<%BANNER%>

Regulation of the Steroidogenic Acute Regulatory Protein (StAR) by cAMP and Transforming Growth Factor-Beta (TGF-Beta) D...


PAGE 1

R E E G G U U L L A A T T I I O O N N O O F F T T H H E E S S T T E E R R O O I I D D O O G G E E N N I I C C A A C C U U T T E E R R E E G G U U L L A A T T O O R R Y Y P P R R O O T T E E I IN (StAR) B B Y Y cAMP A A N N D D T T R R A A N N S S F F O O R R M M I I N N G G GROWTH FACTOR-BETA (TGF-BETA) D E E P P E E N N D D E E N N T T P P A A T T H H W W A A Y Y S S By REBECCA JANNET KOCERHA 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 2005

PAGE 2

Copyright 2005 by Rebecca Jannet Kocerha

PAGE 3

iii ACKNOWLEDGMENTS There are many people I would like to th ank, but without my advisor, Dr. Nancy, Denslow, I would not have had the opport unity to work on such an exciting and interesting PhD project. I am so grateful and appreciative to Nancy for many things, including her scientific creativity, mentoring skills, and friendship, none of which I will forget. I would also like to thank my committee, Dr. Flanegan, Dr. Purich, Dr. Bungert, and Dr. James. Collectively, they really help ed me to understand what it takes to have a successful project and their advice is something I will take with me throughout the rest of my career. I feel honored to have had such a talented group of scientists on my committee. The entire Denslow laboratory, Protein Co re, Education Core, and Hybridoma Core made my PhD experience such a pleasure. Ke vin Kroll taught me so much about fish and helped immensely to make the ovarian follic le cultures a success. Alfred Chung was integral in the development of the LMB St AR antibody by synthesizing the peptides. Dr. Stan Stephens and Dr. Andy Ottens were so helpful with the mass spec analysis. Dr. Tara Sabo-Attwood was always there for me to bounce ideas through many lengthy conversations and I feel very fortunate to ha ve her as a very good friend. I would also like to thank the newest member of the la b and my friend, Mindy Prucha, for doing such a great job in continuing the StAR project and for all the enthusiasm and energy she

PAGE 4

iv brings to the lab. Jason Blum, Natalia Reye ro, Iris Knoebl, and Nicole Mullally were also helpful resources throughout my time in the Denslow lab. Last, but of course not leas t, I want to sincerely tha nk my parents and family for their endless love and support. There truly ar e no words to describe the love, gratitude, and appreciation that I have for them and always will.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABBREVIATIONS...........................................................................................................xi ABSTRACT......................................................................................................................x ii CHAPTER 1 INTRODUCTION........................................................................................................1 Literature Review.........................................................................................................1 Steroidogenic Acute Regulatory Protein (StAR)..................................................1 Identification of the StAR protein..................................................................1 Mode of action...............................................................................................2 Protein-protein interactions with StAR..........................................................4 Mutations and associated diseases.................................................................5 Promoter.........................................................................................................6 Endogenous regulators and signaling pathways.............................................9 Regulation by environmental contaminants.................................................11 Endocrine Disruption...........................................................................................11 Largemouth Bass as a Model..............................................................................13 Ovarian Follicles.................................................................................................15 Cholesterol and Steroidogenesis..........................................................................16 -Sitosterol..........................................................................................................17 Transforming Growth Factor-Beta (TGF).......................................................19 Research Objectives....................................................................................................21 2 MATERIALS AND METHODS...............................................................................27 Animals.......................................................................................................................2 7 LMB StAR mRNA Expression..................................................................................27 Cloning of StAR..................................................................................................27 Development of LMB Real-Time PCR Assay for mRNA Quantitation.............31

PAGE 6

vi Seasonal Study.....................................................................................................33 LMB Ovarian Tissue Cultures.............................................................................34 LMB Ovarian Follicle Cultures...........................................................................34 LMB StAR Protein Quantitation................................................................................35 Protein Expression Vector...................................................................................35 Bacterial Protein Induction..................................................................................36 Protein Purification..............................................................................................38 Development of StAR Antibody.........................................................................38 Western Blots......................................................................................................39 Transcriptional Regulation of LMB StAR.................................................................40 Cloning of the Promoter......................................................................................40 Promoter Analysis...............................................................................................43 LMB SF-1 Cloning..............................................................................................43 Promoter Deletion...............................................................................................43 Mutagenesis of Putative Transcri ption Factor Binding Sites..............................44 Culturing of Y-1 Cells.........................................................................................45 Transfection Assays.............................................................................................45 GFP Quantitation.................................................................................................46 Luciferase Measurements....................................................................................46 Mouse StAR Real-Time PCR Assay...................................................................47 Statistics..................................................................................................................... .47 3 REGULATION OF STAR IN LARGEMOUTH BASS OVARIAN FOLLICLE CULTURES................................................................................................................61 Introduction.................................................................................................................61 Results........................................................................................................................ .63 Cloning of StAR Protein.....................................................................................63 Seasonal Expression............................................................................................63 Regulation of StAR mRNA Expre ssion in LMB Ovarian Cultures....................64 cAMP Induction of LMB StAR..........................................................................64 -sitosterol Exposures.........................................................................................65 TGFExposures.................................................................................................65 Antibody Development.......................................................................................66 Western Blot Detection of E ndogenous LMB StAR Protein..............................67 Discussion...................................................................................................................68 4 TRANSCRIPTIONAL REGULATION OF THE LMB STAR PROMOTER..........87 Introduction.................................................................................................................87 Results........................................................................................................................ .89 Cloning of the StAR Promoter............................................................................89 Identification of the Tran scriptional Start Site....................................................90 Identifying Transcriptional Response Elements..................................................90 Cloning of LMB Steroidogeni c Factor -1 (SF-1)................................................91 Optimization of Transfection Assays..................................................................91 dbcAMP Exposures.............................................................................................92

PAGE 7

vii Promoter Deletion Experiments..........................................................................93 Site-Directed Mutagenesis Experiments.............................................................93 TGFRegulation of Y-1 Cell Endogenous Mouse StAR mRNA.....................95 Discussion...................................................................................................................95 5 CONCLUSIONS AND FUTURE DIRECTIONS...................................................114 REFERENCES................................................................................................................119 BIOGRAPHICAL SKETCH...........................................................................................130

PAGE 8

viii LIST OF TABLES Table page 2-1. Primers for 5 and 3 RACE.....................................................................................48 2-2. Thermocycler conditions for 5 and 3 RACE.........................................................49 2-3. Primers used to fix nucleotide mist akes in full length StAR cDNA sequence........50 2-4. Thermocycler conditions for LMB StAR promoter cloning....................................51 2-5. Thermocycler conditions for cloning of LMB SF-1................................................52 2-6. Primers for promoter mutagenesis...........................................................................53 2-7. Thermocycler conditions for promoter mutagenesis with QuikChange-XL protocol.....................................................................................................................54

PAGE 9

ix LIST OF FIGURES Figure page 1-1. Alignment of mammalian StAR promoters.............................................................22 1-2. Fish ovarian follicle..................................................................................................23 1-3. General pathway for steroidogenesis.......................................................................24 1-4. Structures of -sitosterol and cholesterol. .............................................................25 1-5. TGFsignaling pathway. ......................................................................................26 2-1. Rapid amplification of cDNA ends (RACE). ..........................................................55 2-2. Sample standard curve for real-time PCR................................................................56 2-3. Map of pET-28b vector............................................................................................57 2-4. Location of peptide used for antibod y development is indicated by a green box. 58 2-5. Promoter cloning......................................................................................................59 2-6. Map of pGL3 basic vector (Promega)......................................................................60 3-1. PCR amplification of LMB StAR............................................................................72 3-2. Alignment of LMB StAR cDNA with other species................................................73 3-3. Seasonal expression of LMB StAR..........................................................................75 3-4. Dose response of LMB ovar ian tissue cultures to dbcAMP....................................76 3-5. cAMP induction of ovarian follicles........................................................................77 3-6. Dose response exposure of ovarian follicles to TGF............................................78 3-7. PCR amplification of entire LMB StAR cDNA.......................................................79 3-8. Bacterial expression of LMB StAR.........................................................................80

PAGE 10

x 3-9. Purification of StAR.................................................................................................81 3-10. Identification of bacterially expresse d StAR with Q-STAR mass spectrometry.....82 3-11. Identification of bacterially expr essed StAR by LCQ mass spectrometry...............83 3-12. ELISA with LMB StAR anti-ser um and purified StAR protein..............................84 3-13. Western blot detection with LMB St AR antibody and purified StAR protein........85 3-14. Western blot detection of StAR in dbcAMP exposed LMB tissue cultures............86 4-1. Digested LMB ovarian genomic DNA for promoter cloning................................100 4-2. Cloning of LMB StAR promoter...........................................................................101 4-3. 5 and 3 untranslated region (UTR ) for LMB StAR and identification of transcription start site.............................................................................................102 4-4. Putative transcription re sponse elements identified in the LMB StAR promoter..103 4-5. Cloning of LMB SF-1............................................................................................104 4-6. Optimization of ratio for transfectio n reagent (Fugene6) and promoter DNA concentration..........................................................................................................105 4-7. Optimization of transfection timepoint..................................................................106 4-8. Quantitation of DNA transtection with GFP. 107 4-9. Dose response exposure of Y-1 cells to dbcAMP..................................................108 4-10. Creation of promoter deletion a nd site-mutagenesis constructs.............................109 4-11. Promoter deletion analysis.....................................................................................110 4-12. Exposures of promoter site-mut agenesis constructs to dbcAMP...........................111 4-13. StAR promoter mutation analysis with TGFregulation.....................................112 4-14. Endogenous mRNA regulation of StAR in Y-1 cells by TGF............................113

