, 1999).

Insulin-like Growth Factor I (IGF-I)

IGF-I promotes cell recruitment to injured muscle by coordinating a regenerative response

to muscle injury. Treatment of isolated satellite cells with IGF-I increases their proliferation

rates in response to the JAK/STAT pathway. This growth factor induces both proliferation and

subsequent differentiation of satellite cells via the type I receptor (Allen and Boxhorn, 1989;

Adams and McCue, 1998; Kamanga-Sollo et al., 2004). Activation of myoblast proliferation is

mediated through the MAPK pathway and induction of differentiation signals through the

phosphatidylinositol-3 -kinase (PI3K) pathway. In overloaded skeletal muscles, IGF-I peptide

levels increase (Adams and Haddad, 1996). In vivo evidence demonstrates that when the tibialis

anterior (TA) muscle of a rat is infused with IGF-I, there are measurable increases in muscle

protein, muscle DNA content, and absolute weight of the treated muscle (Adams and McCue,









1998). Interestingly, IGF-l is up-regulated in regenerating muscles and in aged rats and

application of IGF-I rescued approximately 46% of lost muscle mass and increased the

proliferation potential of satellite cells from atrophied gastrocnemius muscle (Chakravarthy et

al., 2000). In mdx~ mice (dystrophin null), high levels of muscle-specific IGF expression resulted

in an increase of approximately 40% in muscle mass. A subsequent increase in force generation

also was observed along with an elevation of signaling pathways associated with muscle

regeneration and protection against apoptosis (Barton et al., 2002). IGF-I serves as a strong

mitogen of satellite cells even in the presence of a strong growth inhibitor, TGFP, yet the

presence of TGFP is able to inhibit IGF-I-mediated satellite cell differentiation (Allen and

Boxhorn, 1989). Additionally, IGF-I increase the magnitude of the proliferative response

elicited by FGF2 and stimulate differentiation when treated alone (Greene and Allen, 1991).

Fibroblast Growth Factors (FGFs)

In muscle tissue, there are several FGFs expressed and released in response to injury

(Anderson et al., 1995). FGF2 and FGF6 are potent enhancers of muscle precursor cell (MPC)

expansion and satellite cells by increasing proliferation and promoting muscle regeneration

(Johnson and Allen, 1993; Lefaucheur and Sebille, 1995; Floss et al., 1997; Sheehan and Allen,

1999; Yablonka-Reuveni et al., 1999). FGF receptors (FGFR) 1, 2, 3, and 4 are expressed in

proliferating rat satellite cells with FGFR1 and 4 being the most prominent (Sheehan and Allen,

1999). FGF2 is a heparin-binding growth factor that increases satellite cell proliferation and

PCNA expression (Johnson and Allen, 1993). Treatment with FGF2 elicits a greater mitogenic

response than IGF-I or TGFPl and is as potent as HGF in stimulating satellite cell proliferation

(Sheehan and Allen, 1999). Combinations of FGF2 and HGF are additive with regard to









proliferation, and after injury, disrupted myofibers express FGF2, particularly in regions of

hyper-contraction (Anderson et al., 1995).

FGF2 is present in newly formed myotubes and inj section of FGF2 into the TA muscle of

male mdve mice during the first round of spontaneous necrosis results in enhanced satellite cell

proliferation. This is due to increasing the number of satellite cells that enter the cell cycle but

inhibits the differentiation of satellite cells (Lefaucheur and Sebille, 1995). The FGF6 knockout

in certain genetic backgrounds exhibits a reduced regenerative capacity after crush injury and

when they are interbred with mdx~ mice, a severe dystrophic phenotype is observed. FGF6 is up-

regulated in response to skeletal muscle injuries and helps completely restore experimentally

damaged skeletal muscle (Floss et al., 1997). Higher levels of FGF2 and FGFR1 are expressed

in the pectoralis major of turkey. Faster proliferating satellite cells from pectoralis major of

turkey express higher levels of FGF2 and FGFR1 by comparison to slower proliferating cells.

They also show a greater mitogenic response to FGF2, suggesting that FGFs may play a role in

proliferative rates of muscle cells (McFarland et al., 2003).

Hepatocyte Growth Factor (HGF)

HGF, also called Scatter Factor (SF), is a critical activator of satellite cells found initially

in crushed muscle extract (CME) (Tatsumi and Allen, 2004; Tatsumi et al., 2002). Direct

inj section of HGF into muscle results in activation of quiescent satellite cells, even in the absence

of trauma, and when an anti-HGF antibody is incubated with CME, the activation capacity of the

CME is abolished (Tatsumi et al., 1998; Tatsumi et al., 2002). This growth factor binds to the c-

Met receptor and signals through the PI3K pathway to promote cell survival and MAPK

pathways to stimulate the mitogenic effect (Allen et al., 1995). Placenta, liver, and muscle all

express HGF (Brand-Saberi et al., 1996). In addition, HGF is present in basal lamina of skeletal

muscle fibers, which provides a reservoir of HGF within skeletal muscle (Tatsumi and Allen,









2004). The HGF (-/-) mouse is embryonic lethal, and without HGF signaling, skeletal muscle

cells cannot migrate from the somite during embryogenesis (Schmidt et al., 1995; Bladt et al.

2002).

Transforming Growth Factor Beta (TGFP) Superfamily

The TGFP superfamily is involved in cellular proliferation, differentiation, migration, and

apoptosis. TGFP is one of the most potent negative regulators of proliferation and differentiation

of satellite cells (Florini et al., 1986; Greene and Allen, 1991; Rao and Koht, 1995; Stewart et al.,

2003; Allegra et al., 2004). Inhibition of myoblast fusion is dose-dependent and reversible

(Florini et al., 1986; Stewart et al., 2003).

The TGFP superfamily can be divided into three groups: the TGFPs, the activins/inhibins,

and the bone morphogenic proteins (BMPs). All three groups of growth factors signal through

serine/threonine kinase receptors (Massague et al., 1994). In the presence of growth factors,

ligands bind to a Type II receptor dimer located on the plasma membrane, which causes auto-

phosphorylation of the Type II dimer, recruitment of a Type I receptor dimer, and subsequent

phosphorylation of this dimer (Wrana et al., 1992; Attisano et al., 1993; Ebner et al., 1993;

Wieser et al., 1993; Wrana et al., 1994). This phosphorylation event recruits the receptor-

regulated Smads or R-Smads, which then undergo phosphorylation (Aoki et al., 2001). The R-

smads form a complex with the common-partner Smads (Co-Smads), or Smad4. The R-

Smad/Co-Smad complex translocates to the nucleus and binds to the DNA altering transcription

of target genes (Derynck et al., 1996; Liu et al., 1996; Meersseman et al. 1997; Nakao et al.,

1997). Additional co-activators and co-repressors lend regulation to the system (Wotton et al.,

1999).










Specificity of TGFP signaling is mediated by the different types of Type II and Type I

receptors present in the target cell and ligand affinities for these receptors (Wrana et al., 1992;

Attisano et al., 1993; Ebner et al., 1993; Wieser et al., 1993; Wrana et al., 1994). Additionally,

R-Smads (Smads 1, 2, 3, 5, and 8) are ligand-specific. Smads are cytoplasmic when inactive and

transfer to the nucleus upon phosphorylation (Derynck et al., 1996; Liu et al., 1996; Meersseman

et al. 1997; Nakao et al., 1997). I-Smads or inhibitory Smads (Smad6 and Smad7) bind to the

receptor and prevent phosphorylation and signaling activities (Imamura et al., 1997; Nakao et al.,

1997; Horiki et al., 2004). Negative regulation of TGFP signaling is achieved by follistatin,

which binds activin and prevents receptor docking (Nakamura et al., 1990). BMPs (2, 4, and 7)

form a trimeric complex between ligand, receptor, and follistatin to inhibit BMP actions (lemura

et al., 1998). Table 1-1 summarizes the receptor and R-Smad specificity for TGFP superfamily

members (ten Dijke et al., 1994a; ten Dijke et al., 1994b; Koening et al., 1994; Macias-Silva et

al., 1998; Yamashita et al., 1995; Rosenzweig et al., 1995; Liu et al., 1995; Nohno et al, 1995).

Myostatin

Myostatin (MSTN), also called growth and differentiation factor 8 (GDF8) is a member of

the TGFP superfamily and is responsible for maintaining muscle size. Mouse devoid of MSTN

possesses a larger muscle mass characterized by hypertrophic fibers. M~STNis expressed in

multiple tissues with the greatest mRNA levels found in muscle (Grobet al., 1997; Kambadur et

al., 1997; McPherron and Lee, 1997). MSTN circulates in the blood in an inactive form until the

pro-domain is cleaved away by a furin enzyme (McPherron et al., 1997; Lee and McPherron,

2001; Zimmers et al., 2002). Treatment with MSTN inhibits proliferation of muscle precursor

cells. Fluorescence Activated Cell Sorting (FACS) analysis determined that MSTN prevents

progression of myoblasts from G1 to S-phase transition of the cell cycle. Sub sequently,









upregulation of p21 and a decrease of Cdk2 protein and activity is observed, which results in an

accumulation of hypophorylated Rb and arrest in G1 (Thomas et al., 2000). This same cell cycle

arrest is observed in MSTN treated satellite cells. MSTN is thought to be required for

maintaining satellite cells in their quiescent state, until inhibited by a stimulus such as injury

(McCroskery et al., 2003). In MSTN-deficient satellite cells, a higher number of satellite cells

are activated compared to wildtype counterparts and addition of MSTN to myofiber explant

cultures inhibits satellite cell activation (McCroskery et al. 2003).

In theM2ST1V knockouts, hypertrophy and hyperplasia are observed (McPherron et al.,

1997). These animals exhibit an increased number of satellite cells versus wild type

counterparts, and the satellite cells present in the knockout animals have an increased

proliferation rate (McCroskery et al., 2003). Gene ablation also results in enlarged hearts of the

mutant animals. Myostatin signals through the Activin Receptor II and transgenic animals

carrying a dominant negative receptor have a three-fold increase in muscle mass compared with

wild type animals (Lee and McPherron, 2001).

Bone Morphogenic Proteins (BMPs)

BMPs are the largest group of family members in the TGFP superfamily (Reddi and

Huggins, 1972; Wozney et al., 1988). As the name implies, BMPs induce bone or cartilage

formation ectopically and initiate osteoblast differentiation (Urist, 1965; Gitelman et al., 1995).

BMPs are 30-3 8kDa homodimers that are synthesized as prepropeptides of approximately

400-525 amino acids. BMPs inhibit the myogenic differentiation of C2C12 cells, and convert

their differentiation pathway into that of osteoblast lineage cells (Katagiri e al., 1994).

Additionally, BMPs play a role in somite development by abrogating premature initiation of

myogenesis in the presomitic mesoderm (Pourquie et al., 1996). A BMP inhibitory signal is









believed to prevent the premature expression ofhyoD before somites are formed (Linker et al.,

2003). In pre-myogenic cells, BMP2, 4, and 7 have dose-dependent effects with low

concentrations maintaining a Pax3-expressing proliferative population and delaying

differentiation. Conversely, high concentrations of these BMPs prevent muscle development

(Amthor et al., 1998). In vivo, BMP2 expression in skeletal-derived cells prevents myogenic

differentiation and promotes osteogenic differentiation (Musgrave et al. 2001).

BMPs mediate non-osteogenic processes. During the developmental and differentiation

processes of the embryo, BMPs regulate epithelial-mesenchymal interactions, cell fate

specification, dorsoventral patterning, apoptosis, and the secretion of extracellular matrix

components (Vainio et al., 1993; Amthor et al., 1998; Weaver et al., 1999; Angerer et al., 2000;

Higuchi et al., 2002; Tiso et al., 2002). More specifically, in vertebrates, BMP2, 4, and 7 are

found to direct the development of neural crest cells into their ultimate phenotypes (Wilson and

Hemmati-Brivanlou, 1995; Miya et al., 1997).

In terms of signaling, there are Type I and Type II receptors specific for BMPs and

Smads1, 5, and 8 are the downstream molecules phosphorylated by the ligand-receptor

complexes (see Table 1-1). At the transcriptional level, BMPs regulate target genes such as,

Runx2, osteopontin, osteonectin, and osteocalcin specific for osteogenesis (Ahrens et al., 1993).

Smad6 is an I-Smad. It functions by binding to type I BMP receptors thus preventing the

activation of BMP Smads 1/5/8 (Imamura et al., 1997). Smad6 overexpression in chondrocytes

results in delayed differentiation and maturation (Horiki et al., 2004).