PAGE 11

xi ABBREVIATIONS 3-B-HSD = 3-beta-hydr oxy-steroid-dehydrogenase ACTH = adrenal corticotrophic hormone AhR = aryl hydrocarbon receptor bp = base pair cAMP = cyclic adenosine monophosphate COUP-TF = chicken ovalbumin upstream promoter transcription factor CRE = cAMP response element CREBP = cAMP response element binding protein DAX-1 = dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 DHP = 17alpha, 20B-hydroxy-4-pregnen-3-one ELISA = enzyme linked immunosorbent assay EMSA = electromobility shift assay ERE = estrogen response element GFP = green fluorescence protein GnRH = gonadotropin releasing hormone Kb = kilobase LCAH = lipoid congenital adrenal hyperplasia LH = luteinizing hormone LMB = Largemouth Bass MALDI = matrix assisted laser desorption ionization PBR = Peripheral Benzodiazepine Receptor PCR = polymerase chain reaction PKA = protein kinase A PKC = protein kinase C RACE = rapid amplification of cDNA ends RAR = retinoic acid receptor RARE = retinoic acid response element ROR = retinoic acid related receptor SBP = StAR binding protein SF-1 = steroidogenic factor 1 StAR = Steroidogenic Acut e Regulatory Protein TGF= transforming growth factor beta

PAGE 12

xii 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 R R E E G G U U L L A A T T I I O O N N O O F F T T H H E E S S T T E E R R O O I I D D O O G G E E N N I I C C A A C C U U T T E E R R E E G G U U L L A A T T O O R RY Y P P R R O O T T E E I I N N (StAR) BY c A A M M P P A A N N D D T T R R A A N N S S F F O O R R M M I I N N G G G G R R O O W W T T H H F F A A C C T T O O R R (TGF-BETA) DEPENDENT PATHWAYS By Rebecca Jannet Kocerha August 2005 Chair: Nancy Denslow Major Department: Biochemistry and Molecular Biology StAR is the rate-limiting step in steroi d production and is tr anscriptionally down regulated by toxin exposure. StAR trans ports cholesterol acr oss the mitochondrial membrane for metabolism into steroids. We cloned the entire coding region of largemouth bass (LMB) StAR and used this sequence to develop a real-time PCR assay to quantify StAR mRNA leve ls in LMB ovarian follicle cultures. Exposure to dbcAMP and TGF-beta, two potent signaling mol ecules known to regulate mammalian steroidogenesis, modulate LMB StAR. TGF-beta down regulates and dbcAMP upregulates StAR mRNA. A polyclonal antibody specific to LMB StAR was developed to measure protein levels by western blot. To further analyze the regulation of LMB StAR, a 3 kb portion of the promoter was cloned. In silico analysis of this segment with other StAR promoters available in the data base showed potential conserved regulatory sites that imply control by a wide range of transcription factors. The 3 kb promoter

PAGE 13

xiii segment was transfected into Y-1 cells, a m ouse adrenalcortical cell line and tested with dbcAMP and TGF beta. The 3 kb construct responded positively to dbcAMP but was not significantly impacted by TGF-B exposure co mpared to the 1.8 kb length promoter. Mutation of potential regulatory sites in th e promoter, including ER E (estrogen response elements), ROR (retinoic acid related recep tor), and COUP-TF (chicken ovalbumin upstream promoter) sites were tested for thei r role in cAMP and TG F-beta signaling. Together, these data suggest th at one way toxins may repress steroid synthesis, and more specifically StAR, is thr ough TGF-beta signaling.

PAGE 14

1 CHAPTER 1 INTRODUCTION The Steroidogenic Acute Regulatory Protei n (StAR Protein) is the rate-limiting step in steroidogenesis and can be re gulated by endogenous and exogenous agents, including environmental toxins (Walsh et al ., 2000). Determining the regulation of StAR is imperative since homeostasis of steroid pr oduction is vital for various cell signaling and metabolic pathways. The aims of this project are to exam ine both the normal and atypical regulation of StAR in largemouth ba ss, with a specific focus on delineating the signaling cascades of cAMP and TGF, two potent molecules that can control steroid production. Literature Review Steroidogenic Acute Regulatory Protein (StAR) Extensive studies in mammalian models have confirmed StAR transports cholesterol across the mitoc hondrial membrane. After tr ansport, cholesterol is metabolized by the side chain cleavage enzyme to pregnenolone and then ultimately to steroid hormones (Stocco and Clark, 1996). Identification of the StAR protein Researchers had known for many years that de novo protein synthesis was required for steroid synthesis; however, the identity of the protein involved in facilitating the transport of cholesterol across the mitochondria eluded them until 1995. A 30 kDa protein in rat adrenal cells stimulated by ACTH, now known as StAR, was first observed when the [35S] methionine labeled proteins were electrophoresed through a 2D gel.

PAGE 15

2 Cycloheximide, a protein synthesis inhib itor, blocked steroi d production without impairing the activity of the side chain cleavag e or the delivery of cholesterol to the outer mitochondrial membrane (Stocco and Clark, 1997). The 30 kDa protein was purified from ACTH stimulated MA-10 mouse leyd ig cells using detergent solubilization followed by separation of the proteins by 1D a nd 2D gels. Bands at the 30 kDA location were excised and digested with trypsin fo r microsequence analysis. Degenerate oligonucleotides for PCR amplification of the 30 kDa protein were designed based on the microsequencing results to obtain a 400-base pair partial product. The full coding sequence for StAR from MA-10 cells was then screened from a cDNA library using the 400-base pair product as a probe (Clark et al., 1994). Since th e original identification in adrenal and leydig cells, StAR has also b een located in the brain, kidney, and heart (Young et al., 2001; Pezzi et al., 2003). Mode of action StAR is a mitochondrial pr otein synthesized as a 37 kD a precursor protein in the cytosol of mammalian cells (Clark et al., 1995) Upon stimulation, th e 37 kDa precursor is targeted via its signal sequence to the mitochondria. As the precursor protein is imported into the mitochondrial inner compar tment, the proteins signal sequence is removed by a matrix processing protease and co ntact sites are formed between the outer and inner mitochondrial membranes. The precu rsor protein is furt her processed by the mitochondrial intermediate processing peptide to remove the targeting sequence, forming the mature 30 kDa protein (Stocco and Clar k, 1997). The cytoplasmic 37 kDa protein has a half-life of around 10-15 minutes; how ever, the 30 kDa inactive protein has a longer half-life of up to several hours (Christenson and Strauss, 2000).

PAGE 16

3 Two current models suggest StAR trans ports cholesterol by either acting on the outside of the mitochondria as a molten gl obule (a protein with extensive secondary structure but disorganized tertiary struct ure, potentially allo wing for hydrophobic amino acids to be exposed) or as an intermem brane shuttle. FRET (flouresence resonance energy transfer) data indicate that StAR unde rgoes a conformational change to a molten globule once it interacts with the outer mitochondrial membrane, enabling it to bind cholesterol (Christenson et al., 2001). Additionally, recombinant mammalian StAR protein lacking the first 62 amino acids is localized to the surface of the outer mitochondria and is prevented from entering th e intermembrane space. It remains active and steroid production continues, suggesting that StAR functi ons by binding to the outer membrane (Bose et al., 1999). The second, but increasingly dubious, m odel shows StAR acting primarily to shuttle cholesterol between the outer and inner mitochondria membranes, across the intermembrane space (Mathieu et al., 2002). This theory was fueled by data showing contact sites between the two mitochondr ial membranes when cholesterol is bound (Thomson, 1998). It is during the formation of these contact sites that cholesterol is thought to be transported across the mito chondrial membranes to be metabolized. Cholesterol is specifically encompas sed or bound by the START (StAR-related lipid-transfer) domain located towards the Cterminus of StAR. The START domain contains about 210 amino acids with a hydrophob ic core where cholesterol binds (Strauss et al., 2003). Crystal structure of the ST ART domain from the MLN64 protein, another lipid transporter protein, suggest s there would have to be a conformational change in the hydrophobic core of StAR for a molecule of c holesterol to enter a nd bind under natively

PAGE 17

4 folded conditions (Tsujishita and Hurle y, 2000.) Furthermore, although the START crystal structure shows room for one molecule of cholesterol, liga nd binding assays with recombinant StAR and increasing titrations of fluorescent cholesterol from 5-100 nM gave a sigmoidal-shaped binding curve (Pet rescu et al., 2001). The binding studies suggest there are two cholesterol binding site s, necessitating a conf ormational change of StAR for sterol binding and transfer, furt her negating the shuttle model. Studies show that StAR prot ein can be degraded by prot easomes. In the presence of a proteasome inhibitor, MG132, there is accu mulation of the cytosolic, 37 kDa form of StAR in human or rat granulosa cells (Taj ima et al., 2001). There is even evidence suggesting that StAR could be subjected to degradation by different proteases in a biphasic manner, ensuring removal of residual protein that escaped the initial protease (Granot et al., 2003). COS cells chased for 15 minutes with 35S methionine prior to treatment with MG132 showed th at degradation of StAR wa s prevented for the first 2 hours. The protein, however, began degrad ing after two hours of MG132 treatment, suggesting that StAR is subjected to degr adation by multiple proteases (Granot et al., 2003). Protein-protein interactions with StAR Studies have indicated there are a couple of putative protein interacting partners for StAR. FRET studies showed that PBR (peripheral-type benzodiazepine receptor) associates with StAR at the mitochondrial membrane. A PBR-StAR association would, in theory, make sense because PBR is necessa ry for cholesterol transport (West et al., 2001). Immunoprecipitation experiments showed that StAR also interacts, in vivo with HSL (hormone-sensitive lipase). HSL medi ates the availability of unesterified

PAGE 18

5 cholesterol for steroid synthesi s (Shen et al., 2003). Rats we re injected with ACTH to induce StAR expression and anti-HSL antibodi es were used to immunoprecipitate HSL from the adrenal glands. The HSL-immunopr ecipitate complexes were separated on an SDS-PAGE gel and the presence of StAR in the complex was detected with anti-StAR antibodies. Additionally, a recent study used a yeast two-hybrid system to identify another protein that binds StAR, wh ich they named SBP (StAR bi nding Protein). Binding of StAR to SBP results in increased steroidogene sis (Sugawara et al., 2003). A recombinant form of StAR (N-62 StAR) lacking the firs t 62 amino acids in which the mitochondrial import signals are located was used as the ba it for the yeast assay. Interaction of SBP with N-62 StAR suggests the binding occu rs in the cytoplasm or at the outer mitochondrial membrane. Northern blot an alysis suggests that SBP may be found in several tissues, including the gonads, liver lung, and kidney. It is possible that identifying protein interacting pa rtners will help to elucidate the mode of action for StAR mediated cholesterol transport. Mutations and associated diseases StAR knockout mice and humans with LCAH (lipoid congenital adrenal hyperplasia) disease exhibit similar symptoms of severely repressed steroid synthesis (Hasegawa et al., 2000). Charact eristic of LCAH patients is the presence of large adrenal glands with high levels of cholesterol or chol esterol esters. Death can result in infancy if patients are not treated with hormone repl acement. A series of various nucleotide insertions and deletions in StAR DNA are attributed to LCAH and these can vary among the afflicted individuals. Th is disease just emphasizes th e importance of StAR (Stocco and Clark, 1996).

PAGE 19

6 Promoter Characterizing the response elements in the StAR promoter is important for determining specific proteins and protein-DNA interactions used to regulate its transcriptional activity. The promoter fo r StAR has been sequenced for several mammalian species, including human, rat, mous e, pig, sheep and cow (Sugawara et al., 1997; Reinhart et al., 1999; Ru st et al., 1998). In these sy stems, the StAR promoter contains a TATA box and several recognizab le response elements for transcription factors. Some of the most prevalent response elements identified in mammalian species bind C/EBPs, GATA-4, SF-1, DAX-1, AP-1, and Ah R (Manna et al., 2004; Sugawara et al., 2001; Sandhoff and McLean, 1999) and are conserved across species (Figure 1-1). Interestingly, although StAR is a cAMP-depe ndent regulated gene, a perfect consensus site for CRE (cAMP response element) has no t been recognized in any of the StAR promoters sequenced. However, studies have shown that when wild type CREB (cAMP response element binding protein) is transfected into seve ral mammalian cell lines, there is an increase in StAR prom oter activity and mRNA expressi on (Stocco et al., 2001). It is suggested that CREB may play a role in the absence of a consensus CRE by binding indirectly to a non-consensus site It is also possible that CREB regulates an activator of StAR expression such as SF-1. One of the most abundant response elemen ts in the mammalian StAR promoter is SF-1 (steroidogenic factor-1), a known inducer of steroidogenic tran scriptional activity. SF-1 is an orphan nuclear receptor transcript ion factor that has been sequenced from many species, including mammalian and fish system s (Yaron et al., 2003). SF-1 also is called AD4-BP (adrenal 4-bi nding protein) and is the mammalian homolog of the Drosophila factor FTZ-F1. FTZ-F1 regulates transcription of the fushi tarazu homeobox

PAGE 20

7 gene in fly embryos. Four transcripts are encoded by the SF-1 gene, including ELP1, ELP2, ELP3, and SF-1, partly generated through alternate splicing (Naw ata et al., 1999). SF-1 is expressed in all st eroidogenic tissues such as the adrenal, gonads, and the placenta. SF-1 knockout mice implicate the im portance of SF-1 in cellular functions. The knockout mice lack adrenal glands and gonads which leads to lethal adrenocortical insufficiency (Hammer and Ingraham, 1999.) It is not exactly known how ligands lead to the activation of SF-1; however, there are a lot of studies showing post-translational modification of SF-1 occurs. SF-1 does have a consensus site for protein kina se A (PKA) phosphorylation (Bertherat, 1998). Further studies have shown that inhibition of mitogen activated protein kinase (MAPK) can decrease SF-1 responsive genes (Hammer and Ingraham, 1999). SF-1 binds as a monomer to its response element and has two zinc fingers which helps it bind to its consensus site (Ito et al ., 2000). Up to 6 SF-1 binding sites have been identified in the mammalian StAR promoter, with the nearest element typically located only about 40 base pairs away from the transc ription start site. The first two SF-1 sites closest to the star t site appear to be conserved across species (Reinhart et al., 1999). SF-1 sites have been found in the promoter of ar omatase for several fish species, including goldfish, medaka, and zebrafish (Callard et al., 2001; Tchouda kova et al., 2001). Aromatase is the final protein in the steroi dogenic pathway involved in the conversion of testosterone to estradiol (Calla rd, 2001). SF-1 sites in promot ers of genes in fish studied to date have the general consensus seque nce of PyCAAGGPyPyPur with the exception that zebrafish have a purine instead of a pyrim idine for the first nucleotide (Kazeto et al., 2001; Honda et al., 1993). It appears that one way in which SF-1 regulates StAR

PAGE 21

8 transcriptional activity is by interacting with other transcription factors, including C/EBP, AP-1, and SP1 (Reinhart et al., 1999; Shea-Eat on et al., 2002). Prom oter regulation can, therefore, be very complex and involve tr anscription factors ac ting individually or cooperatively to exert their actions. RAR and RXRs are part of the steroid-thyroid hormone receptor subfamily and each are encoded by three genes, , and RAR mediates activation by heterodimerizing with RXR and it is the he terodimer which binds RARE (retinoic acid response element) (Pfahl, 1993). Typically, the RARE has a direct repeat of an AGGTCA core motif which is separated by 2 or 5 nucleotides (Bastien and RochetteEgly, 2004). ROR (retinoic acid receptor-related orpha n receptor) is also a member of the nuclear hormone receptor superfamily. ROR can bind to the RORE (ROR response element) as a monomer or homodimer. If ROR binds as a monomer, it recognizes a 6 base pair A/T rich region followed by an AGGTCA motif. To bind as a homodimer, a direct repeat of the RORE separated by 2 nuc leotides is necessary (Boukhtouche et al., 2004). ROR has been well characterized to acti vate gene transcription in the absence of a ligand; however, a recent study shows that ch olesterol is an ROR ligand (Kallen, 2002). ROR alpha transcriptional activity is represse d in U20S osteosarcoma cells depleted of cholesterol with st atins, a family of drugs that inhi bit cholesterol synthesis (Boukhtouche et al., 2004). Although RORs are known to be involved in tissue development or differentiation like some other nuclear receptors, there is s till much to be known about the genes that it regulate s (Jarvis et al., 2002).

PAGE 22

9 To date, only DAX-1 (dosage-sensitive se x reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) is well doc umented to repress St AR transcription (Jo and Stocco, 2004). When Y-1 mouse adrenal cell s were co-transfected with 2 Kb of the rat StAR promoter and a vector encodi ng DAX-1, basal and 1mM dbcAMP stimulated luciferase activity were repressed by at least 40% (Sandhoff and McLean, 1999). Although the exact mechanism is still being elucidated, it has been shown in mammalian systems that DAX-1 binds to a hairpin struct ure in the promoter rather than at a consensus site (Stocco et al., 2001). COUP-TF (chicken ovalbumin upstream pr omoter-transcription factor) is also known to downregulate steroidogene sis; however, its role in the regulation of StAR is just beginning to be investig ated (Buholzer et al., 2005; Shib ata et al., 2004). A recent study showed that mRNA induction of St AR by angiotensin II was completely suppressed by overexpression of COUP-TF in bovine adrenal glomerulasa cells (Buholzer et al., 2005). COUP-TF are nucl ear orphan receptors that can form homodimers and bind to response elements w ith variations of an AGGTCA core motif, which includes the RARE (Tran et al., 1992). COUP-TF can also si lence the activity of other transcription factors like RXR by hetero dimerizing with them and thereby limiting their availability for other binding partners (Berrodin et al., 1992). COUP-TF can also interact synergistically with corepressors like N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor) (Shibata et al., 1997). Endogenous regulators and signaling pathways StAR has been shown to be upregulat ed by cAMP, forskolin, GnRH, ACTH, and cholesterol containing lipoprot eins, including both LDL a nd HDL (Clark and Stocco,

PAGE 23

10 1996; Reyland et al., 2000). The signaling ca scade triggered by cAMP and other related inducers is complex and encompasses many di fferent proteins and crosstalk between various pathways. It has been confirmed that cAMP modulat es StAR through a PKA (protein kinase A) mediated event. PKA activation can regulate StAR directly and in directly. Indirectly, PKA can induce transcriptional activation by phosphorylating SF-1, a tr anscription factor commonly known to regulate StAR (Aesoy et al., 2002). There are multiple SF-1 sites throughout the mammalian StAR promot er (Sandhoff et al., 1998). Additionally, there are PKA sites with in the protein sequence of StAR. In vitro studies in mammalian cells showed specifically that mutation of a serine in a protein kinase A (PKA) site at amino acid positi on 195 resulted in 40% less steroidogenic capacity (Arakane et al., 1997). Studies with a normal and PKA mutant cell line showed that phosphorylation of StAR mo st likely stabilizes the protei n and therefore results in a dose response increase to dbcAMP as seen by western blot (Cla rk et al., 2001). [35S]methionine incorporation into StAR was shown in the PKA mutant cell line (Kin-8), suggesting that PKA can act post-translationally. Less is known about endogenous downregulat ors of StAR. Some studies have shown PGF-2 (prostag landin factor), TGF(transforming growth factor beta), and glucocorticoids can repress StAR transcri ption (Sandhoff and McL ean, 1999; Brand et al., 2000; Huang and Shirley, 2001). When de xamethasone, a glucocorticoid, was added to follicles cultured from LH (luteinizing hormone) treated rats, StAR activity was impaired but side chain cleavage was not (H uang and Shirley, 2001). This is potentially