Noggin and other cystine knot-containing BMP antagonists associate with BMPs to block

their signaling. Noggin overexpression in osteoblasts, results in osteoporosis in mice (Devlin et

al., 2003; Wu et al., 2003). Noggin is a secreted peptide, which is expressed in condensing









cartilage and immature chondrocytes. Ablation of Noggin is embryonic lethal at 18.5 days post

coitum (dpc) and results in severe hyperplasia of the cartilage with multiple j oint fusions, severe

defects in somitogenesis, and multiple skeletal defects (Brunet et al., 1998; McMahon et al.,

1998). Noggin is found to have different affinities for different BMP family members making its

regulation of BMP-mediated processes more complex. For example, in the Noggin null

transgenic, different bones have varying responses to the de-repression of BMP signaling

(Zimmerman et al., 1996; Chang and Hemmati-Brivanlou, 1999). Depending on location and/or

embryonic origin of the bones, one observes inhibition, delay or acceleration of ossification

processes (Tylzanowski et al., 2006).

Another BMP negative regulator is Tob, which suppresses the activity of receptor-

regulated Smads (1/5/8) (Bradbury et al., 1991; Fletcher et al. 1991; Yoshida et al., 2000). Tob

null mice exhibit enhanced BMP signaling and increased bone formation (Yoshida et al., 2000).

Negative BMP regulation also is achieved by mechanisms that target elements of BMP signaling

for degradation. The Hect domain E3 ubiquitin ligase, Smurfl, targets Smad1 and 5 for

degradation, in addition to interacting with and mediating the degradation ofbone-specific

transcription factors such as Runx2 (Zhao et al., 2003). Smurfl can also target type I BMP

receptors for degradation by interacting with Smad6. They form a complex that can be exported

from the nucleus where it interacts with the BMP receptors to promote degradation (Murakami et

al., 2003). Furthermore, when Smurfl is overexpressed in osteoblasts, postnatal bone formation

is inhibited (Zhao et al., 2004).

While BMPs are capable of redirecting muscle mesenchyme cells to differentiate into bone

tissue and stimulate bone formation in vivo (Urist, 1965), previous reports have demonstrated

that mutations of BMP family members results in disruption of skeletal development (Kingsley









et al., 1992; Storm and Kingsley, 1999). Moreover, BMPs have opposing activities that are

concentration-dependent. In the presence of low BMP levels, myogenic precursor cells are

maintained in a proliferative state in developing limb bud, while high BMP levels induce cell

death. Thus, BMPs can both stimulate and restrict muscle growth suggesting that a

concentration gradient of BMPs is needed for the correct determination and maintenance of the

myogenic program (Amthor et al., 1998; Amthor et al., 2002). While BMPs signal through the

Smads, a multitude of different transcription factors are recruited, accounting for the diversity of

functional BMP responses. One set of genes involved in skeletal development are the Hox

family of transcription factors. In analyses of gain- or loss-of-function and naturally occurring

mutations of Hox genes, BMPs play a central role in embryonic skeletal patterning (Manley and

Capecchi, 1997; Yueh et al., 1998). Furthermore, several of the Hox genes interact with Smads

and there is evidence that Hox gene expression may be regulated by BMPs further suggesting

that BMPs are a critical mediator of skeletal myogenesis (Ladher et al., 1996; Tang et al. 1998;

Liu et al., 2004).

BM~P6, a member of the BMP subfamily, was originally isolated from phage plaques of a

hgl0-based cDNA library derived from 8.5-dpc murine embryos. The murine library was

hybridized with a 32P-labeled partial Xenopus laevis Vg-1 cDNA under low-stringency

conditions (Derynck et al., 1988). Northern blot analyses of murine tissues illustrated that BM~P6

was present in muscle (Lyons et al., 1989). The human and bovine homologs were isolated from

bone and designated BM~P6 (Celeste et al., 1990). Expression predominates in mature

chondrocytes during endochondral ossification and BMP6 treatment stimulates chondrogenic

and osteogenic phenotypes in vitro and induction of cartilage and bone formation in vivo

(Gitelman et al., 1994; Gitelman et al., 1995; Yamaguchi et al., 1996). Adult muscles express









BMP6 (Lyons et al., 1989) and BMP6 expression increases in Raf-arrested myoblasts (Wang et

al., 2004). In keratinocytes, treatment with BMP6 significantly decreases DNA synthesis

(D'Souza et al., 2001) and triggers differentiation programs (Tennenbaum et al., 1996). In these

differentiated keratinocyte cultures, E2F5 protein levels are significantly increased (D' Souza et

al., 2001). Treatment of keratinocytes with TGFPl causes reversible cell cycle arrest without

activating the differentiation program (Pierce et al., 1998; Dicker et al., 2000).

BMP Function in vivo: Lessons Learned from Knockouts

A compilation of the various BMPs and receptors targeted knockouts and their resulting

phenotypes are presented in Table 1-2. BM~P5 null mice are called short ear mice, because these

animals have reduced ear size in comparison to wild type animals, in addition to exhibiting

reduced vertebral processes, and a reduced number of ribs and sesamoid bones (Green, 1968;

Kingsley et al., 1992; King et al., 1994). Mice with a homozygous BM~P7 deletion are found to

die at birth due to renal failure because of hypoplastic/dysplastic kidneys (Dudley et al., 1995;

Luo et al., 1995; Wawersik et al., 1999). BM~P6 null mice are viable and fertile and exhibit no

maj or defects, except for a delay in ossification of the developing sternum (Solloway et al.,

1998). It is believed that BM~P2 may be functionally compensating for BM~P6 ablation since

BM~P2 and BM~P6 are required for some overlapping or redundant functions (Solloway et al.,

1998).

Utilization of BMPs in a clinical setting was demonstrated in various therapeutic

interventions such as, bone defects, non-union fractures, spinal fusion, osteoporosis, and root

canal surgery. Multiple studies utilizing BMP2 demonstrate its ability to promote healing of

severe long bone defects in rats, rabbits, dogs, sheep and non-human primates (Murakami et al.,

2002). Adenoviral administration of BMP2 mixed with a bioresorbable polymer and









mesenchymal stems cells was able to repair bone defects (Chang et al., 2003). RhBMP2

administered systemically increased mesenchymal stem cell activity and reversed age-related

bone loss due to ovariectomies in different mouse models (Turgeman et al., 2002) demonstrating

a potential application of BMPs in osteoporosis treatment. Interestingly, rhBMP2 has been used

as a complete bone graft substitute in spinal fusion surgery and in some cases BMP2 has been

more efficacious in promoting successful bone fusion as compared to autogenous bone grafts.

BMP2 was used in other fusion applications such as intervertebral and lumbar posterolaterial

fusions (Sandhu, 2003). BMPs can be used in dental applications since BMP2 induces bone

formation around dental implants used in periodontal reconstruction. It has been suggested that

BMPs could serve as an alternative to root canal surgeries (Schwartz et al., 1998; Cochran et al.,

1999).

Current BMP6 Studies

Recently, BMP6 was able to induce matrix synthesis and induce differentiation of bovine

ligaments fibroblasts providing another potential source of chondrocytes for tissue repair

(Bobacz et al., 2006). Mesenchymal stem cells (MSCs) treated with BMP6 in combination with

parathyroid hormone (PTH) and vitamin D(3) increased osteocalcin production. Osteocalcin is

used as a marker for bone formation and in these MSCs, BMP6 enhanced calcium formation

(Sammons et al., 2004). TGFP and BMPs may act in an antagonistic manner towards each other.

TGFP inhibits chondrocyte maturation relatively early in differentiation by down-regulating

bmp6, ihh (Indian hedgehog), and colX(collagen type X), which are genes specifically expressed

by hypertrophic chondrocytes (Vortkamp et al., 1996; Ferguson et al., 2004).

BMPs are implicated in the development of cancers of the GI tract, breast, and prostate

(Howe et al., 2001; Pouliot and Labrie, 2002; Brubaker et al., 2004). Currently, much focus is










being placed on TGFP superfamily members as targets for cancer therapy due to their ability to

suppress tumor progression by inhibiting growth of neoplastic tissues. BMP6 in the early stages

of carcinogenesis inhibits benign and malignant skin tumor formation (Wach et al., 2001).

BMP6 exerts an anti-proliferative and pro-apoptotic effects in multiple myeloma (Kawamura et

al., 2000; Hj ertner et al., 2001; Kersten et al., 2005).

Ras/Raf in Skeletal Muscle

A key regulator of signal transduction pathways controlling cell proliferation,

differentiation, and oncogenesis is Ras, a monomeric guanine nucleotide-binding protein with

intrinsic GTPase activity (Marshall, 1996). Ras is a molecular switch that cycle between a GTP-

bound active and a GDP-bound inactive state (McCormick, 1993). Oncogenic H-Ras inhibits the

differentiation of muscle cells independent of their continued proliferation (Olson et al., 1987).

Ras inhibits myogenesis by disrupting MRF function, resulting in the inhibition of differentiation

of muscle cells independent of proliferation (Ramocki et al., 1998). Ras proteins are localized at

the cytoplasmic face of the plasma membrane and activated by a large number of extracellular

stimuli, such as growth factors and hormones (McCormick, 1993; Bar-Sagi, 2001). A family of

Ras effector molecules specifically binds to and is activated by Ras-GTP, which then carries out

the downstream functions of activated Ras (Marshall, 1996). Some effectors of Ras are the Ral

GTPase signaling pathway involved in the G1 to S progression (Ramocki et al., 1998) and the

PI3K/AKT pathway involved in cell survival (Murphy et al., 2002). All of the major Ras

effectors have been tested for a role in the Ras-mediated inhibition of skeletal myogenesis but

none of them are able to duplicate the effects of oncogenic Ras (Ramocki et al., 1997; Weyman

et al., 1997).









The Raf Family

The Raf family consists of three serine/threonine specific kinases and are the best

characterized effectors of Ras. Members include A-Raf, B-Raf, and c-Raf, also called Raf-1

(Marshall, 1996). There are three conserved regions (CR1-3) within Rafs consisting of two N-

terminal regulatory domains (CR1 and CR2) and a C-terminal catalytic kinase domain (CR3).

Rafs lead to the activation of the extracellular signal-related kinase (ERK) pathway, which

mediates cellular proliferation and differentiation (Marshall, 1996).

Inactive Raf is cytosolic and translocates to the plasma membrane following activation by

Ras (Chong and Guan, 2003). The activation of Raf is important in mediating growth factor

gene expression and a maj or function of Raf protein kinase is to phosphorylate MEK 1 and

MEK2. Subsequently, ERK1 and ERK2 are phosphorylated on tyrosine and threonine residues.

These activated ERKs then either phosphorylate numerous cytoplasmic targets or migrate to the

nucleus to activate transcription factors such as c-fos and Elk1 (Huang et al, 1993). The

Raf/MEK/ERK signaling cascade is required for cell cycle progression and overexpression

causes cell transformation (Kolch et al., 1991; Cowley et al., 1994; Mansour et al., 1994). The

sustained activation of this pathway is implicated in differentiation with prolonged ERK

activated leading to differentiation of PC12 cells (Qui and Green, 1992). Raf-activated MEK-

ERK cascades are likely participants in apoptosis because potent ERK activation can protect

cells from apoptosis (Le Gall et al., 2000).

Signaling through the Raf/MEK/ERK controls several aspects of myogenesis.

Overexpression of a constitutively active Raf and MEK results in reduced muscle gene

transcription and these myoblasts are unable to form myocytes (Dorman and Johnson, 1999;

Dorman and Johnson, 2000; Samuel et al., 1999; Winter and Arnold, 2000). Down-regulation of

ERK activity by overexpression of MAPK phosphatase I (MKP-I), or through the use of










synthetic inhibitors, upregulates muscle specific gene transcription (Bennett and Tonks, 1997;

Dorman and Johnson, 1999). By contrast, other reports have found no effect of ERK activity on

muscle gene expression, myoblast fusion, and myoblast differentiation (Weyman et al., 1997;

Jones et al., 2001). Myoblasts expressing low levels of Raf differentiate more efficiently than

control cells again suggesting that ERKl/2 promotes myogenesis (Wang et al., 2004). In

differentiating C2 myoblasts, ERKl/2 activity increases MyoD expression and transcriptional

activity (Gredinger et al., 1998). Activated ERK1 is associated with myoblast proliferation and

activated ERK2 is associated with myoblast differentiation (Sarbassov et al., 1997). ERK1 and

ERK2 are both ubiquitously expressed in most tissues and similar in structure. ERK1 null mice

are viable, fertile, and of normal size (Nekrasora et al., 2005), whereas, ERK2 null mice are

embryonic lethal (Yao et al., 2003).

E2F Family

The maj or function of the E2F factors is cell cycle progression, in addition to playing roles

in metabolic activities such as proliferation, differentiation, and apoptosis (Fujita et al., 2002).

Genes expressed at the G1/S transition contain E2F-binding sites within their promoters

(DeGregori et al., 1995). E2F members are conserved throughout evolution from invertebrates

to mammals and there are currently nine family members, which demonstrate tissue-specific

activities (Dynlacht et al., 1994; D' Souza et al., 2001). Two major groups, activators and

repressors, comprise the E2F family. E2F l, E2F2, and E2F3a are involved in positive cell cycle

control and S-phase entry of quiescent cells. These members preferentially bind pRb with

expression peaking in late G1 and association with E2F-regulated promoters during the G1-S

transition. Ectopic expression of these members results in S-phase induction in serum-starved

cells (DeGregori et al., 1997; Lukas et al., 1996; Leone et al., 1998; Humbert et al., 2000).