PAGE 24

11 one example of how steroids can regulate th emselves and that StAR may be subject to feedback inhibition by downstream steroids. Regulation by environmental contaminants Several recent studies have shown that StAR expression can be downregulated by environmental contaminants, in cluding phthalates, which are pl asticizers (Shultz et al., 2001; Barlow et al., 2003), the pesticide Roundu p, the herbicide Lindane (Walsh et al., 2000), and the insecticide Dimethoate (Walsh et al., 2000). Studies have shown that these toxins could impact transcription or post-transcription activity. Experiments using MA-10, mouse leydig cells, showed Lindane and Dimethoate inhibit both mRNA and protein expression while not repressing overa ll protein synthesi s or inhibiting PKA activity (Walsh et al., 2000). Also, both mi croarray and real-time PCR analysis showed di-butyl phthalate to repre ss StAR in fetal rat testes, further suggesting a negative regulation of transcription by environmental co ntaminants (Shultz et al., 2001; Barlow et al., 2003). The regulation of StAR by paper mill contaminants, which is known to cause repression of steroid levels, ha s not been investigated (McM aster et al., 1996). Several studies have linked the altered steroid levels in fish to -sitosterol (Lehtinin et al., 1999; MacLatchy et al., 1995). Inte restingly, StAR can transport -sitosterol as efficiently as cholesterol under in vitro conditions (Kallen et al., 1998) It is currently unknown how these contaminants could specifically regula te the promoter, even though several of the toxins listed above decrea sed StAR mRNA expression. Endocrine Disruption There is substantial evidence that humans and wildlife exposed to chemicals in the environment can exhibit alterations in st eroidogenic capacity, leading to changes in

PAGE 25

12 secondary sex characteristics, gonad weight and production and size of eggs (McMaster et al., 1995; Sepulveda et al., 2001.). The consequences of steroid imbalance are so significant and universal that the Environmental Protecti on Agency (EPA) Office of Research and Development decided to incorpor ate endocrine disruption as one of its top six research priorities. The EPA establis hed several long-term goals, which included providing a better understanding of the scie nce underlying the e ffects, exposure, assessment, and risk management of endocrine disruptors; determining the extent of the impact on humans, wildlife, and the envi ronment; and finally, supporting the EPAs screening and testing program. Environmental contaminants can imp act reproduction and steroidogenesis by mimicking the actions of endogenous androge ns or estrogens, or by affecting their synthesis or metabolism. Cellular signali ng pathways can converge to mediate the response to the endocrine disruptors, which includes endogenous signaling molecules like cAMP and TGFas well as critical transcription factors like SF-1, ER, COUP-TF, and RAR. Some environmental toxins that have been shown to impact steroidogenesis in fish include PCB (Spies and Rice, 1988), polyaroma tic hydrocarbons (PAH) (Spies and Rice, 1988), phthalates (Barlow et al., 2003), R oundup (Walsh et al., 2000), and paper mill effluents (McMaster et al., 1996), among othe rs. Extensive studies on several fish species exposed to paper mill toxins, includi ng the white sucker in Lake Superior and largemouth bass (LMB) in Florid a, show males have decreased testosterone and females have decreased estrogen levels (McMaste r et al., 1995; Sepul veda et al., 2001; MacLatchy and Van Der Kraak, 1995). Severa l of the compounds present in paper mill

PAGE 26

13 effluent have been identified, including resi n acids, dioxins, abietic acid, and phytosterols (MacLatchy and Van Der Kraak, 1995; McMaster et al., 1996; Sepulveda et al., 2001). While paper mill effluent is a complex mixture of chemicals, attention has been focused on -sitosterol. -sitosterol is a phytosterol released by paper mills into rivers and lakes. Althought published results suggest -sitosterol acts through the estrogen receptor (Gutendorf and Westendorf, 2001), other studies suggest -sitosterol affects the transport of cholesterol across the mitochondrial memb rane (MacLatchy and Van Der Kraak, 1995; MacLatchy et al., 1997). Studies sh ow that goldfish injected with -sitosterol exhibit decreased steroid levels a nd expression of the P450 side chain cleavage enzyme, an enzyme involved in the conversion of choles terol to pregnenolone (MacLatchy et al., 1997). However, when ova rian tissue cultures from -sitosterol exposed fish are treated with a membrane permeable form of chol esterol, steroid prod uction is recovered (MacLatchy et al., 1997). This study imp licates StAR as a site of regulation by sitosterol since it binds and transports chol esterol to the inner mitochondrial space for steroid production. Ovarian follicles were cultured from goldfish that were exposed in vivo to sitosterol to test whether th eir steroidogenic ability was im paired by the toxic treatment (MacLatchy and Van Der Kraak, 1995.) Culturing follicles has also been used to study mammalian StAR function (Huang and Shirley, 2001) and it is a very relevant and physiological assay to study regulation of steroi dogenic proteins for many different types of model species. Largemouth Bass as a Model Fish are often surrounded by many environm ental contaminants in the water, therefore, are a good model system to study the effects of toxins on reproduction in

PAGE 27

14 vertebrates. Valuable information on the sp ecific mode of cellu lar regulation by these toxins can be gathered. In particular, LMB exposed to chemical contaminants discharged from the Palatka paper mill into the St Johns River have already been studied extensively and have been shown to e xhibit decreased steroid levels of 17estradiol (E2) and 11-ketotestosterone as well as othe r reproductive anomalies (Sepulveda et al., 2001; Sepulveda et al., 2003). Additionally, the gonads were smaller in weight and underdeveloped for fish closer to the source of contamination (Sepulveda et al., 2001). LMB can bioaccumulate the environmental toxins in their tissues and organs since they survive on a diet of other fish, crabs, frogs, snakes, mice, turtles, and birds ( http://www.go4bass.com/largemouth.html .) LMB are particularly useful for studying exposure to environmental contaminants be cause they are a freshwater species found across the country. Their reproductive cycle extends for se veral months, usually from November through April, which allows for an extended period for experiments. Because LMB are annual spawners, their reproductive cycles are fairly synchronous making controlled experiments more feasible. Some fish, such as zebrafish, spawn several times in a year and do not have synchronized cycles. Our laboratory has preliminary data indicating LMB exhibit seasonal changes in E2 and testosterone. Steroid levels were correlated with the corresponding stage of ovarian follicle ma turation during a one year time span. LMB can release up to 100,000 eggs from the matured ovarian follicles per year ( http://www.go4bass.com/largemouth.html .)

PAGE 28

15 Ovarian Follicles Ovarian follicles provide an essential sour ce of steroid biosynthesis in females. The term follicle refers to the oocyte surr ounded by an internal gr anulosa cell layer and external thecal cell layer, wh ich contains fibroblasts, coll agen fibers, and thecal cells (Figure 1-2). The thecal and granulosa layers are separated by a basal lamina (Babin, 1986) and both cell types are able to produce ster oids. The thecal cells, in fish, are what primarily have been found to form testoste rone since biochemical and ultrastructural studies show these cells contain 3-be ta-hydroxy-steroid -dehydrogenase (3-HSD) (Kusakabe et al., 2003). 3-HSD is an enzyme invol ved in the conversion of pregnenolone to progesterone, and progesterone can then be metabo lized to testosterone. Granulosa cells contain the P450 aromatase enzyme, which converts testosterone to E2 (Nagahama et al., 1995). It ha s been shown in fish that synthesis of testosterone by thecal cells increases during vitello genesis (Nagahama et al., 1995). Vitellogenesis is defined as the hepatic synthesis and secretion of vitellogenin (VTG), an egg yolk precursor, followed by uptake of VTG into the oocyte from the bloodstream by receptor-mediated endocytosis. After endocytosis, VTG is cleaved by specific cathepsins to form yolk proteins (W allace and Selman, 1990). The production of VTG is stimulated by E2 (Skipper and Hamilton, 1977). Ovarian follicles go through several stages of devel opment which involves many complex processes. At the most immature stage, oocyte cell cycles are arrested at prophase of meiosis and are called primordial follicles (Wallace and Selman, 1990). The phase during which the oocyte grows and gr anulosa cells prolif erate is called the primary-follicle stage. During this stage, the theca cells begin to differentiate and continued follicle development becomes re liant on gonadotrophins, with FSH (follicle

PAGE 29

16 stimulating hormone, also called GTHI in fish ) levels being very elevated (Kagawa et al., 2003). Upon completion of follicle growth, LH (l uteinizing hormone, also called GTHII in fish) levels rise for the final maturation and ovulation of the oocyte. Gonadotropin stimulates the production of a steroid, DHP (17 20-dihydroxy-4-pregnen-3-one), which is involved in the maturation by bindi ng directly to a recep tor on the oocyte (Pang and Ge, 2002). DHP is made in the granulosa cells from 17 -hydroxyprogesterone, which is made in the thecal cells, showi ng the interconnection between the two cells. Following maturation, the oocyte is released and is either fertilized or undergoes atresia, a process shown to involve apoptosis (Manabe et al., 2004). Completely developed ovarian follicles in LMB are about 1.4 to 1.5 mm in diameter, but that diameter range can vary across species. Ovarian follicles have been cultured fr om various species, including mammals and fish, to study the regulation of ster oidogenic enzymes by endogenous and exogenous substances (Petrino and Shuetz, 1986; Babi n 1986). Follicle diameter, stage of maturation, and quantity of cultured follicles are all parameters that can be selected in this assay Another benefit of using cu ltured ovarian follicles is that the contact between granulosa and thecal cells remains int act, which more closely resembles an in vivo system (figure1-2). It has been show n that there is increased ster oidogenesis in co-cultures of granulosa and theca cells than when either cell type is cultured alone (Shores et al., 2000). Cholesterol and Steroidogenesis Cholesterol is the b ackbone for steroid hormones a nd can be derived exogenously from dietary sources or endogenously synthe sized in the small intestine or liver.

PAGE 30

17 Typically, hepatic or intestin al cholesterol production supplies 2 to 3 times the amount that is absorbed from food (Lu et al., 2001) In addition to playing a vital role in metabolic homeostasis, cholesterol is a major component of the plasma membrane, helping to ensure the integrity of cellular stru cture. That same cholesterol can also be extracted from the membranes for steroi d production (Pichler and Riezman, 2004). Multiple fates exist for cholesterol after being synthesized or absorbed, but it is primarily packaged into lipoproteins for in tracellular transport. Lipoproteins are categorized as chylomicrons, LDL (low de nsity lipoprotein), VLDL (very low density lipoprotein), and HDL (high densit y lipoprotein) based on their ro les in sterol transport Chylomicrons shuttle dietary c holesterol from the small intestine to the peripheral tissues; VLDL and LDL transfer endogenou sly derived sterols from th e liver to tissues. HDL provides one of the only ways to clear chol esterol from the body by returning cholesterol from the tissues back to the liver (Ginsberg, 1998). The liver metabolizes cholesterol into bile acids that can either be further broke n down by microorganisms in the large intestine and excreted in urine, or re-used by the body to aid in fat digestion. There are a couple of proteins which are cr ucial to maintaining appropriate levels of cholesterol. HMG-CoA reductase is the rate-limiting enzyme in cholesterol biosynthesis and is therefore a target of many pharmaceuticals to alleviate symptoms of high blood pressure, atherosclerosis, as well as other diseases (Rosanoff and Seelig, 2004). The steroidogenic acute regulatory protei n (StAR Protein) is the rate-limiting step in cholesterol metabolism and it is just begi nning to be investig ated (Figure1-3). -Sitosterol -sitosterol is a phytosterol that shares stru ctural similarity with cholesterol (Figure 1-4). It is discharged from paper mills into waterways upon processing of paper

PAGE 31

18 products. -sitosterol makes up about 65% of the phytosterols present in paper mill effluent. The other phytosterols include cam pesterol, stigmasterol, and sitostanol (MacLatchy and Van Der Kraak, 1995; McMaster et al., 1996; Sepulveda et al., 2001). Unlike cholesterol, there is no endogenous production of the phytosterols in vertebrates and they can only be obtained through di etary sources (Salen et al., 1970). -sitosterol is metabolized to pregnenol one and steroid hormones; however, studies suggest -sitosterol metabolism is less efficient than cholesterol (B ennett et al., 1969; Aringer et al., 1979; Werbin et al., 1960). Absorbed phytosterols circulate in lipoprotein particles but the rates of absorption for the different phytos terols vary. There is evidence that phytosterols may accumulate in steroidogenic tissue, including the ovary, testis, and adrenal gland of animals (Moghadasian and Frohlich, 1999). Absorption into steroidogenic tissues suggests th at phytosterols, like cholesterol, can serve as precursors to steroid hormone synthesis. -sitosterol is used clinically to lower chol esterol. Studies have shown that plasma cholesterol levels are significantly lowere d by 10 days in humans fed a diet of 20 g/g body weight of phytosterols (Jones et al., 1998) Although only about 5% of phytosterols are absorbed, studies have shown that -sitosterol competes with cholesterol for uptake into bile acid micelles (C ompassi et al., 1997). In cel ls incubated with micelles containing either -sitosterol, choles terol, or both, -sitosterol was shown to decrease the movement of cholesterol from the plasma memb rane into the cell (Fie ld et al., 1997). It is thought that -sitosterol displaces cholesterol from the bile acid micelles. Bile acids, metabolites of cholesterol, are necessary for c holesterol absorption (Sirtori et al., 1991). If cholesterol is displaced from the bile ac id micelles by sitosterol, the absorbability of

PAGE 32

19 cholesterol by cells is diminished (Field et al., 1997). Phytosterols lower overall plasma cholesterol levels by inhib iting intestinal cholesterol absorption or by preventing recirculation of bile acids. Although -sitosterol is used to treat patient s with high cholesterol levels, high plasma concentrations of phytosterols in animals may have deleterious effects on reproductive organs. Rats injected with 0.5 to 5 mg/kg body weight per day of sitosterol exhibited a diminished sperm count and testes weight (Moghadasian and Frohlich, 1999). In addition studies have s hown that steroid levels are decreased in goldfish when given a singl e injection of 5, 10, or 100 g/g -sitosterol (MacLatchy and Van der Kraak, 1995). Pregnenolone, a metabo lite in the conversion of cholesterol to steroid hormones, is also decreased with -sitosterol exposure. Transforming Growth Factor-Beta (TGF) Although the exact mechanism of how -sitosterol represses st eroidogenesis is still unknown, it has been shown that -sitosterol upregulates TGFin prostate cells (Kassen et al., 2000). There is also evidence that TGFmay be regulated by dioxins, also found in the environmental (Dohr et al., 1994), s uggesting a possible signa ling pathway used by toxins to repress st eroidogenesis. TGF, through SMAD proteins, has even been shown to modulate the arylhydrocarbon receptor, a tran scription factor whic h is regulated by dioxins and for which a response element has been found in the human StAR promoter (Wolff et al., 2001). TGFis a powerful cytokine involved in cell signaling and has been shown to inhibit steroidogenesis in adre nocortical, trophoblast, and testic ular cells (Liakos et al, 2003; Luo et al., 2002; Gautier et al., 1997). Se veral recent studies have even shown the

PAGE 33

20 StAR Protein to be a major target for TGFregulation. Transfecti on studies in a human adrenocortical cell line, H295R, showed that 1.3 Kb of the human StAR promoter is inhibited by about 25% when cells are treated with 1ng/ml TGFfor 24 hours (Brand et al., 1998, 2000). The authors then tested seve ral deletions of the 1.3 Kb promoter with TGFand showed that all th e deletions were downregul ated except the 0.085 Kb construct. The study suggests the regulation of StAR by TGFis mediated by elements located between 0.085 Kb and 0.15 Kb upstream of the transcriptional start site in the mammalian promoter. It was also shown th at mutating the various SF-1 sites did not alleviate the downregulation of the promoter, however, no other transcription factors were examined. There is substantial research outlining th e specific signaling cascades that TGFtriggers. Two serine/threoni ne receptor tyrosine kinase s, Type I and Type II, are assembled and dimerized when TGFor other related ligands are bound. The dimerization leads to Type I receptor activation by phosphoryl ation in a glycine/serine rich domain. The active receptor can then phosphorylate and activate SMAD proteins (Figure 1-5). SMAD proteins are the essential link in TGFsignaling (de Caestecker, 2004). Research has specifically show n SMAD3 involvement in TGFinhibition of human StAR transcriptional activity in adreno cortical cells. Over expression of a wildtype SMAD3 protein in the cells pote ntiated the inhibito ry action of TGFon StAR mRNA levels, whereas, expression of a mu tant SMAD3 alleviated some of that repression (Brand et al., 1998). SMADS en compass a large and diverse family of proteins that can either activate or repre ss gene expression. A primary way in which

PAGE 34

21 TGFsignaling can be terminated is by ubi quitinylation of SMADS in the nucleus, which targets the proteins for proteosome -mediated degradation (Lee et al., 2003). Research Objectives The goals of this project were to exam ine cellular signaling mechanisms involved in regulation of LMB StAR by envi ronmental toxins. cAMP and TGFare two potent signaling molecules known to regulate steroidog enesis in mammalian species, therefore, their role in LMB StAR regulation was examin ed. The overall hypothesis of this project was that LMB StAR transcription and post-tr anslation activity are upregulated by cAMP and downregulated by TGF. The project was divided into three specific aims to meet the overall objective. Specific aim 1 was cloning the LMB StAR cDNA and development of a specific polyclonal antibody. Specific aim 2 was to deve lop LMB ovarian follicle cultures. The follicle assays were used to measure changes in mRNA by real-time PCR and protein by western blot detection with the anti-S tAR antibody upon exposure to cAMP or TGF. Specific aim 3 was to comprehensively exam ine the transcriptional regulation of LMB StAR. For aim 3, the promoter was clone d and used in transfection assays in conjunction with promoter deletion and s ite-directed mutagenesis experiments for functional transcrip tional response element analysis.

PAGE 35

22 Figure 1-1. Alignment of mammalian StAR prom oters. Line-up of the first hundred base pairs for the sheep, pig, human, and rat s howed conservation of several critical transcriptional binding sites, in cluding SF-1, GATA, and C/EBP.

PAGE 36

23 Figure 1-2. Fish ovarian follicle. The ovary of a fish is composed of many individual follicles, where steroids are produced by the theca and granulosa cells, located below the epithelial membrane. Vitellogenin, made in the liver, is the egg yolk precursor for the growing follicle.

PAGE 37

24 Figure 1-3. General pathway fo r steroidogenesis. Choleste rol is the backbone for all steroid hormones. Cholesterol is me tabolized in the mitochondria to pregnenolone. The various steroids are then formed from pregnenolone.

PAGE 38

25 Figure 1-4. Structures of -sitosterol and cholesterol. -sitosterol is the plant equivalent of cholesterol with the only structural difference being an additional ethyl group (outlined in red) for -sitosterol.

PAGE 39

26 Figure 1-5.TGFsignaling pathway. Binding of TGFto the Type II receptor kinase induces dimerization of the Type II a nd Type I receptor. A cascade of phosphorylation and signaling events mediated by SMAD proteins occurs following the receptor dimerization, ultim ately activating transcription of many different genes.