E2F3b, E2F4 and E2F5 are classified as repressor proteins, due to their negative control of cell









cycle progression. E2F4 and E2F5 predominately bind pl07 and pl30 pocket proteins

(DeGregori et al., 1997; Leone et al., 2000). E2F6, E2F7, and E2F8 also are classified as

repressors but through a mechanism independent of pocket protein interaction because they lack

the Rb-binding sequence at the C-terminus (Campanero et al., 2000). Members of the E2F

family demonstrate differential mRNA expression throughout the cell cycle. For example, E2F 1

and E2F2 mRNA increase in late G1, and peak at the G1/S transition, although E2F2 mRNA

expresses at a lower level than E2Fl1. During quiescence, E2F3 and E2F5 mRNA are expressed,

with E2F 1 barely present. In early-to-mid G1, E2F3 and E2F5 mRNA levels rise (Sardet et al.,

1995; Pierce et al., 1998).

Members of the E2F family are grouped based on their homologous DNA binding domain

(DBD). All of the E2Fs, except E2F7 and 8, and both DP proteins have a conserved DBD and a

dimerization domain. E2F4 and E2F5 demonstrate a 72% amino acid identity to each other and

a 35% amino acid identity to E2Fl1-3 (Vaishnav et al., 1998). E2F4 and E2F5 make up a

subclass of the E2F family since their N-terminus lacks the cyclin A-binding domain found in

the other members. The repressive E2Fs contain the Rb-binding sequence and have nuclear

export sequences instead of nuclear localization sequences in the N-terminus (Helin et al., 1993;

Sardet et al., 1995).

E2F Signaling Mechanism and Involvement of Pocket Proteins

The E2F proteins signal via the formation of active heterodimer complexes with DP

proteins (Helin et al., 1993). E2Fs can dimerize with either DP1 or DP2, except for E2F7 which

binds DNA in a DP-independent manner (Ormondroyd et al., 1995). These E2F complexes

promote gene expression required for G1 progression and DNA replication (Chen et al., 2004).

The mammalian Rb family of proteins (pRb, pl07, and pl30) is also referred to as the

pocket proteins. The pocket protein motif allows for interactions with cellular proteins that










possess a LXCXE peptide motif (Lee et al., 1998). The LXCXE motif is present in E2Fs, the D-

type cyclins (Gill et al., 1998), and HDACs. The pocket domain is separated into two

functionally conserved regions, the A and B pockets. The spacer region is specific to each

pocket protein and contains binding sites for cyclin/CDK complexes in pl07 and pl30 but not in

pRb. pl30 and pl07 contain approximately 50% amino-acid identity and 30-35% identity to

pRb (Ewen et al., 1991; Hannon et al., 1993). Overexpression of cDNAs coding for pocket

proteins induces growth arrest at G1 (Classon et al., 2000).

Rb was the first tumor suppressor gene cloned and loss of Rb function serves as a hallmark

of oncogenic progression. pRb is a major G1 checkpoint and inhibits S-phase entry. pRb

promotes terminal differentiation, cell cycle exit and tissue specific gene expression (Dunaief et

al., 1994; DeCaprio et al., 1989; Dick et al., 2000).

Cell Cycle Repression

Pocket protein dephosphorylation occurs from anaphase to G1 or in response to growth

inhibitory signals (Ludlow et al., 1993). Phosphorylation events can also lead to permanent

inactivation of the pocket protein and possibly target it for degradation (Ma et al., 2003). The

type of functional effect that pocket proteins have on a cell is dependent on what type of co-

accessory proteins interact with the target gene (Stevaux et al., 2005). To repress gene

transcription needed for the G1 to S-phase transition, the pRb binds directly to the transactivation

domain of E2F. pRb also recruits chromatin remodeling factors such as histone deacetylase I

(HDAC1), SUV39H1, hBRM, and BRGl. These factors act on the surrounding nucleosome

structure to remodel it, and promote histone acetylation/deacetylation and methylation events

(Shao and Robbins, 1995). HDAC1 is recruited to E2F complexes by pRb to function in

repressing cyclin E gene expression (Magnaghi-Jaulin et al., 1998). SUV38H1 is a

methyltransferase that methylates K9 of histone H3 and cooperates with pRb in the repression of









E2F-responding promoters (Rea et al., 2000). hBRM and BRG1 are the mammalian homologs

of SNF2/SWI2, which are yeast chromatin remodeling complexes and they associate with pRb

(Strober et al., 1996; Kang et al., 2004).

E2Fs and Pocket Proteins in Skeletal Muscle

pRb is required for muscle differentiation and for transcription of myogenic bHLH factors.

Functional Rb is required for the activity of MyoD-mediated transcriptional activation of

myogenic genes (Gu et al., 1993). In models of inactive Rb, myoblast differentiation was

inhibited in vitro and terminally differentiated myotube nuclei were able to reenter the cell cycle.

In the absence of pRb, skeletal muscle cells exhibited ectopic DNA synthesis and/or apoptosis,

and pRb null mice exhibit severe defects in skeletal muscle (Schneider et al., 1994; Novitch et al,

1996; Zacksenhaus et al., 1996; Novitch et al., 1999; de Bruin et al., 2003; Wu et al., 2003).

Interestingly, E2Fs in complex with pl30 accumulate in cells such as, myoblasts, and

melanocytes undergoing terminal differentiation (Shin et al., 1995). In rat L6 myoblasts, pl07 is

normally involved in regulation of E2F proteins during cell cycle progression. Exponentially

growing L6 myoblasts demonstrate complexes of E2F and bound pl07 throughout the cell cycle.

During the differentiation of L6 cells, pl07 levels are reduced and pl30 levels are greatly

increased, suggesting that pl30 is a differentiation-specific regulator of E2F activity (Kiess et al.,

1995).

In summary, skeletal myogenesis is regulated by a multitude of growth factors and

signaling events. Since most studies have focused on the role of BMPs in embryonic

development, little is known about its role in postnatal muscle. E2Fs are critical mediators of

cell cycle progression. Satellite cells are requisite for growth and repair of skeletal muscle. It is

not well understood how satellite cells are activated to enter the cell cycle nor how they exit the

cell cycle to return to quiescence. E2F5 appears to have some role in Raf-arrested myoblasts and









BMP6 expression is high in these Raf-arrested cells. Therefore, these data will further assess the

role of BMP6 and E2F5 in regulation of skeletal myogenesis and/or satellite cell biology.









Table 1-1. Receptor and R-Smad specificity for TGFP superfamily members
Ligand Type II Receptor Type I Receptor R-Smad
Activin Act-R-II/IIB ALK4 Smad2,3


ALK5
ALK 1

ALK3
ALK6
ALK2


Smad2,3
Smad l,5,8

Smad l,5,8
Smad l,5,8
Smad l,5,8


TGFS


BMP


TGFPRII


BMPRII
ActRII/IIB










Table 1-2. TGFS
Genes
Ligands
Bmp2


Bmp3

Bmp4




Bmp5

Bmp6
Bmp7

Bmp8a

Bmp8b

Bmp 11 (Gdfl1)

Bmpl2 (Gdf7)

Bmp15
Gdfl
Gdf5


Gdf8
(Myostatin)
Gdf9
Nodal



Leftv1
Leftv2

Activinpa

Activinpb

Inhibina
MIS

Tgfp l


superfamily transgenic knockouts*
Phenotypes

Delayed primitive streak, small allantois,
lack of amnion, heart defects, decreased
number of PGCs.
Increase bone density

Lack of allantois and PGCs, posterior
truncation, heart defects, and lack of
optic vesicle; heterozygotes-cystic
kidney, craniofacial malformations,
microphthalmia,
Short ear phenotype including defects in
skeleton, lung, and kidney
Delayed sternum ossification
Skeletal defects, kidney agenesis, eye
defects
Defects in spermatogenesis and
epididymis
Defects in PGC formation, testis cord
formation, and spermatogenesis
Defects in A-P patterning of axial
skeleton
Hydrocephalic abnormalities growth
defects in seminal vesicle
Subfertile due to defects in oogenesis
Defects in left/right asymmetry
Brachypodism shortenedd skeleton in
limbs and reduced number of digit
bones)
Skeletal muscle hypertrophy

Sterile due to defects in oogenesis
No gastrulation (lack of primitive
streak): anterior neural pattern defects:
placenta defects (increased number of
trophoblast giant cells)
Abnormal left-right axis (left isomerism)
Extended streak, excessive mesoderm,
left isomerism
Lack whiskers and low incisors and cleft
palate
Defects in eyelid development and
female reproduction
Ovarian cancer
Pseudo-hermaphrodites (female
reproductive tract present in males)
Hyperactive immunity and defects in
angiogenesis


References


Zhang and Bradley, 1996: Ying and
Zhao, 2001

Bahamonde and Lyons, 2001; Daluiski
et al., 2001
Winnier et al., 1995: Dunn et al., 1997:
Funrta and Hogan, 1998: Lawson et al.,
1999; Ying et al., 2000


Green, 1968: Kingsley et al., 1992:
King et al., 1994
Solloway et al., 1998
Dudley et al., 1995: Luo et al., 1995:
Wawersik et al., 1999
Zhao et al., 1998

Zhao et al., 1996: Ying et al., 2000;
Yao et al., 2002
McPherron et al., 1999

Lee et al., 1998: Settle et al., 2001

Yan et al., 2001
Rankin et al., 2000
Storm et al., 1994: Storm and Kingsley,
1996

McPherron et al., 1997

Dong et al., 1996
Conlon et al., 1994: Varlet et al., 1997:
Lowe et al., 2001; Ma et al., 2001;
Zhou et al., 1993

Meno et al., 1998
Meno et al., 1999

Matzuk et al., 1995b

Vassalli et al., 1994

Matzuk et al., 1992
Behringer et al., 1994

Shull et al., 1992: Dickson et al., 1995










Table 1-2 continued.


Genes
Tgf32


Tgfp33
Receptors
Alk1
Alk2 (ActrlA)

Alk3 (BmprlA)



Alk4 (ActrlB)

Alk6 (BmprlB)


BmprlI
TgfbrlI

ActrlIa


ActrlIb


Phenotypes
Perinatal lethality due to multiple defects
in heart, lung, limb, spinal column, eye,
inner ear, and urogenital system
Cleft palate

Defects in embryonic angiogenesis
Defects in mesoderm formation as a
result of defective visceral endoderm
Defects in epiblast proliferation and no
mesoderm formation in null mutants,
impaired cardiac and limb development
in conditional mutants
Defects in epiblast differentiation and
lack ofmesoderm formation
Defects in seminal vesicle development,
female reproduction, and limb skeletal
formation
Defects in gastrulation/lack of mesoderm
Defects in vasculogenesis and
hematopoiesis
Deficiency in reproduction due to
suppressed FSH and mild defects in
skeletal development
Defects in axial patterning and left-right
asymmetry (45% right isomerism)


References
Sanford et al., 1997


Proetzel et al., 1995

Oh et al., 2000
Gu et al., 1999; Mishina et al., 1999

Mishina et al., 1995: Ahn et al., 2001;
Gaussin et al., 2002


Gu et al., 1998

Baur et al., 2000; Yi et al., 2000; Yi et
al., 2001

Beppu et al., 2000
Oshima et al., 1996

Matzuk et al., 1995a


Oh and Li, 1997


*Adapted from Zhao, G.H. 2003. Consequences of Knocking out BMP signaling in the mouse.
Genesis 35:43-56. Table 1, pgs.45-46.











CHAPTER 2
MATERIALS AND METHODS

Myoblast Cell Culture

C2C12 skeletal muscle satellite cells (Blau et al., 1985) were grown in Dulbecco's

modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% v/v

penicillin/streptomycin, and 0.1% v/v gentamycin. 23A2 skeletal muscle cells (Konieczny and

Emerson, 1984) were maintained in Basal Medium Eagle (BME) supplemented with 15% FBS,

1% v/v penicillin/streptomycin, 0.1% v/v gentamycin reagent solution, 1% v/v L-glutamine.

23A2RafERDD myoblasts were derived from the parental 23A2 myoblasts and stably express a

tamoxifen-inducible chimeric Raf protein. The estrogen receptor fused to the Raf kinase domain

is unstable in the absence of the estrogen analog 4-hydroxytamoxifen (4HT). Addition of 4HT

binds to the estrogen receptor and allows for a dose-dependent increase in Raf protein expression

and kinase activity that initiates downstream ERKl/2 activation (Wang et al., 2004).

23A2RafERDD myoblasts were maintained in the same media as 23A2 cells with the addition of

10mM puromycin (Wang et al., 2004). All culture media, supplements, and sera were purchased

from Invitrogen, Carlsbad, CA. For induction of stable Raf expression, cells were washed with

phosphate buffered saline (PBS), treated with 10 Clg/ml protamine sulfate (CalBioChem, San

Diego, CA) in serum-free BME for 10 minutes. Cells were starved in serum free BME for one

hour and treated with 1CIM 4-hydroxytamoxifen (4HT; Sigma, St. Louis, MO) in 2% FBS BME.

Cells for immunofluorescence were cultured on 35 mm glass-bottomed plates (World Precision

Institute, Sarasota, FL) coated with 10% v/v BD Matrigel Matrix HC (BD Biosciences, San Jose,

CA).









Growth Factor Treatment, and BrdU Pulsing and Fixation

C2C12 cells were seeded on gelatin-coated tissue culture plates at a concentration of 1x105

cells/well in a 6-well cluster. Myoblasts were treated in the absence and presence of human

recombinant BMP6 (R&D Systems, Minneapolis, MN) at final concentration of 25 ng/ml for 48

hours in 2% FBS DMEM. When necessary, cells were pulsed with 10 C1M bromodeoxyuridine

(BrdU) during the last thirty minutes of treatment and then fixed in 70% Ethanol (EtOH) at 40C

for 30 minutes.

Plasmids and Transfections

Semi-confluent myoblasts were transfected by calcium phosphate precipitation formation.

In brief, each well received DNA precipitate containing 1 Clg of luciferase reporter, 0.5 Clg of

activator plasmid, 0.5 Clg of kinase, and 50 ng of pRL-tk, a Renilla luciferase plasmid used as a

monitor of transfection efficiency. The luciferase reporter plasmids used in the study were a

multimerized BMP response element (BRE-luc) (Korchynskyi and ten Dijke, 2002), the internal

response element of the quail troponin I gene (Tnl-luc) (Johnson et al., 1996), a multimerized

TGFP responsive element (3TP-luc) (Wrana et al., 1992), an Immunoglobulin E box reporter

plasmid (C1E5-Luc) (Johnson et al, 1996), and E2F-TA-luc (Clontech, Mountain View, CA).

Activator plasmids used were CMV-E47 (Page et al., 2004), CMV-E2F5 (OriGene Technologies

Inc., Rockville, MD), and pEM-MyoD (Page et al., 2004). Cells were maintained in growth

media or differentiation-permissive medium supplemented with human recombinant BMP6, and

TGFP (R&D Systems, Minneapolis, MN) for 48 hours prior to lysis and measurement of

luciferase and P-galactosidase or Renilla luciferase activities (Promega, Madison, WI). Final

concentrations of BMP6 were 1, 10, 25, or 100 ng/ml. TGFPl was supplemented at 10 ng/ml.









BrdU Staining and BrdU Incorporation

Cells were fixed in 70% ethanol for 10 minutes at room-temperature, followed by

incubation in 2N Hydrochloric acid (HC1) for 1 hour at 370C. Cells were washed well with

Phosphate Buffered Saline (PBS) and then incubated in blocking buffer (PBS containing 5%

horse serum (HS), and 0.1% Triton X-100) for 1 hour. Antigen was detected by incubation with

Biotynylated Anti-BrdU IgG (H+L) (BA-2000) at 1:100 (Vector Laboratories, Burlingame, CA)

for 1 hour, followed by incubation with HRP-Streptavidin at 1:100 (Vector Laboratories,

Burlingame, CA) for 1 hour. Antibodies were diluted in blocking buffer (PBS containing 2%

Horse Serum, and 0. 1% Triton X-100). Visualization of BrdU staining was accomplished by

addition of one part 3,3' -Diaminobenzidine tetrahydrochloride (DAB) and five parts Nickel

Chloride (NiC1) in the presence of H202. Percentages of BrdU incorporation were calculated by

dividing the number of BrdU positive nuclei by the total number of nuclei. The averages of a

minimum of six microscopic fields at 200X per treatment are shown.

Immunofluorescent E2F5 Staining

23A2RafERDD CellS were permeabilized for twenty minutes (lX PB S, 0.01% Triton X-

100), incubated in blocking buffer for one hour, and then incubated overnight at 40C with E2F5

antibody (sc-999) (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 in blocking buffer

(lX PBS, and 2% Horse Serum). Next, cells were incubated with Alexafluor 488 conjugated

anti-rabbit diluted 1:100 in blocking buffer (Molecular Probes, Carlsbad, CA) for 45 minutes at

room temperature. Secondary antibody controls also were included to demonstrate the

specificity of an antibody's expression patterns. Hoechst dye (1 Clg/ml final concentration) was

included as a nuclear stain. Representative photomicrographs at 630X under oil immersion were

captured with a Nikon 1200DMX digital camera.









RNA Isolation and Nylon Arrays

Total RNA was isolated from C2C12 myoblasts and myofibers and 23A2 myofibers with

the use of a Stratagene RNA Easy Kit. Equal amounts (3 ug) of pooled RNA were reverse

transcribed with Superscript II (BD BioScience, San Jose, CA) in the presence of biotin-UTP.

Biotinylated cDNAs were used to probe a RT2 Gene Expression Assay, a nylon mini-arrays

containing 96 genes coding for various members of the TGFP superfamily, their receptors,

signaling intermediates, and transcriptional regulators (SuperArray Bioscience Corporation,

Frederick, MD). Array membranes were pre-hybridized with 100 Cpg/ml heat-denatured salmon

sperm DNA in GEAhyb Hybridization Solution (SuperArray Bioscience Corporation, Frederick,

MD) for 2 hours at 600C. Biotinylated probes were added to arrays in GEAhyb solution to

hybridize overnight (O/N) at 600C. Following hybridization, the membranes were washed twice

in 2X SSC (saline sodium citrate), 1% SDS (sodium dodecyl sulfate) and twice in 0.1X SSC,

0.5% SDS for 15 minutes each at 600C. After washing, membranes were blocked with

GEAblocking solution Q for 40 minutes at room temperature. Alkaline phosphatase-conj ugated

streptavidin (AP) was diluted 1:8,000 with Buffer F and incubated with the membrane for 10

minutes at room temperature. Membranes were washed four times with lX Buffer F for 5

minutes each and then rinsed twice with Buffer G and visualized with CDP-Star

chemiluminescent substrate for 2-5 minutes at room temperature (SuperArray Bioscience

Corporation, Frederick, MD) and exposure to X-ray film (XAR-5, Kodak).

Western Blots

C2C12 myoblasts were differentiated in the presence or absence of 100 ng/ml BMP6.

After 48 hours, the cells were lysed in 4X SDS-PAGE sample buffer (250 nM Tris pH 6.8, 8%

SDS, 40% glycerol, 0.4% P-mercaptoethanol) and protein concentrations were measured (Bio-









Rad; Bradford, 1976). Equal amounts of protein were electrophorectically separated through

denaturing gels and transferred to nitrocellulose membrane. The blots were incubated with 5%

nonfat dry milk or 5% bovine serum albumin (BSA) in TBST (10mM Tris, pH 8.0, 150nM NaC1,

0.1% Tween 20) to block nonspecific binding sites. Primary antibodies were diluted in blocking

buffer, and the blots were incubated overnight at 40C with shaking. Antibodies and dilutions

included the following: anti-myogenin (F5D ascites, Developmental Hybridoma Bank,

University of Iowa, 1:5,000), anti-myosin heavy chain (MF20 hybridoma supernatant,

Developmental Hybridoma Bank, University of Iowa, IA, 1:5), anti-troponin T (R & D Systems,

Minneapolis, MN, 1:2,500), anti-desmin (D3, hybridoma supernatant, Developmental

Hybridoma Bank, University of Iowa, IA, 1:10), anti-phospho-Smadl1/Smad5/Smad8 (Cell

Signaling Technology, Beverly, MA, 1:1,000), anti-p38 (1:5,000), and anti-phospho-p38

(1:5,000) (Cell Signaling Technology, Beverly, MA, 1:2,000). After three times of washing with

TBST for 15 minutes each, the blots were reacted with the appropriate peroxidase conjugated

secondary antibody diluted 1:5,000 in blocking buffer for 60 minutes at room temperature.

Visualization of protein bands was accomplished by chemiluminescence (ECL, Amersham

Biosciences, Piscataway, NJ) and exposure to X-ray film (XAR-5, Kodak).

Alkaline Phosphatase Staining

C2C12 myoblasts were treated for 0, 1, 24 or 48 hours with 100 ng/ml BMP6 or vehicle

alone (control). Cultures were fixed with 4% paraformaldehyde and alkaline phosphatase (ALP)

activity measured by reaction with 165 Cpg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and

330 Cpg/ml Nitro Blue Tetrazolium (NBT) color development substrates (Promega, Madison, WI)

for 18 hours at 37 C. Representative photomicrographs at 100X were captured under bright field

conditions with a 1200DMX digital camera (Nikon).









p38 Inhibition Assays

Myoblasts were treated with 10 CIM SB202190 (Upstate Biotechnology, Charlottesville,

VA), an inhibitor of p3 8 for 1, 24, and 48 hours. SB202190 is a cell permeable pyridinyl

imidazole that potently inhibits p3 80/SAPK2a and p3 8P/SAPK2b. This specific p3 8 inhibitor

does not effect ERK activity or SAPK/JNK MAP kinases. After treatment, cells were Eixed with

4% paraformaldehyde and assessed for ALP activity measured by reaction with NBT + BCIP or

lysed and assayed for Tnl-Luc activity.

Apoptosis Analysis

Myoblasts were treated with 25 CIM staurosporine (Sigma, St. Louis, MO) for 3 hours.

Cells were lysed in 4X SDS-PAGE sample buffer and equal amounts of protein were

electrophorectically separated through denaturing gels and transferred to nitrocellulose

membrane. The blots were incubated with 5% nonfat dry milk in TBST and primary antibodies

were diluted in blocking buffer. The blots were incubated overnight at 40C with shaking.

Antibodies and dilutions included the following: anti-Poly (ADP-ribose) polymerase (PARP)

(1:1000, Cell Signaling Technology, Inc., Danvers, MA) and anti-Bcl-2 (1:500, BD Biosciences,

San Jose, CA). After three times washing for 15 minutes each with TBST, the blots were reacted

with the appropriate peroxidase conjugated secondary antibody diluted 1:5,000 in blocking

buffer for 60 minutes at room temperature. Visualization of protein bands was accomplished by

chemiluminescence and exposure to X-ray film.

Statistics

All data presented represents at least three independent experiments with a minimum of

two to three replicates per treatment group. All numerical data were compared to appropriate

controls and each other as indicated for each experiment and analyzed following the General









Linear Models (GLM) Procedures of the Statistical Analysis System (SAS) (SAS, 1988).

Differences between treatments were calculated using predicted differences between the least-

squares means of treatment divided by standard deviation. Classes were designated as cells,

treatment (trt), replicates (rep), and relative luciferase units (rlu). The statistical model included

rlu equals treatment and replicates (rlu = trt rep) as main effects. Data was presented as Means +

SEM. Treatments were considered significantly different when P < 0.05.










CHAPTER 3
DIFFERENTIAL EXPRESSION OF TGFP SUPERFAMILY MEMBERS DURING
SKELETAL MYOGENESIS

Objective

Our previous work demonstrated that activated Raf, a key regulator of the MEK/ERK

pathway inhibits myogenesis based on its overall signaling intensity (Ramocki et al., 1997;

Dorman and Johnson, 1999; Page et al., 2004; Wang et al., 2004). In myoblasts exhibiting

strong Raf/Extracellular-Signal Regulated Kinase (ERK) signaling, TGF/73, GDF8, and BM~P6

are up-regulated, suggesting that these TGFP family proteins may serve as autocrine inhibitors of

differentiation (Wang et al., 2004). These proteins are members of the TGFP superfamily, which

consist of the BMP, activin, and TGFP subfamilies. Skeletal myogenesis is intricately regulated

by differential gene expression of these ligands. TGFP1 and GDF8 are inhibitors of proliferation

and differentiation (Florini et al., 1986; Greene and Allen, 1991; Rao and Kohtz, 1995; Stewart

et al., 2003; Allegra et al., 2004). Ablation ofM~STN in mouse and the naturally occurring

mutations found in some Belgium Blue cattle result in greater muscle mass (Grobet al., 1997;

Kambadur et al., 1997; McPherron and Lee, 1997). Much less is known about BMP effects on

myogenesis. The objective of these experiments was to examine TGFP signaling components

during myogenesis in embryonic myoblasts and a postnatal satellite cell derivative.

Differential Transcriptional Activity in Myoblasts versus Myofibers

Autocrine activity during the transition from myoblasts to myofibers was examined in

C2C 12 cells transfected with a TGFP response element promoter reporter (3 TP-Lux) (Wrana et

al., 1992), a BMP responsive reporter gene (BRE-Luc) (Korchynskyi and ten Dijke, 2002) or a

muscle reporter gene (Tnl-Luc). Myoblasts were maintained in differentiation-permissive

medium for 48 hours prior to lysis, and luciferase activity measured. As expected, Tnl-Luc









levels were barely detectable in myoblasts and increased approximately 12-fold during the

conversion to myofibers (Figure 3-1). Evaluation of BMP and TGFP-driven transcription varied

in myogenic cells. In myoblasts, 3TP-Lux relative activity is decreased during differentiation.