PAGE 40

27 CHAPTER 2 MATERIALS AND METHODS The overall goals of this project were to characterize the expression of the largemouth bass (LMB) Steroidogenic Acute Regulatory Protein (StAR) and then specifically examine its regula tion at the transcriptional le vel. Cloning the LMB StAR coding region and a portion of the promoter re gion were the initial steps in achieving the goals of this project. Th e sequence information was used for development of assays, including real-time PCR, tran sfections, and western blots, to quantitate changes in LMB StAR transcription and transl ation. StAR expression was examined in response to various endogenous and exogenous chemicals, including cAMP and transforming growth factor beta (TGF), central molecules involved in cell signaling pathways. Animals Largemouth bass ( Micropterus Salmoides ) were used for all tissue and ovarian follicle cultures and were purchased from American Sport Fish Hatchery (Montgomery, AL). All fish were housed at the Center for Environmental a nd Human Toxicology at University of Florida in accordance with th e National Institute for Health (NIH) Guide for the Care and Use of Laboratory Animals. LMB StAR mRNA Expression Cloning of StAR Total RNA was isolated from larg emouth bass ovarian tissue using the RNeasy Kit with spin columns (Qiagen). The quality of RNA was verified by examining 3-5 g on a formaldehyde based gel and looking for the presence of two bands, with the 28S rRNA

PAGE 41

28 band being twice the intensity of the 18S rRNA band. 3 g of RNA was reverse transcribed into cDNA with 200 units of Supe rscriptII enzyme (Invitrogen). Basically, the reverse transcription reaction involved heating the RNA and 150 ng random hexamers at 70 C for 10 minutes. A master mix of 10 mM DTT, 0.2 mM dNTP mix (stock has 2.5 mM each nucleotide), and 5X buffer [ 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 ] was diluted to 1X in a 50 l final reaction volume. After adding the master mix, the reaction was heated at 42 C for 2 minutes. The reaction was then stopped to add 200 units of SuperscriptII which was then followed by continued heating at 42 C for 50 minutes. A final extension at 70 C for 15 minutes completed the reaction. LMB StAR was PCR amplified from the ovarian cDNA using partially degenerate primers designed with the web program CODEHOP ( http://blocks.fhcrc.org/codehop.html ) and an alignment of va rious mammalian sequences in the database, including human, pig, horse, and cow. Sequences for the forward and reverse primers were 5TGGA GCAGATGGGCGANTGGAAYCC3 and 5TTGATGATGGTCTTGGGCADCCANCCYTT3, respectively. The cDNA was amplified using 10 picomoles (pmol) of each primer, 1 Unit of amplitaq, and 1.5 mM MgCl2, with 10X PCR buffer [500 mM KCl and 100 mM Tris-HCl (pH 8.3)] diluted to 1X in a 20 l final volume. The PCR reacti ons used a primer annealing temperature of 60.9 C for 45 cycles in a Perkin Elmer 9600 model thermocycler. The PCR reaction was run out on a 1% agarose ge l and the predicted 350 base pair band was gel purified by excising the ethidium bromide stained bands from two 20 l loaded lanes under low UV light and then using the gel purificati on kit and protocol from Qiagen.

PAGE 42

29 The gel purified DNA was ligated into a pGEM-T cloning vector. The amount of DNA to ligate into the vector was calculated by multiplying 50 ng of vector by the size of the DNA to be inserted and then dividing th at number by the size of the cloning vector, which was 3 Kb for pGEM-T. The concentration of the gel purified product was quantified using a DNA mass ladder. A molar ratio of both 1:1 and 3:1 for insert to vector was used. Ligation was done at 16 C overnight with a reac tion containing the gel purified product (see calculation above) 3 units T4 DNA ligase (Promega), 50 ng pGEM-T vector (Promega), and 2X ligation buffer (Promega-proprietary) diluted to 1X in a final volume of 10 l. The ligation product (3 l) was transformed into 50 l of DH5 cells (Invitrogen). Basically, the transformation involved incuba ting the cells and lig ation product on ice for 20 minutes, then heat shocking the cells for 45 seconds at 42 C followed by immediate cooling on ice for 2 minutes. The cells were then shaken in 950 l of LB broth for 1 hours at 230 rpm before being plated on ampicillin (100 g/l) LB/agar plates for overnight incubation at 37 C. Colonies were screened for ligation by PCR with M13 primers. Positive clones were minipreped us ing the Qiagen spin column kit and sent for sequencing at the University of Fl orida ICBR DNA Sequencing Core. RACE (rapid amplification of cDNA ends) were used to sequence the remaining coding regions of LMB StAR using the SMART RACE protocol from Clontech/BD Biosciences (Figure 2-1). Briefly, first-strand cDNA for 5 RACE was prepared by reverse transcribing 1 g of total RNA is olated from LMB ovary using 1 l of Powerscript Reverse Transcriptase (Clontech, proprietary), 1 l of 5 RACE primer (proprietary concentration; 5 (T)25VN3), 1 l SMART oligo

PAGE 43

30 (5AAGCAGTGGTATCAACGCAGAGTACGCGGG-3 ), 1.3 mM DTT, and 2 l of 5X First Strand Buffer (250 mM Tris-H Cl, pH 8.3; 375 mM KCl; 30 mM MgCl2) in a final volume of 15 l for a 1.5 hour incubation at 42 C. The reverse tran scriptase reaches the 5 end of the mRNA and adds several dC re sidues, which serves as a template on the opposite strand for an oligo that contains 3 G residues (Smart II oligo) at its 3 end. To make cDNA for 3 RACE, the same prot ocol was followed for that of 5RACE, however, the following primer [5AAGC AGTGGTATCAACGCAGAGTAC(T)30VN3] in proprietary concentrations was used and the SMART oligo was not added to the reaction mix. The 5RACE and 3 RACE cDNA that wa s synthesized was then used in PCR reactions with gene specific primers and prim ers to the Smart II oligo. The primers and thermocycler conditions used for 5 and 3 RA CE are listed in Table 2-1 and Table 2-2. All gene specific primers, designed using the Oligo program, were between 23 and 28 nucleotides in length, 50-70% GC content, a nd a melting temperature of greater than 65 C. Nested gene specific primers were also designed to help eliminate non-specific PCR amplification. All PCR reac tions contained 1 l of 50X Advantage 2 Polymerase Mix (Invitrogen) and 0.2 mM dNTP stock mix in a final volume of 50 l. Initial PCR reactions were diluted 1:50 in Tricine-EDTA (10 mM Tricine-KOH-pH 8.5; 1 mM EDTA) and 1 l of the diluted DNA was used for nested/secondary PCR amplifications. For 3RACE, a gene specific primer and primer to the poly A adaptor oligo (Invitrogen) that was attached during revers e transcription was used for PCR amplification. 5RACE amplification used a primer (Invitrogen) to the 5cDNA adaptor oligo and gene specific primers. Products were cloned into the pGEM -T vector and plated onto LB/AMP agar

PAGE 44

31 plates as described in the above section. All sequence information was verified by at least 3 different clones. Development of LMB Real-Time P CR Assay for mRNA Quantitation Quantitation of LMB StAR mRNA levels in ovarian tissue and follicle cultures was done by real-time PCR using Taqman technol ogy. RNA for all Taqman reactions was extracted from about 5 mg (10 follicles) of bass ovarian follicles using RNA STAT-60 reagent (Tel-TEST). The ovarian follicles were homogenized in 500 l of RNA STAT60 using a polytron followed by addition of 150 l of chloroform. Af ter centrifugation at 12,000 x g for 15 minutes, the upper aqueous RNA containing layer was removed for an additional RNA STAT-60/chloroform extraction. The RNA was then precipitated with 250 l of 100% isopropanol overnight at -20 C and then pelleted by a 30 minute centrifugation at 12,000 x g at 4 C. The RNA pellets were washed twice with 75% ethanol (made with DEPC treated water) and then resuspended in 25 l of RNAsecure, a reagent that helps minimize RNase activity (Ambion). All of the RNA (all samples contained less than 10 g total RNA) was trea ted with 2 units of DNA-Free (Ambion) for an hour to remove traces of contaminati ng chromosomal DNA. Quality of RNA was checked on an agarose gel for the presen ce of 28S and 18S ribosomal bands. 3 g of each RNA sample was reverse-tr anscribed into cDNA using 25 units of Stratascript enzyme (Stratagene), 1500 ng random primers, 1mM of each dNTP (4 mM total dNTP), along with 10X Stratascript buffer (Stratagene) diluted to be 1X in a final reaction volume of 25 l. In short, RNA, random primers, and water were added first, then all tubes were heated at 65 C for 5 minutes followed by cooling at room temperature. Then, 4 l of a master mix of dNTPs, buffer, and Stratascript enzyme was

PAGE 45

32 added to each reaction followed by incubati on at 42 C for 1 hour. The enzyme was inactivated by heating the reacti ons at 90 C for 5 minutes. The primers for real-time PCR using Taqman technology were designed using the Primer Express program (Applied Biosystems ) based on the original sequence obtained for LMB StAR. All PCR reactions used 10 pmoles of the forward primer, 5ACCCCTCTGCTCAGGCATTT 3, and 10 pmoles of the reverse primer, 5GGGCTCCACCTGCTTCTTG3, to amplify 0.12 g of reverse-transcribed RNA using universal thermocycler parameters as recommended by Applied Biosystems. For the real-time PCR assays, 2X Sybr Green r eagent (Applied Biosystems) was used at a concentration of 1X in the final reaction vol ume. Sybr Green is a fluorescent dye that binds to double stranded DNA. Therefore, as more amplification occurs, more fluorescence is detected. A dissociation cu rve was run with the LMB StAR Taqman primers to check for amplification specificity after the PCR cycles are completed (Figure 2-2). A dissociation curve is generated by raising the temperat ure and obtaining a specific melting point for separation of double stranded amplified StAR DNA, which is represented by a steep loss of fluorescence signal. A standard curve for real-time PCR quantitation was developed with known amounts of the StAR plasmid. Standard cu rves typically were performed in 10X dilutions from 8.1 X 106 to 8.1 X 101 copy numbers of plasmid and samples with unknown amounts of StAR were quantitated by ex trapolation to the st andard curve. A sample standard curve and amp lification curve is shown in Figure 2-2. The slope for the standard curve should be around -3.3, which indicates perfect doubling per cycle.