Conversely, BMP-directed transcriptional activity doubled in myofibers as compared to

myoblasts (Figure 3-1). The different reporter levels observed in myoblasts and myofibers

suggests that autocrine loops are present that may contribute to distinct stages of myogenesis.

Differentiation TGFP Gene Expression in Myoblasts and Myofibers

Numerical changes in reporter activity suggested differential expression of TGFP proteins

during myogenesis. RT2 Gene Expression Assays were used to quantify changes in relative

TGFP superfamily member gene expression profies in myoblasts and myofibers. Total RNA

was isolated from 23A2 (embryonic myoblasts) and C2C12 (adult satellite cells) myofibers and

evaluated by ethidium bromide impregnated formaldehyde agarose gels. Distinct 18S and 28S

bands demonstrate intact RNA (Figure 3-2A). Equal amounts of RNA were reverse transcribed

in the presence of biotin-UTP (Figure 3-2B). The TGFP miniarray was hybridized with

biotynlated cDNA according to manufacturer' s recommendations (SuperArray). Results

demonstrate Statl, Noggin, and Runx1 transcripts are more abundant in 23A2 myofibers than

C2C12 myofibers. By contrast, Bnp3, Bnprla, Itgb5, and Igfbp3 expression is greater in

C2C12 myofibers than 23A2 myofibers (Figure 3-3). Appendix A provides a complete list of

genes and spotting locations.

Total RNA was isolated from proliferating C2C12 myoblasts or differentiated myofibers

and relative gene expression was determined by nylon array. Results demonstrate that BMP

ligands and receptors, TGFP ligands, and Runxs are expressed differentially during the transition

from myoblasts to myofibers. Runx1 mRNA levels are low in mononucleated myoblasts and









increase in multinucleated myofibers; Runx2 mRNA levels are high in myoblasts and low in

myofibers and TGFP3 is low in myoblasts and increases in myofibers (Figure 3-4).

The BMP ligands are inductive and inhibitory in myogenesis (Tzahor et al., 2003).

Expression ofBM~P1, 2, 3, 4, and 6 were detectable in both myoblasts and myofibers. Closer

examination of these BMPs shows that BMP3, BMP4 and BMP6 mRNA levels are greater in

mononucleated myoblasts than multinucleated myofibers. BM~P2 expression does not appear to

differ between myoblasts and myofibers. BMP1 mRNA slightly increases from myoblast to

myofibers (Figure 3-5). Relative amounts ofBM~P1, 3, 4, and 6 messages differ in myoblasts

versus myofibers suggesting these genes may be involved in distinct stages of muscle formation.

BM~P6 expression is greater in myoblasts than myofibers. Autocrine regulation of BMP6

was examined in C2C12 myofibers. Results demonstrated that BM~P6 levels, and other BMP

ligands, are not affected by ectopic BMP6 treatment (Figure 3-6). In summary, differential gene

expression of TGFP superfamily members is detected in different myoblast cell lines and during

various stages of myogenesis. BMP6 mRNA is abundant in myoblasts but does not up-regulate

it own expression.

Discussion

Assessment of autocrine activity during the transition from myoblast to myofiber by

evaluation of BMP-responsive, TGFP-responsive or muscle specific reporter elements

demonstrated the presence of autocrine loops that may contribute to distinct stages of

myogenesis. Tnl-Luc activity drastically increased during the conversion of myoblasts to

myofibers, which confirmed successful differentiation. TGFP-sensitivity was high in myoblasts

and low in myofibers, supporting the idea that a TGFP-like protein is inhibiting differentiation.

Conversely, BMP-sensitivity was low in myoblasts and high in myofibers. The different reporter









levels observed in myoblasts and myofibers further suggests that there are differential responses

for TGFP and BMPs at different stages of skeletal myogenesis or myoblasts versus myofibers.

23A2 cells are embryonic myoblasts that were derived from C3H10T1/2 embryonic

fibroblasts treated with 5-Azacytidine (Konieczny and Emerson, 1984). C2C12 are adult

satellite cells derived from murine limb muscle (Blau et al., 1985). While both of these

immortalized cell lines exhibit myogenic properties, there are also marked differences between

23A2 and C2C12 cells. Comparison of embryonic myoblasts and adult satellite cells

demonstrated detectable gene expression differences. Transcripts for Statl, Noggin, and Runx1

are greater in embryonic myoblasts than adult satellite cells. Stat1 is involved in modulating

anti-proliferative and growth arrest signals by inducing expression of cell cycle inhibitors,

p21wAFl/CIP and pro-apoptotic signals (Durbin et al., 1996; Meraz et al., 1996). The Stat1

knockout mouse demonstrates no overt developmental abnormalities although the mice have a

significant increase in bone mineral density and bone mineral content (Xiao et al., 2004).

Greater levels of Stat1 in 23A2 myofibers may explain why ALP induction is greater in C2C12

than 23A2 myoblasts since it may act as a negative regulator of osteogenic activity.

Conversely, Bnp3, Bnprla, Itgb5, and Igfbp3 transcripts are greater in adult satellite cells

than embryonic myoblasts. Bnp3 is the most abundantly expressed member of the BMP

subfamily in both mononucleated myoblasts and multinucleated myofibers assessed by nylon

array. BMP3 is also the most abundant BMP in adult bone and a maj or component of

osteogenin, which does have osteogenic activity (Wozney and Rosen, 1993; Luyten et al., 1989).

However, recombinant BMP3 is unable to induce an osteogenic response in multiple cell lines

(Bahamonde and Lyons, 2001; Daluiski et al., 2001). Knockout models demonstrate that Bnp3

is a negative determinant of bone density with null mice exhibiting twice as much trabecular









bone as wildtype counterparts. Additionally, BMP3 inhibits BMP2-responsiveness in

osteoprogenitor cells and acts as an antagonist of osteogenic BMPs by activating the

TGFP/activin pathway, which would antagonize BMP signaling (Bahamonde and Lyons, 2001;

Daluiski et al., 2001). Therefore, the abundant gene expression of BMP3 would be important for

skeletal muscle formation because high levels ofBmp3 play an essential role in modulating

osteogenic BMPs. Specifically, Bmp3 would help inhibit osteogenic conversion of myoblasts

through competition for signaling components similar to both the TGFP/activin and BMP

pathways such as Smad4 (Heldin et al., 1997).

Gene expression profies for myoblasts and myofibers demonstrated differential gene

expression such as Runx1 and TGF/73 are greater in myofibers than myoblasts and Runx2 is

greater in myoblasts than myofibers. Results suggest that these genes play a role in cell origin

and regulate distinct stages of skeletal myogenesis. Runx2 (or Cbfal) is the master regulator for

bone development and the Runx2 knockout mouse demonstrates a complete lack of bone

formation and chondrocyte hypertrophy in most of the skeleton (Shum and Nuckolls, 2002).

Based on nylon array results, Runx2 is greater in myoblasts than myofibers and this may be a

function of the ability of myoblasts to undergo transdifferentiation in response to BMP6.

Alternatively, myofibers and fibroblasts do not undergo transdifferentiation, which may be due

to lower levels of Runx2. Nylon arrays demonstrated that there are differential gene expression

profiles between 23A2 and C2C12 myofibers. Additionally, both lines were found to undergo

transdifferentiation in response to ectopic BMP6 treatment, but to different magnitudes which

also demonstrates differences in the molecular signaling of these immortalized lines.

In conclusion, these nylon arrays allow for a glimpse of differential gene expression of

embryonic myoblasts and postnatal satellite cells, and different stages of skeletal myogenesis.









Verification of these results by Real-Time PCR analyses are needed with specific primers for

each uniquely expressed gene. While previous literature demonstrates a role for these BMPs in

embryonic development and somite patterning (Winnier et al., 1995; Zhang and Bradley, 1996;

Dunn et al., 1997; Solloway et al., 1998; Daluiski et al., 2001; Ying and Zhao, 2001), the role of

BMPs in adult myogenic cells has not been highly researched and this would determine which

TGFP superfamily members, specifically BMP genes, are important for molecular regulation of

myogenic differentiation and fusion.






















* *





bhc


gl4
a 12





4


h


3TIP
luc


BRE
luc


luc


TnI
luc


3TP
luc


BRE
luc


Figure 3-1. Differential transcriptional activity in myoblasts versus myofibers. C2C12
myoblasts (1 x 105) were transfected with 1 Cpg of 3TP-luc (TGFP-specific), BRE-luc
(BMP-responsive), or Tnl-luc (Muscle-specific). Cells were maintained in growth
medium (myoblasts) or differentiation-permissive medium (myofibers) for 48 hours.
Cells were lysed, and luciferase and Renilla luciferase activities were measured.
Means and SEM are from three independent experiments. Different letters indicates a
significant difference, P<0.05.


d


MWYOBLA ST


MWYOFIBER


























23A2 biotin-cD>NA IJ g a

C2C12 biotin-cDNA .g g. 1$

1:20 1:80 1:320 1:1280 1:5120 dilution

Figure 3-2. Intact RNA and cDNA probe synthesis. Total RNA was isolated from 23A2 and
C2C12 myofibers. 15 Clg was electrophoretically separated through formaldehyde
agarose gels. RNA integrity was visualized with ethidium bromide, 18S and 28S
ribosomal RNA are noted (A). One Clg of RNA was reverse transcribed in the
presence of biotin-UTP. Serial dilutions of biotin cDNA were spotted to nylon and
incubated with streptavidin HRP. Chemiluminescence demonstrates biotin-UTP
incorporation (B).


23A2 C2C12


18S

















se *P7
e as ~Itgb




Figre -3 Gene exrsso prfie in emroniad a teltecl myofes ToalN a









Figu Sgnfian g-. ene exprsiproiession dfemrenic arindicated in thel yolumnbetwee TtheRN two
Msolae rpogenetic Poeibyni ReeporieA la, Collal Prclagen, tye I, alph 1,fr Itb5,
Inernd betual 5, 1gfp(3, Insli n-like gevrowt fatosrbindin prtin 3,e pRunx1, Rubitnt
re .Bitnlated trncrpi n s fact re 1,a d Statl, ea GP/M Signal tasdcr n aci at r of
tSprrasriptiFolon in 1.rdztotebosweeicbtdwt vd






















M~ M
rt a :

rt "It2 :


Gene Classification
-- Activin Receptors
- BMP Ilganids
- BMP liganids and receptors
- BMP antagonists & collagens
- TGFPs
- GDFs
- ids and SMAD target genes
- Inhlbmns
- SMADs
ECMs and Development

-T~GFB ligands and receptors
H-ousekeeping genes


I E



g r


Symbol Le gend
S= Runx2 (Cbfal)

S= Runx1

= TGF63


Figure 3-4. Differential gene expression in myoblasts and myofibers. Total RNA was isolated
from C2C12 myoblasts (A) or myofibers (B) and equal amounts (3 Cpg) of RNA were
reverse transcribed in the presence of biotin-UTP. Biotinylated cDNAs were used to
probe a TGFP/BMP Signaling Array (SuperArray). Following hybridization, the
blots were incubated with avidin-peroxidase and visualized by chemiluminescence.
Representative blots are shown. Gene Classifieation Groups are indicated on the
right of the diagram. Abbreviations: BMP, Bone Morphogenic Protein, TGFP,
Transforming Growth Factor beta, GDFs, Growth and Differentiation Factors, Ids,
Inhibitor of DNA binding, SMAD, MAD homolog, ECMs, Extracellular Matrix
Molecules.


g n

27 g
g :


au asa


ES 22















.Myablasts





Myofib ers






SG= GAPDH


Figure 3-5. Relative BMP gene expression in myoblasts and myofibers. Total RNA was
isolated from C2C12 myoblasts and myofibers and equal amounts (3 Gig) of RNA
were reverse transcribed in the presence of biotin-UTP. Biotynlated cDNAs were
used to probe a TGFP/BMP signaling array (SuperArray). Following hybridization,
blots were incubated with avidin-peroxidase and visualized by chemiluminescence.
Representative subsections of blots for BMP 1, 2, 3, 4, 5, 6, 10, and 15 are shown.
(A) Myoblasts; (B) Myofibers; (C) Schematic location of genes.














C control





B.


BMP6




C.



G = GAPDH


Figure 3-6. BMP6 does not undergo autocrine gene regulation. C2C 12 myoblasts were
differentiated for 48 hours in the absence (A) or presence (B) of 100 ng/ml BMP6.
Total RNA was isolated and equal amounts (3 Gg) were reverse transcribed in the
presence of biotin-UTP. Biotynlated cDNAs were used to probe a TGFP/BMP
signaling array (SuperArray). Following hybridization, blots were incubated with
avidin-peroxidase and visualized by chemiluminescence. Schematic location of BMP
genes (C).









CHAPTER 4
IMPACT OF BMP6 ON SKELETAL MYOGENESIS

Objective

In Raf-arrested myoblasts, removal of TGFP1 biological activity does not restore the

myogenic program suggesting that another factor is mediating this inhibition (Wang et al., 2004).

Ectopic GDF8 treatment does not inhibit 23A2 myoblast differentiation. BMP6 mRNA is

present in myoblasts and absent in myofibers suggesting an inhibitory action during myogenesis

(Derynck, 1989). The balance of proliferation, cell differentiation, and apoptosis mediates the

pool of myoblasts available for skeletal myogenic maintenance. The objective of these

experiments was to measure BMP6 effects on myoblast proliferation, differentiation, and

apoptosis.

Inhibition of Skeletal Myogenic Differentiation by BMP6

To further examine the role of BMP6 in embryonic myoblasts and satellite cells, 23A2

myoblasts and C2C12 satellite cells were transiently transfected with Tnl-Luc and treated with 0

or 100 ng/ml BMP6 for 48 hours in differentiation media prior to lysis and measurement of

luciferase activity. Results show that treatment with 100 ng/ml BMP6 resulted in more than an

80-fold inhibition of muscle specific reporter (Tnl-Luc) activity in both myogenic cell types

(Figure 4-1, P<0.05). Due to the significant biochemical inhibition of differentiation by BMP6,

assessment of morphological changes was performed. C2C 12 myoblasts were differentiated in

the presence of 100 ng/ml BMP6 for 48 hours. Subsequently, cells were Eixed and

immunostained for MyHC. Results show very few multinucleated fibers in response to BMP6

treatment (Figure 4-2A). Total cell lysates were isolated from a second set of C2C12 myofibers

treated in an analogous manner. Equal amounts of protein (10Clg) were analyzed by Western

blot for contractile and regulatory protein expression. Inhibition of the muscle contractile protein









MyHC, in addition to, myogenin, and troponin T protein markers was observed in response to

BMP6 treatment (Figure 4-2B). Thus, BMP6 inhibits the complete differentiation program.

Dose-Dependent Effects of Recombinant BMP6 on Myoblasts

BM~P 2, 4, and 7 have opposing activities that are concentration-dependent during

embryonic muscle growth (Amthor et al., 1998; Amthor et al., 2002). Due to the high

concentration of BMP6, a retrospective dose-response experiment was performed (1 ng/ml to

50 ng/ml BMP6). Biochemical differentiation was measured following transient transfection of

Tnl-Luc into C2C12 myoblasts. Cells were treated for 48 hours in differentiation media with

increasing concentrations of BMP6. Analysis of luciferase reporter activity shows an

approximate 20% decrease of Tnl-Luc activity at 25 ng/ml (P<0.05) and an approximate 50%

decrease at 50 ng/ml (P<0.001) (Figure 4-3A). Parallel plates were lysed for protein analyses.

Equal amounts of total cell proteins were separated by SDS-PAGE and transferred to

nitrocellulose. Blots were probed with anti-MyHC and anti-myogenin. Results demonstrate that

MyHC protein expression begins to decrease at 50 ng/ml BMP6 (Figure 4-3B). Interestingly,

myogenin protein expression begins to decrease at 10 ng/ml BMP6, further at 25 ng/ml and is

completely absent in response to 50 ng/ml BMP6 (Figure 4-3B).

Induction of ALP Activity in Response to BMP6

BMPs induce bone or cartilage formation ectopically (Urist, 1965; Gitelman et al., 1995).

BMP2 inhibits the myogenic differentiation of C2C12 cells, by converting their differentiation

pathway into that of osteoblast lineage cells (Katagiri et al., 1994). To determine if the block to

differentiation is associated with transdifferentiation, assessment of alkaline phosphatase (ALP)

activity of myofibers treated with BMP6 was measured. C2C12 myoblasts were treated with

vehicle only or increasing amounts of BMP6 in differentiation media for 48 hours. Cells were









Eixed and histologically stained for ALP activity, a marker enzyme of osteogenic cells. As

shown in Figure 4-4, as little as 5 ng/ml BMP6 was sufficient to induce ALP activity.

To determine if the effect of BMP6 is unique to the committed myoblast, C3H10T1/2

fibroblasts were treated with 0, 1, 10, and 100 ng/ml BMP6 in differentiation media for 48 hours.

Cells were Eixed and histologically stained for ALP activity. Results indicated that BMP6 does

not cause ALP induction in C3H10T1/2 fibroblasts (Figure 4-5).

BMP6 Induces Rapid Transdifferentiation in Myoblasts

C2C 12 myoblasts were treated with 100 ng/ml BMP6 for 24 and 48 hours. Sub sequently,

cells were Eixed, and ALP activity was measured. Results show that ALP activity is observed as

early as 24 hours and further increases at 48 hours (Figure 4-6). C2C 12 cells treated with vehicle

alone also demonstrated a slight induction of ALP at 24 and 48 hours indicating endogenous

ALP activity.

BMP6 Does Not Alter Proliferation Rates of Myoblasts

Cell cycle withdrawal or inhibition of proliferation is required for differentiation and

fusion of myofibers. To determine if the block to myogenic differentiation by BMP6 is due to

altered cell proliferation, C2C12 myoblasts were treated with 25 ng/ml BMP6 for 48 hours.

Cells were pulsed with BrdU during the final thirty minutes of the treatment interval, fixed with

methanol, and immunostained for BrdU (Figure 4-7A). The numbers of nuclei were not different

between control and BMP6 treated cells (Figure 4-7B, P<0.05). Therefore, exposure of

myoblasts to BMP6 does not alter cellular proliferation.

BMP6 is not Anti-Apoptotic

Previous reports indicate that BMP2 and 4 promote cell survival in pluripotent

mesenchymal cells by inhibiting TNF-mediated apoptosis (Chen et al., 2001). BMP6 was

demonstrated to partially restore survivability in human mesenchymal stem cells (hMSCs)









induced by the BMP antagonist sclerostin (Sutherland et al., 2004). Bcl-2 is an anti-apoptotic

protein expressed abundantly in satellite cells (Krajnak et al, 2006). The protective actions of

BMP6 were examined in C2C12 myoblasts treated with staurosporine. C2C12 satellite cells

were treated with increasing concentrations of BMP6 for 48 hours and Bcl-2 protein content

measured by Western blot. Results demonstrate no changes in Bcl-2 protein concentration

following BMP6 treatment (Figure 4-8A). Subsequently, myoblasts were treated for 3 hours

with BMP6, 25 C1M staurosporine or BMP6 and staurosporine. Cells were lysed and assayed for

Poly (ADP-ribose) polymerase (PARP) protein expression by Western blot analysis. Results

demonstrate that staurosporine stimulates PARP cleavage, a hallmark of apoptosis (Figure 4-8B).

BMP6 does not prevent PARP cleavage in C2C12 myoblasts treated with staurosporine, thus

precluding an anti-apoptotic function (Figure 4-8B).

In summary, BMP6 significantly inhibits the complete differentiation program as observed

by biochemical suppression of muscle specific reporter activity, and morphological disruption of

myofiber formation and muscle-specific protein expression. This inhibitory effect is dose

dependent and results in rapid transdifferentiation of committed mesodermally-derived

myoblasts to an osteogenic lineage. BMP6 does not inhibit differentiation by promotion of a

proliferative state. Nor does BMP6 serve as an anti-apoptotic factor in myoblasts.

Discussion

BM~P6 is greater in myoblasts than myofibers, and up-regulated in Raf-arrested myoblasts.

Therefore, the function of BMP6 on proliferation, differentiation, and apoptosis in skeletal

myogenesis was evaluated. Treatment of 23A2 and C2C12 myoblasts with 100 ng/ml BMP6

significantly inhibited muscle specific activity (Tnl-luc). Muscle fiber fusion and muscle

specific protein expression of myosin heavy chain (MyHC), myogenin and Troponin T are also









decreased in response to BMP6 treatment. Results suggest that BMP6 inhibits the complete

differentiation program.

Other members of the TGFP superfamily can also inhibit differentiation. One of the most

notable examples is GDF8 or myostatin. M~yostatin is mutated in double muscled cattle breeds,

Belgium Blue and Piedmontese (Grobet et al., 1997; Kambadur et al., 1997; McPherron and Lee,

1997). It is predominately expressed in the muscle and negatively regulates myogenic

proliferation. The myostatin knockout mouse also demonstrates two times larger muscle mass as

compared to wild type counterparts (Thomas et al., 2000). Therefore, TGFP superfamily

members are critical mediators of skeletal myogenesis.

BMP6 is a morphogen, and morphogens are characterized by exhibiting different effects at

different levels or concentrations. In pre-myogenic cells, BMP2, 4, and 7 have dose dependent

effects with low concentrations maintaining a Pax3-expressing proliferative population and

delaying differentiation. Conversely, high concentrations of these BMPs prevent muscle

development (Amthor et al., 1998). In the presence of low BMP levels, myogenic precursor

cells are maintained in a proliferative state in developing limb bud, while high BMP levels

induce cell death. Thus, BMPs can both stimulate and restrict muscle growth (Amthor et al.,

1998; Amthor et al., 2002). This suggests that a concentration gradient of BMPs is needed for

the correct determination and maintenance of the myogenic program (Centrella et al., 1994;

Alliston et al., 2001; Reddi, 1994). 23A2 myoblasts treated with increasing concentrations of

BMP6 were measured for muscle specific reporter activity and MyHC and myogenin protein

expression. Results demonstrated that muscle reporter activity is significantly decreased at both

25 ng/ml and 50 ng/ml. MyHC protein expression began to decrease at 50 ng/ml BMP6 and

myogenin decreased at 10 ng/ml BMP6. This suggests that BMP6 exhibits dose dependent









effects on contractile and regulatory muscle proteins. Smad1 and Smad5 binding sites are found

within the promoter of myogenin, which allow BMP6 to induce downstream Smad signaling.

C2C12 cells transiently transfected with Smad1 and Smad5 were able to induced ALP activity

and decrease myogenin/chloramphenical acetyltransferase (myogenin-CAT) activity. Although

in NIH3T3 fibroblasts, Smad1 and Smad5 decreased myogenin-CAT but did not induced ALP

activity, which demonstrates that Smad1 and Smad5 are involved in the BMP signaling that

inhibits myogenic differentiation and induces transdifferentiation. Furthermore, the conversion

of these two differentiation pathways is regulated independently at the transcriptional level

(Yamamato et al., 1997). Additionally, high levels of BMP6 impact or repress stages of skeletal

myogenesis prior to terminal differentiation.

Promotion of osteogenic differentiation by BMP2 expression in skeletal muscle-derived

C2C12 cells (Musgrave et al. 2001) demonstrated that myoblasts can undergo

transdifferentiation from a myogenic to osteogenic cellular lineage in response to BMP. ALP

staining measures osteogenic activity and ectopic BMP6 treatment of C2C12 myoblasts

demonstrated a dose dependent induction of ALP activity. Additionally, when C2C12 myoblasts

were treated for 24 and 48 hours, induction of ALP activity in response to BMP6 treatment

demonstrated a time dependent transdifferentiation of myofibers into an osteogenic lineage.

The substantial induction in the numbers of ALP positive cells in C2C12 cultures maintained in

low serum at 24 and 48 hours also suggests a basal level of osteogenic activity in C2C12

myofibers. These cells are mononucleates and not myofibers. Comparison of gene transcripts

between myoblasts and myofibers by nylon array suggests that greater transcripts ofRunx1 could

explain why these cells demonstrate a basal level of osteogenic activity. Furthermore, BM~P1, 3,

and 4 transcripts are expressed in both untreated and BMP6 treated myoblasts, but only BM~P4









displays osteogenic activity, which could contribute to endogenous ALP activity of C2C12

myoblasts. BMPs 2, 4, 6, 7, and 9 are characterized as osteogenic BMPs, with BMPs 2, 7, 6, and

9 displaying the highest osteogenic activity when applied ectopically in vitro and in vivo (Luu et

al., 2007).

BMP 1 is a metalloprotease that regulates deposition of fibrous extracellular matrix (ECM)

in vertebrates and does not display osteogenic activity (Bond and Beynon, 1995). It provides

procollagen C proteinase (PCP) activity to cleave the C propeptides of procollagens I-III to yield

the major fibrous components ofECM (Kessler et al., 1996; Li et al., 1996; Suzuki et al., 1996;

Scott et al., 1999). In bone, BMP1 co-purifies with TGFP-like BMPs from osteogenic extracts

of bone and is believed to coordinate the deposition of ECM with the activation of certain BMPs

in early development and later in the development of bone and other tissues (Wozney et al.,

1988; Scott et al., 2000). Furthermore, BMP3 would not contribute to endogenous ALP activity

because as mentioned previously, recombinant BMP3 does not display osteogenic activity and

acts as an antagonist of osteogenic BMPs (Bahamonde and Lyons, 2001; Daluiski et al., 2001).