PAGE 46

33 All Taqman reactions were normalized to 18S rRNA using universal thermocycler conditions as recommended by Applied Biosys tems. Each 25 l reaction contained 1.25 l of 18S rRNA master mix containing pr oprietary amounts of primers and probes (Applied Biosystems, catalog #4310893E), 0.12 g of reverse transcribed RNA, and 2X Taqman universal primer mix (Applied Biosystems, catalog # 4304437) diluted down to 1X in the final reaction volume. All real-time PCR calculations were base d on converting the c oncentration of the the StAR plasmid used in standard curve to copy number. The conversion to copy number was done since the concentration of the plasmid DNA includes the cloning vector plus StAR DNA, which would not accurately reflect the concentr ations for StAR. Converting to copy number ensures that the pl asmid is considered as one unit, including both the vector and insert, and amplification is therefore a reflection of that unit. All quantities from real-time PCR were measured as copy number of plasmids amplified. The calculation for determining amount of c opy number used to develop the standard curve is : ul copies / (g/mol) plasmid the of weight molecular (g/ul) ion concentrat plasmid X l) (copies/mo 10 X 623 Seasonal Study Adult LMB between 2 and 3 years old were maintained in freshwater ponds at the USGS facility in Gainesville, FL. Female fish were caught by electroshock bi-weekly over a five month time span. Ovarian tissue wa s carefully removed a nd immediately flash frozen with liquid nitrogen for l ong-term storage at -80 C.

PAGE 47

34 LMB Ovarian Tissue Cultures Ovarian tissue cultures from LMB were cu ltured to detect changes in mRNA levels after exposure to various chemicals. The ova rian tissue was carefully dissected into 2030 mg pieces, rinsed with culture media, a nd immediately placed in 1 ml of Dulbeccos Modifed Eagles Medium Nu trient Mixture (DMEM) w ith F-12 Ham containing LGlutamine and 15mM HEPES (Sigma) supplemented with 1.2 grams of sodium bicarbonate and 1% antibiotic/antimycotic so lution (ABAM). This media has the same osmolality as LMB plasma, about 295 mOsmol/kg (Bowman thesis, 2001). Cultures were equilibrated in a chilled incubator at 21-22 C w ith 5% CO2 for 24 hours prior to exposure with appropriate chemical to be studi ed. All experiments were carried out in 24 well culture plates, which were placed on a sl ow moving shaker during the exposures. LMB Ovarian Follicle Cultures Ovarian follicles were isolated from the ovary and cultured for further examination of LMB StAR mRNA expression. Follicles we re individually disse cted and measured with a micrometer to control size and stag e of follicles incubated. Follicles were incubated in 500 l of the DMEM culture me dia and equilibrated for 24 hours prior to exposure. Ten follicles were incubated per we ll of a 24 well cell culture plate. Follicles and culture were removed from the culture plate post exposure by using a BSA (bovine serum albumin) coated wide boar 1 ml pipet tip and placed in a 1.5 ml microcentrifuge tube. Follicles were gently pelleted to the bottom of the tube by centrifugation for 5 minutes at less than 3000 RPM. Follicles were washed once with 1X PBS (phosphate buffered saline) and then frozen at -80 C until RNA isolation. The viability of follicles was tested by adding 10% Alamar Blue reagent (Biosource) to the cultures and looki ng for reduction of Resazurin (blue and

PAGE 48

35 nonfluorescent) to resorufin (p ink and highly fluorescent). Changes in the color from blue to pink for metabolically active cells can be detected with spectrophotometry readings at A570 and A600. LMB StAR Protein Quantitation Protein Expression Vector There is no commercial antibody available for StAR that cross reacts with any fish species, therefore, we develope d a polyclonal antibody for wester n blot detection. First, a protein expression vector was constructed by amplifying the entire coding region in one piece using 10 pmol of AT GCTACCTGCAACCTTCAAACTGTG as the forward primer and 10 pmol of TCAGCAGGCGTGAGCCATCTCCA TA as the reverse primer. The annealing temperature was 72 C for 7 cycles followed by 67 C for 42 cycles. The PCR reaction contained the primers 1 l of 10 mM dNTP mix, 2 l of 25 mM Mg(OAc)2, 0.15 g of LMB ovarian cDNA, 50X Advantage Genomic Polymerase Mix diluted to 1X [Clontech: 5-6 units/l Tth DNA polymerase, 0.5g/l TthStart an tibody, 50% glycerol, 10 mM Tris-HCl (pH 7.5), 230 mM KCl], a nd 10X PCR reaction buffer [400 mM TrisHCl (pH 9.3), 150 mM KOAc, 0.2% Triton X-100] diluted to 1X in a final volume of 50 l. The entire coding region was ligated into the pET-28b vector (Novagen) (Figure 23) and transformed into DH5 cells (Invitrogen) using simila r procedures as outlined in the section for cloning of StAR. Expressi on with the pET-28b vector produces proteins with a 6 histidine tag on to N-terminus to ultimately allow for purification with an appropriate affinity column. Amplification of the full length StAR result ed in 8 amino acid mistakes that were fixed using the QuikChange Kit (Stratagen e). Primers to change the appropriate

PAGE 49

36 nucleotides were designed using Stratage nes website program for site directed mutagenesis (Table 2-3). The primers were de signed to have the targeted mutation near the middle, a minimum of 40% GC content, an d a 3 end that terminates with one or more G or C bases. Complementary revers e primers were designed against each forward primer. The QuikChange protocol basica lly involved a PCR r eaction using 125 ng of each relevant forward and reverse primer, 38 ng of StAR plasmid template, 1 l of a proprietary dNTP mix, and 2.5 U/l of Pfu Turbo DNA pol ymerase in a 50 l final volume. Reactions were amplified in a thermocycler for 12 cycles of 95 C at 30 seconds, 55 C for 1 minute, and 68 C for 7 mi nutes. The parent st rand is digested by DpnI, leaving the corrected produc t to be transformed in DH5 cells. The transformed constructs were minipreped with the Qiag en miniprep spin kit and DNA was sent for sequencing to verify that the relevant nucleot ides were fixed. Since pET-28b is a lowcopy number plasmid, concentration yields fr om minipreps were maximized by using 3 ml of bacterial culture and by eluting the pur ified DNA from the spin column with 70 C water. The final, completely corrected construct was transformed into BL21 (DE3) cells (Novagen) for bacterial expression. Bacterial Protein Induction The LMB StAR expression construct was grown in 30 ml of LB broth with 24 g/ml kanamycin for 4 hours at 37 C with c onstant shaking at 280 rpm. The bacterial cultures reached a desired dens ity after the 4 hours with A600 spectrophotometry readings between 0.6 to 0.8. StAR protein expression was then induced in the cultures with 1 mM or 3 mM IPTG for an additional 4 hours with 1ml aliquots taken hourly. Bacterial cells were pelleted by spinning down the 1 ml of culture at 10,000 x g for 1 minute, discarding the supernatant. The pellet was resuspended in 100 l of 1X phosphate buffered saline

PAGE 50

37 (PBS) along with 1 l of benzonase to lesse n the viscosity. 100 l of 4X SDS buffer (250 mM Tris-HCl pH 6.8, 8% SDS, 10% 2-mercaptoethanol, 300 mM DTT, 40% glycerol, and 0.02% bromophenol blue) was then added to the protein sample and heated at 85 C for 3 minutes for denaturation. Th e denatured protein samples were run on a 412% Bis-Tris NuPAGE gel (Novex) with MES running buffer for 30 minutes. The gel was stained with Colloidal Coomassie Bl ue Stain (Genomic Solutions) overnight followed by de-staining for 2 hours. The bands on the protein expression gel that were induced by IPTG compared to the controls were excised and ultimately subjec ted to digestion with trypsin for definitive identification of largemouth bass StAR peptide fragments in the gel. The in-gel trypsin digestion protocol involved washing the gel pieces with 50% aceton itrile (ACN) 3 times while vortexing for 15 minutes followed by dehydration of the gel with 100% ACN until gel piece turns white. The gel was then rehydrated with 100 mM ammonium bicarbonate (ABC) for 5 minutes. The proteins in the gel piece were reduced with 45 mM DTT for 30 minutes at 55 C. DTT is a reducing ag ent that separates proteins which are linked by disulfide bonds for more effective analys is by mass spectrometry. To prevent the cysteine residues in the separa ted peptides from recombining, they are alkylated with 100 mM iodoacetatamide for 30 minutes in the dark at room temp. The gel piece was then washed for 15 minutes 3 times with 50% ACN/50 mM ABC while vortexing. The gel was completely dried in a speed vac prior to digestion with 12.5 ng/ l Trypsin (Promega) prepared in 50 mM ABC pH 8.4, 5 mM CaCl2 on ice for 45 minutes. The enzyme solution was then removed and replaced with just the buffer for in cubation overnight at 37 C prior to analysis by mass spectrometry.