Conversely, exposure of C3H10T1/2 fibroblasts to a BMP6 dose-response curve did not

induce ALP activity at any dosage for 1, 10, and 100 ng/ml BMP6. The differential responses

observed in C2C12 and C3H10T1/2 cells suggest that BMP6-mediated transdifferentiation is

specific to lineage restricted cells. Similar results of a significant ALP induction in C2C 12 cells

by BMPs (2, 4, 6, 7, and 9) was reported by other groups. C2C12 cells demonstrated the most

potent ALP induction (Ebisawa et al., 1999). Therefore, the origin of the cell line may determine

if the cells will respond to BMPs and/or the overall magnitude of the ALP response. Yang et al.,

(2003) did demonstrate induction of ALP activity and stimulation of osteoblast marker genes by

300ng/ml of recombinant BMP6, whereas, our experiments never used a dosage higher than









100ng/ml BMP6. Another group demonstrated an induction of ALP activity in C3H10T1/2

fibroblasts in response to 100 ng/ml BMP2, suggesting that other BMP family members or

higher dosages of BMP6 may have different impacts on fibroblasts. Additionally, BMP2-

mediated ALP induction in C3H10T1/2 fibroblasts was increased in the presence of HGF (Imai

et al., 2005). Therefore, the addition of HGF with BMP6 might result in a detectable ALP

induction in C3H10T1/2 fibroblasts.

Comparison of BMP2 and BMP6 finds that both are expressed in skeletal muscle cells but

are in different classes of the BMP subfamily (Lyon et al, 1989). BMP2 is a member of the DPP

class along with BMP4, whereas, BMP6 is a member of the 60A class along with BMP5, 7, 8A,

and 8B (Gitelman et al., 1997). Structurally, BMP2 contains 3 exons, two of which encode the

precursor protein (Feng et al., 1994). The non-coding exon refers to an exon located on the 5'

flanking region of the DNA that has been shown to serve as an alternate promoter, which

suggests that BMP2 is regulated in both a developmental and tissue-specific manner (Gitelman et

al., 1994). BMP6 is composed of 7 coding exons with the mature protein encoded by 3 full

exons and a portion of the fourth exon and is also developmentally regulated. While BMP2 and

BMP6 are members of the same subfamily, there is little similarity in the localization of the

intron-exon structures further demonstrating differences at the structural level (Gitelman et al.,

1994).

In vivo analyses demonstrates that BM~P6 null mice are viable and fertile and exhibit no

maj or defects in known BMP6-expressing tissues, except for a delay in ossification restricted to

the developing sternum (Solloway et al., 1998). It is believed that BM~P2 may be functionally

compensating for BM~P6 ablation since BM~P2 and BM~P6 are required for some overlapping or

redundant functions (Solloway et al., 1998). Based on nylon arrays, BM~P6 is greater in









myoblasts than myofibers, yet BM~P2 and BM~P4 transcripts don't appear to differ in myoblasts

and myofibers. Conversely, when the muscles of athymic nude rats were inj ected with

adenoviral vectors for BMP6 (AdBMP6), a rapid tissue calcifieation was observed. The

induction of bone was produced through mechanisms similar to both intramembranous and

endochondral ossifieation pathways and AdBMP6 was even more potent than the prototypical

adenoviral vector AdBMP2 (Jane et al., 2002). These studies utilized titers of BMP6 that would

not mimic physiological conditions but do demonstrate the powerful osteogenic activity of

BMP6. BM~P2 null mice exhibit multiple developmental defects including a delayed primitive

streak, small allantois, lack of amnion, heart defects and a decreased number of primordial germ

cells (Zhang and Bradley, 1996; Yin and Zhao, 2001). Therefore, BMP2 and BMP6 appear to

have different biological functions.

It has also been suggested that receptor oligomerization determines BMP2 signaling

pathways. Nohe et al., (2002) demonstrated that binding of BMP2 to preformed receptor

complexes activates the Smad pathway. Conversely, BMP2-induced recruitment of receptors

activates a Smad-independent pathway, which results in the induction of ALP activity via p38

MAPK (Nohe et al., 2002). These different receptor complexes may also recruit different

adaptor proteins such as, XIAP (Yamaguchi et al., 1999), BRAM-1 (Kurozumi et al., 1998), and

FKBPl2 (Wang et al., 1996). BMP2 treatment of C3H10T1/2 cells stimulates ERK1 and ERK2

during osteoblastic differentiation (Lou e al., 2000), and ERK activation can inhibit nuclear

translocation of Smad1, which would block the Smad pathway (Kretzschmar et al., 1997). Our

experiments demonstrate a strong Smadl/5/8 activation by BMP6 and only a slight p38

activation suggesting that BMP6 induces ALP activity through a different signaling mechanism

than BMP2.









Characterization of BMP6 and BMP2 in C2C12 cells demonstrates that BMP6 strongly

binds to activin receptor-like kinase (ALK)-2 or ActR-I (Ebisawa et al., 1999). ALK2 forms

complexes with receptors like, BMPR-II or ActR-II. BMP6 can also weakly bind to ALK3,

which also can bind BMP2 (Ebisawa et al., 1999) but BMP2 preferentially binds to BMPRIA

and IB. C2C12 cells only express mRNA for BMPRIA (Akiyama et al., 1997). C3H10T1/2

fibroblasts express both BMPRIA and BMPRIB but BMPRIA expression levels are

endogenously higher. Transfection experiments with BMPRIA and BMPRIB in C3H10T1/2

cells also demonstrate that the dominant role in BMP2 mediated osteogenic development was

mediated by BMPRIA, with BMPRIB only partially influencing osteogenic development (Kaps

et al., 2004). Additionally, type IB and IA BMP receptors appear to transmit different signals

during the specification and differentiation of mesenchymal lineages (Kaps et al., 2004).

Truncation and overexpression of BMP receptors, BMPRIA and BMPRIB, have demonstrated

that overall receptor levels expressed in cells play a critical role in specification and

differentiation of osteoblasts by BMP2 (Chen et al., 1998). Therefore, while BMP2 and BMP6

can interact with the same BMP receptor, they demonstrate the strongest affinity for different

type I receptors. Therefore, differences in signaling could be based on like receptor

oligomerization, cross-talk with other signaling pathways, competition for signaling components,

interaction with additional proteins, and activation of different downstream transcriptional

targets, which would result in different biological responses of BMP2 and BMP6.

Transdifferentiation of myogenic cells to an osteogenic linage results in induction of

specific markers at the transcriptional level. Therefore, measurement of genes such as, Runx1,

Runx2, osteopontin, osteonectin, and osteocalcin (Ahrens et al., 1993) would determine if the

cells were exhibiting more "bone-like" characteristics and what genes are up-regulated in BMP6-









treated myoblasts. Interestingly, based on the nylon array assessment of myoblasts versus fibers,

Runx1 appeared to be greater in myofibers, yet Runx2 appeared to be greater in myoblasts.

Runx2 is required for later stages of chondrocyte and osteoblast differentiation, while Runx1

mediates early events of endochondral and intramembranous bone formation (Smith et al., 2004).

These results would need to be confirmed by Real-Time PCR and may have implications in

transcriptional regulation of the myoblast to myofiber transition and/or transdifferentiation of

myoblasts to osteoblasts. Since myogenic precursor cells (MPCs) also have the ability to form

skeletal muscle, bone, or cartilage, the differential expression of these transcription factors may

demonstrate how these precursor cells determine their ultimate cell fate.

Inhibition of embryonic skeletal muscle differentiation through promotion of a

proliferative state was monitored. Others have suggested that Raf inhibits skeletal myogenesis

by keeping myoblasts in a proliferative state (Samuel et al., 1999). Since BMP6 expression is

high in Raf-arrested myoblasts, it was proposed that BMP6 may also be promoting a

proliferative state of cells. When myoblasts were treated with BMP6, the number of BrdU

positive nuclei or cells in S-phase was not significantly different from untreated myoblasts. The

percent BrdU incorporation was calculated by dividing the number of BrdU positive nuclei by

the total number of cells and taking the average of six Hields. There also was no significant

difference in BMP6 treated percent BrdU incorporation versus control myoblasts. Therefore,

exposure of myoblasts to BMP6 does not inhibit proliferation suggesting that BMP6-mediated

inhibition of differentiation is not mediated through modulation of proliferative events.

Conversely, another group did demonstrate an inhibition of proliferation by [3H] thymidine

incorporation assays in a dose-dependent manner (Ebisawa et al., 1999). Comparatively, this

study did a dose response up to 1000 ng/ml BMP6 and only observed approximately a 20%










decrease at 300ng/ml, whereas our analyses assessed BrdU incorporation at 25 ng/ml, which is

considered a more physiological dose.

Apoptosis is a mechanism of programmed cell death that promotes tissue turnover,

embryonic development, and immunological defense mechanisms (Kerr et al., 1972; Adams,

2000; Siu et al., 2005). The intrinsic and extrinsic signaling pathways are the two principal

pathways involved in apoptosis. The intrinsic route uses cell signaling pathways to alter

mitochondrial function. Permeabilization of the outer mitochondrial membrane results in release

of cytochrome c, thereby forming an apoptosome. This macromolecular complex activates

caspase-9, mediated by apoptotic protease activign factor (APAF)-1 (Roy and Nicholson, 2000).

Conversely, the extrinsic pathway sends 'death ligand' signals of the the Tumor Necrosis Factor

(TNF) or Fas families through appropriate receptors that activate caspase-8 in conjunction with

the adaptor molecule, Fas-associated death domain (FADD). At this point, intrinsic and extrinsic

cascades converge to activate effector caspases (caspase-3 and -7), which cause the proteolytic

degradation of cellular material (Roy and Nicholson, 2000).

BMP2, BMP4, and BMP6 promote cell survival in mesenchymal cells (Chen et al., 2001;

Sutherland et al., 2004). Yet when myoblasts were treated with ectopic BMP6, Bcl-2 protein

expression, an anti-apoptotic marker, did not change in response to increasing amounts of BMP6.

Additionally, BMP6 did not prevent PARP cleavage in response to staurosporine, a common

inducer of apoptosis. This suggests that BMP6 does not have an anti-apoptotic function in

skeletal myoblasts. BMP6 was able to partially protect hMSC cells from sclerostin-induced

apoptosis by decreasing caspase activity (Sutherland et al., 2004). Sclerostin is a BMP

antagonist that binds to BMPs and blocks downstream signaling pathways. Furthermore, the

protective effect of BMP6 observed was only a partial block of sclerostin-mediated apoptosis










suggesting that additional factors are involved (Winkler et al., 2003). Therefore, the protective

effect of BMP6 observed in this scenario may be a result of additional signaling components not

present in myoblasts or is due to an alternative apoptotic signaling cascade specific to Sclerostin

or different from staurosporine-induced apoptosis.

In conclusion, BMP6 inhibits the complete differentiation program in myoblasts. BMP6

treatment results in a rapid transdifferentiation of myoblasts that is specific to a committed

mesodermal derived myogenic cell. BMP6 does not appear to involve modulation of

proliferation rates of myoblasts. Nor does BMP6 appear to demonstrate a survival role in

myoblasts.













120
a n
o 23AS2
S100-
:E~ ~ ~ I iC2C12








2-



0 100

BMP6 (ng/ml)

Figure 4-1. Biochemical inhibition of skeletal myogenesis by BMP6. Myoblasts were
transiently transfected with 2 Clg of Tnl-Luc reporter construct and 50 ng pRL-tk
(Renilla) and treated 0 or 100ng/ml BMP6 in differentiation media for 48 hours.
Cells were lysed and assessed for Tnl-Luc activity in both 23A2 and C2C12
myofibers. Reporter luciferase activity was normalized to the amount ofRenilla
luciferase activity and vehicle only was set to 100%. Data represents the mean and
standard error of the mean (SEM) of three independent experiments. Different letters
indicates a significant difference, P<0.05.

































ca-MyHC


ce-myo ge nm

| a-Troponin T


- -- ~ cl-desmin


c~ -~ulin


Figure 4-2. Inhibition of skeletal myogenesis by BMP6. C2C12 myoblasts were treated with
100 ng/ml BMP6 for 48 hours. Myoblasts were fixed and immunostained for MyHC
expression. Representative microscopic images (200X) are shown (A). Parallel
plates were lysed and analyzed for muscle specific proteins by Western blot (B).
Tubulin expression was used as a loading control.


"-


~


I





















60-





O 1 5 10 25 50
BRIP~6 (ng/ml11)

B.
O 1 5 10 25 50 BMP6 (ng/ml)
,~~9 ggp e ii-MyHC
e. .. a-myogemni




Figure 4-3. BMP6 dose response curve. C2C12 myoblasts (1x105) were transiently transfected
with 2 Clg of Tnl-Luc reporter construct and 50 ng pRL-tk (Renilla). Cells were
maintained in differentiation media supplemented with BMP6 for 48 hours. Cells
were lysed and luciferase activities were measured (A). Reporter luciferase activity
was normalized to the amount of Renilla luciferase activity and the control (vehicle
only) was set to 100%. Data represents the mean and standard error of the mean
(SEM) of three independent experiments. Different letters indicates significance at
p<0.05. Total cell lysates were analyzed by Western blot for MyHC and myogenin
protein expression (B). Tubulin expression was used as a loading control.




































Figure 4-4. BMP6 induction of alkaline phosphatase activity. C2C12 myoblasts were treated
with 0, 5, 10, 25, 50, and 100 ng/ml BMP6 for 48 hours. Cells were fixed and ALP
activity detected colorimetrically. Representative photomicrographs at 200X are
shown.




































Figure 4-5. BMP6 does not induce alkaline phosphatase (ALP) activity in fibroblasts.
C3H10T1/2 fibroblasts were treated with 0, 1, 10, and 100 ng/ml BMP6 for 48 hours.
Cells were fixed, and stained for ALP activity. Representative photomicrographs at
200X are shown.


Ongml Bhlvl%


In ml B1IM 6


10n ml hlVII%


100ndml BlV~II5


;;i"~';; $
lirr
;.!.
L;


t~~ r;
I '


)t,


i


'k'


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ir ~4
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r



t r
I,;
C

,h;C.
;:~vi' i ...C
























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IP;C~


1 hr


24 hrs


Figure 4-6. BMP6 induces rapid transdifferentiation in myoblasts. C2C12 myoblasts were
treated for 1, 24 or 48 hours with vehicle alone (control) or 100 ng/ml BMP-6.
Cultures were fixed with 4% paraformaldehyde and ALP activity measured
colorimetrically. Representative photo-micrographs at 200X demonstrate intense ALP
staining as early as 24 hours of treatment.


48 hrs


















Vphiplp Only Ce *i
Ir

r r
.
r
I r


C 11
I



BMP6 (25nml)


rl


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8


Treatment


Figure 4-7. BMP6 treatment does not alter myoblast proliferation. C2C12 myoblasts were
treated with 25 ng/ml BMP6 in differentiation medium for 48 hours, pulsed with
10 CtM BrdU for 30 minutes, fixed with 70% ethanol at 40C for 30 minutes and
immunostained for BrdU expression (A). Representative photomicrographs at 100X
shown. % BrdU incorporation was calculated by dividing the number of BrdU
positive nuclei by the total number of nuclei (B). The averages of a minimum of six
fields per treatment are shown.













0 1250 100 BMP6 (ng/ml)
.. -- a r-Bcl-2




B.
vEhilE1 STS

+ -+ BMP6

EaI-PARP


I~~lW Er-tubulin


Figure 4-8. BMP6 is not anti-apoptotic. C2C12 myoblasts were placed in differentiation
permissive media with increasing concentrations of BMP6 for 48 hours. Whole cell
lysates were prepared and analyzed for expression of Bcl-2 by Western blot (A).
C2C12 myoblasts were treated with 100ng/ml BMP6 in the absence or presence of
25 C1M staurosporine (STS). Whole cell lysates were assessed for PARP cleavage
and tubulin protein expression (B).









CHAPTER 5
BMP6 SIGNALING DURING SKELETAL MYOGENESIS

Objective

During skeletal myogenesis, BMP6 actions can be mediated by multiple signaling

mechanisms (Figure 5-1). The specifieity of the BMP6 and TGFP1 signaling responses are

achieved by different types of Type I and Type II receptors and R-Smads (Wrana et al., 1992;

Attisano et al., 1993; Ebner et al., 1993; Wieser et al., 1993; Wrana et al., 1994). Traditionally,

BMPs signal through Smads 1/5/8, while TGFP1 signals through Smads 2/3. BMPs also initiate

p38 signaling and within muscle, this regulation is specific to the p3800 isoform. A third, more

novel regulation, is through cross-talk with Notch signaling. The objective of these experiments

was to validate the presence and activation of three putative intracellular signaling cascades

induced by BMP6.

Analysis of BMP Signaling Systems in Myoblasts

The ability of BMP6 and TGFP1 to induce Smad phosphorylation and nuclear translocation

was evaluated. In brief, C2C12 myoblasts were treated with 10 ng/ml TGFP1 and 25 ng/ml

BMP6 for 48 hours. Total cell lysates were analyzed by Western blot for total and phospho-

Smadl/5/8 expression. Myoblasts treated with vehicle alone demonstrated lows levels of

phospho-Smadl/5/8, which were increased in response to both 10 ng/ml TGFP1 and BMP6

(Figure 5-2A). No differences in total Smadl/5/8 or tubulin were evident. Parallel plates of

C2C12 myoblasts treated as described were Eixed and immunostained for phospho-Smadl/5/8.

In both instances, the signaling molecules are located in the nucleus (Figure 5-2B). Therefore,

the archeotypical Smad signaling system is intact and functional in myoblasts. These results also

support previous observations that demonstrated expression of Smadl/5/8 in C2C12 cells.

Subsequent phosphorylation of Smad5 and weak phosphorylation of Smad1 was also observed









by BMP6. Smad8 was found to be constitutively phosphorylated in C2C12 cells (Ebisawa et al.,

1999).

In addition to signaling through the Smad proteins, BMPs also may utilize components of

the MAPK and Janus kinase (JAK)/signal transducers and activators of transcription (STAT)

family of signaling proteins, culminating in activation of JNK or p3 8 (von Bubnoff and Cho,

2001). p38 signaling is a requirement for muscle formation (Zetser et al., 1999; Lee et al.,2002).

The interplay of BMP6 signaling and p38 kinase activity during myogenesis was examined.

C2C12 myoblasts were treated with increasing amounts of BMP6 and assessed for total and

phospho-p38 protein expression by Western blot. Results demonstrate that while levels of total

p3 8 don't change in response to BMP6, phospho-p3 8 is slightly induced in myoblasts treated

with 25 and 50 ng/ml BMP6 (Figure 5-3).

C2C 12 myoblasts were transiently transfected with Tnl-Luc activity and treated with

100 ng/ml BMP6, 10 CIM SB202190 or a combination of both for 48 hours. Results show

100 ng/ml BMP6 significantly inhibited Tnl-Luc activity, as observed previously (Figure 5-4A,

P<0.05). Treatment with 10 C1M SB202190 inhibited Tnl-Luc activity (Figure 5-4A, P<0.05).

Importantly, the combination of BMP6 and SB202190 demonstrated an additive effect indicating

independent pathways (Figure 5-4A, P<0.0001). Morphological observations showed myofibers

in control cells, few myofibers in SB202190 treated, and no myofibers in either BMP6 treated or

the combination of BMP6 and SB202190 (Figure 5-4B).

C2C12 myoblasts were treated with BMP6, SB202910, and BMP6 with SB202910 for 48

hours in differentiation media. Cells were lysed and assayed for MyHC, myogenin, Troponin T,

and desmin protein expression by Western blot. Results demonstrate that muscle specific protein

markers (MyHC, myogenin, and Troponin T) are reduced in the presence of BMP6, p38 inhibitor









(SB202910), and the combination (Figure 5-4). C2C12 myoblasts treated with both BMP6 and

SB202910 demonstrated a more severe inhibition of myogenesis.

The importance of p3 8 signaling during BMP6-mediated transdifferentiation was

examined. Parallel plates of C2C12 myoblasts treated with BMP6, SB202190, and the

combination of BMP6 and SB202190. ALP activity was detected colorimetrically. Results

show that inhibition of p3 8 function does not alter ALP induction in response to BMP6 treatment

(Figure 5-6). This further demonstrates that BMP6 is signaling through an independent pathway

to cause inhibition of differentiation and transdifferentiation.

Impact of Notch Inhibitor on BMP6-Mediated Inhibition of Differentiation

A third signaling mechanism used by BMP ligands involves Notch. Notch is a

transmembrane receptor that is cleaved on the intracellular surface to release a proteolytic

fragment that translocates to the nucleus and affects gene transcription (Nye et al., 1994; Ahmad

et al., 1995). Notch inhibits myogenic differentiation similar to BMP6 (Kopan et al., 1994;

Takahashi et al., 1994). BMP4 repression of muscle gene expression involves Notch activation

(Dahlqvist et al., 2003). The involvement of BMP6 in Notch controlled signaling was examined

in C2C12 myoblasts. In brief, myoblasts were induced to differentiate in the presence or absence

of 25 ng/ml BMP6 and 10 CIM L685,458, a Notch inhibitor, for 72 hours. Cells were fixed and

immunostained for MyHC. Results show that MyHC was expressed in control cells and

significantly inhibited in BMP6 treated (Figure 5-7A and 5-7B, P<0.05). Treatment of C2C12

myoblasts with L685,458 alone does not alter MyHC expression and interestingly, the

combination of BMP6 and L685,458 partially restores MyHC expression (Figure 5-7A and 5-7B,

P<0.05). This suggests that the BMP6-mediated inhibition of differentiation is partially

controlled by the Notch signaling pathway.










In summary, differential BMP- and TGFP-responsiveness and gene expression exists in

myoblasts versus myofibers. This suggests that TGFP superfamily members play different

regulatory roles at various stages of myogenesis as myoblasts undergo terminal differentiation.

Treatment of myoblasts with BMP6 results in a dramatic increase of ALP activity in a dose-

dependent and time-dependent manner. Exogenous BMP6 treatment of skeletal myoblasts

results in inhibition of myoblast differentiation as observed by significant inhibition of muscle

reporter activity, muscle specific protein synthesis, and myoblast fusion. BMP6 treatment does

not alter proliferation rates of myoblasts or promote a proliferative state to inhibit differentiation.

Inhibition of p3 8 activity combined with BMP6 treatment caused inhibition of TnI-Luc activity

that was greater than either treatment alone suggesting an additive effect between BMP6 and

inhibition of p3 8 activity. Interestingly, the combination of BMP6 and Notch inhibition by

L685,458 partially restores MyHC expression in fibers. This suggests that the myogenic

inhibitory effect observed in the presence of BMP6 is partially mediated by functional Notch

signaling.

Discussion

BMPs can be mediated by multiple signaling pathways and these experiments validated the

presence and activation of these cascades in skeletal myoblasts. BMPs signal through

serine/threonine kinase receptors (Massague et al., 1994). In the presence of growth factors,

ligands bind to a Type II receptor dimer located on the plasma membrane, which causes auto-

phosphorylation of the Type II dimer and recruitment and transphosphorylation of a Type I

receptor dimer (Wrana et al., 1992; Attisano et al., 1993; Ebner et al., 1993; Wieser et al., 1993;

Wrana et al., 1994). This phosphorylation event recruits the receptor-regulated Smads (R-

Smads), which then undergo phosphorylation (Aoki et al., 2001). The R-Smads form a complex









with the common-partner Smad (Co-Smad), Smad4. The R-Smad/Smad4 complex translocates

into the nucleus and binds to DNA causing activation of target genes (Derynck et al., 1996; Liu

et al., 1996; Meersseman et al. 1997; Nakao et al., 1997). Co-activators and co-repressors lend

additional regulation to the system (Wotton et al., 1999). BMPs signal through Smadsl/5/8 and

the TGF~s typically signal through Smads2/3. BMP-activated Smads, most importantly Smad5,

are necessary for inhibition of myogenic differentiation and osteoblastic induction in C2C12

cells (Lee et al., 2000). Assessment of functional Smad 1/5/8 activation was observed in the

absence and presence of TGFP1 or BMP6 treatment by immunostaining for phospho-Smadl/5/8

expression. Untreated C2C12 myofibers demonstrated low levels of phospho-Smadl/5/8 protein

expression, which were increased in response to both 10 ng/ml TGFP1 and 25 ng/ml BMP6

observed by Western analyses. Additionally, nuclear phospho-Smadl/5/8 protein expression

was induced in response to 100 ng/ml BMP6 and 25 ng/ml TGF~lby immunoflourescence.

Therefore, induction of phospho-Smadl/5/8 protein expression in response to BMP6 or TGFPI

treatment demonstrates functional Smadl/5/8 activation in C2C12 myofibers and this induction

of phospho-Smadl/5/8 is not due to an increase in total Smadl/5/8 protein expression.

Since both BMP6 and TGFP1 induce functional Smadl/5/8 activation in myofibers, it may

be argued that similar downstream effects could result from either ligand treatment. While

BMP6 induces dramatic transdifferentiation of myoblasts, this same effect is not observed in

response to TGP1 treatment. Previous reports have demonstrated that TGFP1 and BMP6 ligand

bind to different Type II receptors and induce different target genes. For example, transfection

of Smad1 and Smad5 into C2C12 myoblasts and NIH3T3 fibroblasts resulted in decreased

myogenin promoter activity in both lines but ALP activity induction was only observed in

C2C12 myoblasts and not in NIH3T3 fibroblasts. These results demonstrated that these