PAGE 51

38 Protein Purification Purified LMB StAR was obtained by induc ing 10 ml of bact erial culture for 4 hours with the StAR expression plasmid (see section on bacterial protein induction for details). The bacteria were pelleted at 4000 x g for 15 minutes and stored at -80 C until ready for purification. A nickel affinity column (Ni-NTA spin column kit, Qiagen) was used to purify histidine-tagged StAR from the bacterial pell et under denatured condi tions. The bacterial cells were lysed by thawing the pellet for 15 minutes and then resuspending them in a buffer of: 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0. The cells were shaken for 1 hour at room temperature. Cellular debris was removed from the lysate by centrifuging at 10,000 X g for 20 minutes. Spin columns were equilibrated with 600 l of the pH 8 buffer followed by centrifugation at 700 x g for 2 minutes. Histidine tagged proteins were bound to the nickel affinity column by flowing 600 l of the bacterial lysate through the equilibrat ed column at 700 x g for 2 minutes. The columns were washed 3 times with 600 l of a buf fer containing: 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 6.3. Finally, the bound proteins were eluted from the spin column with 200 l of the following buffer: 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 4.5. The purified protein was qua ntitated by protein assay using Coomassie Plus Reagent. Two micrograms of the purified protein was run out on a 4-12% Bis-Tris NuPAGE gel (Novex) and stained overnight in Colloidal Coomassie Blue followed by a 2 hour de-stain. Development of StAR Antibody A polyclonal antibody was made against th e largemouth bass StAR Protein. The antibody was produced by injecting two differe nt rabbits (Cocalico company) with a

PAGE 52

39 synthetic peptide designed to a part of the coding sequence. The antigenic peptide used for rabbit injections, CFLAGMSTQHPKMPEQRG VVR (Figure 2-4), was constructed in an area of the coding region where prolines we re present so the antibodies are able to recognize relatively exposed areas of StAR. The peptide was conjugated to a carrier protein, KLH (keyhole limpet hemocyanin), at the University of Florida Protein Core to help ensure an immunogenic response to the StAR peptide. For the rabbit injections, the following schedule was followed; Day 0 = Prebleed/Initial Inoculation, Day 14 = Boost, Day 21 = Boost, Day 35 = Test Bleed, Day 49 = Boost, Day 56 = Test Bleed. Monthly boosts were subsequently continued for about year to obtain more antiserum. Western Blots The specificity and reactivity of the LMB StAR antibody was tested by western blot using the purified StAR protein as a posi tive control. 1g of purified protein was run on a 4-12% Bis-Tris NuPAGE gel followed by tr ansfer to nitrocellulose membrane at 100 volts for 1 hour. The transfer buffer cont ained 20 mM Tris, 144 mM glycine, and 20 % methanol. The nitrocellulose membrane was blocked for 3 hours, while shaking, with 5% Carnation non-fat dry milk diluted in 1X TBST (25 mM Tris, 0.15 M NaCl, 0.05 % Tween-20, pH 7.6) to help reduce non-specific antibody binding. The membranes were then washed 4 times, at 15 minut es each, with 1X TBST. Washed membranes were then exposed to the primary antibody for 3 hours with vigorous shaking. Dilutions of 1:5000 a nd 1: 15000 primary antibody were tested. Following the incubation, primary antibody was removed by washing 4 times with TBST before incubation with concentrations of 1:20000 and 1:40000 secondary antibody for one hour. The secondary antibody used was a mouse anti-rabbit IgG conjugated to

PAGE 53

40 horseradish peroxidase (Pierc e). Membranes were again thoroughly washed with TBST before antibody binding was detected using chemiluminescence after exposing for 3-4 minutes to 4 ml of each chemiluminescent reagent (Super Signal West Pico Chemiluminescence Kit, Pierce.) Presence of luminescence was captured on Kodak film. Transcriptional Regulation of LMB StAR Cloning of the Promoter Most transcriptional regulation occurs in the promoter, therefore, the first goal for this part of the project was to clone the promoter for LMB StAR using the GenomeWalker Kit (Clontech) (Figure 2-5). The promoter cloning started with isolating high quality genomic DNA that was phenol/c hloroform purified twice from 20 mg of LMB ovarian tissue using the Wizard Kit (Pro mega.). Basically, th e genomic isolation involved lysing the tissue with 600 l of a lysis solution (proprietary, Promega) for 20 minutes at 65 C. Contaminating RNA was degraded by incubation of the lysate for 20 minutes at 37 C with 3 l of an RNase so lution (proprietary, Promega). The solution was chilled on ice with 200 l of a Protein Precipitation Solution (proprietary) and the protein was removed by centrifuging at 14,000 X g for 4 minutes. The supernatant was put into a new tube. Genomic DNA was preci pitated from the remaining supernatant with 600 l of isopropanol and pelleted by centrifuging at 15000 X g for 1 minute. The pellet was washed with 70% ethanol and then air dried for 12 minutes. The pellet was resuspended in 100 l of TE buffer (10 mM Tris-HCl, pH7.3; 1 mM EDTA, pH 8.0) by incubating at 65 C for 1 hour. Residual pr otein was removed from the genomic DNA with a phenol/chloroform extraction followed again by resuspension in TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 7.5).

PAGE 54

41 Genomic DNA (2.5 g) was digested for 20 hours with 4 different restriction enzymes; StuI, EcoRV, DraI, and PvuII to create digested libraries for PCR amplification. Complete digestion of th e genomic DNA was checked by running 5 l on a 0.5% agarose gel stained with ethidium bromide at 70 volts for one hour. The restriction libraries were purif ied with 2 phenol/chloroform ex tractions. An adaptor oligo (Clontech) was ligated onto the various dige sted pieces of genomic DNA by incubating with 3 units of T4 DNA lig ase for 20 hours at 16 C. PCR amplification of the LMB StAR promot er from the restriction libraries was done using a primer to the ligated adaptor oligo and a gene specific primer. Two different sets of gene specific primers were us ed, one set of primers started closer to the 5end of the coding sequence than the other, which gave overlapping sequences and provided for some sequence verification. On e set of gene specific primers used was 5CAGGCAACATCTTACTCAGGACTTTGTC3 (p romoter jk1). It was followed by 5TCACCTTGCTTCACATAAGACATCT CT3 (promoter jk2) fo r the nested reaction. The second construct used 5TTCCACT CCCCCATTTGCTCCATATTT3 (promoter jk3) for the primary PCR reaction and 5CAGGCAACATCTTACTCAGGACTTTGTC3 (promoter jk1) for the nested reaction. All primers were designed using the Oligo Program and were between 26 and 30 nucleotides with a 40-60% GC content, with no more than 3 G and Cs in the last 6 bases of the 3 end. Nested gene specific primers were used to he lp reduce non-specific amplification. All secondary PCR reactions using the nested primers were done using a 1:50 dilution of the primary PCR product in a final volume of 50 l. Thermocycler conditions for the promoter cloning are listed in Table 2-4. PCR products were gel

PAGE 55

42 purified with spin columns and protocol from Qiagen and ligated into the pCR2.1-TOPO vector (Invitrogen) using a similar protoc ol as outlined under the section for StAR cloning. The sequence for the 2.9 Kb promoter fragment (named pSP1 plasmid) was verified with two independe nt PCR reactions. The promoter was cloned into the pGL3 basic vector (Promega) (Figure 2-6) for transfections. The cloning of the promoter i nvolved two phases, first, both the pGL3 and pSP1 plasmids were double digested with XhoI and HindIII promega enzymes and Buffer B (6 mM Tris-HCl, 6 mM MgCl2, 50 mM NaCl, 1 mM DTT, pH 7.5) for 13 hour digestion at 37 C. The XhoI /HindIII fragment vector wa s ligated into the cut pGL3 vector by protocol outlined under StAR cloning section and the plasmid was named pLUCStAR1. The pLUCStAR1 construct, however still had the transl ational start site from the LMB StAR sequence since the primers used for promoter cloning originated in the coding region. Removing the ATG start site from the plasmid was essential since the pGL3 plasmid contains its own ATG site for lu ciferase protein synthesis. The ATG start site was removed from the pLUCStAR 1 with a double digest of HindIII (Stratagene) and BlpI (NEB labs) in buffer #2 (NEB labs = 20 mM Tris-OAc, 50 mM KOAc, 10 mM Mg(OAc)2, 1 mM DTT; pH 7.90) @ 25C. All promoter constructs were obtained using endot oxinfree maxiprep kits (Qiagen). The maxiprep protocol basically involved growing up a single colony of the plasmid in DH5 cells in a 3 ml LB/Ampicillin (100 g/ml AMP) starter culture for 8 hours. The starter culture wa s then added to 100 ml of LB /Ampicillin (100 g/ml AMP) for overnight growth at 37 C. The pl asmid was then purified under endo-toxin free conditions from the DH5 cells by binding of the DNA to an anion-exchange resin. The

PAGE 56

43 plasmid was eluted from the resin unde r high salt conditions (1.6 M NaCl; 50 mM MOPS; 15% isopropanol). The DNA was then concentrated and rid of salt with isopropanol precipitation. All maxipreps were suspended with 2 ml of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). Promoter Analysis The transcription start site for the LM B StAR promoter was identified by sequencing to the end of the 5UTR (untra nslated region) using 5RACE. The 5UTR information was then matched up with genomic sequence to determine where the UTR ends and the promoter begins. Sequence upstr eam of the transcription start site was then analyzed with three different web search engines, MatInspect or V2.2, ProfessionalMatInspector V7.3, and TFSearch, to identi fy putative consensus binding sites. LMB SF-1 Cloning A 246 base pair portion of LMB SF-1 was cloned from ovarian tissue by PCR with a forward primer 5CCAACCGCACCAT CAAGTCNGARTAYCCNG 3 and reverse primer 5GAAGACCATGCAGCGG CKNGCCCANTC 3. The PCR reactions consisted of 10 pmol each primer, 1.5 mM MgCl2, 0.2 mM dNTP mix, 1 unit of amplitaq (Perkin Elmer/Applied Biosystems), 0.45 g cDNA, and 10X PCR buffer [500 mM KCl and 100 mM Tris-HCl (pH 8.3)] diluted to 1X in a 20 l final volume. PCR amplification conditions using a Perkin Elmer 9600 ther mocycler are listed in Table 2-5. Promoter Deletion One deletion of the 2.9 Kb LMB StAR pr omoter was made. The deletion was made via a double digest with EcoRV and BstEII. The EcoRV restric tion site is at the 5end of the promoter and the BstEII is a bout 1000 bp from the 5 end, leaving a 1.86 Kb promoter when digested. The digested promot er construct was analyzed on a 1% agarose

PAGE 57

44 gel and the bands corresponding to the proper deletion pieces were purified using spin columns from a Qiagen kit. Additiona lly, unlike EcoRV, BstEII is not a blunt-end cutting enzyme, which left incompatible ends requiring filling in the ends with Klenow enzyme (NEB labs). For the Klenow reacti ons, 1 unit of Klenow was incubated with 1g DNA and 0.2 l of a 10 mM dNTP mix in a fi nal volume of 20 l. The Klenow reactions were stopped immediately after 15 minutes by flowing the DNA through spin columns from a PCR purification kit (Qiagen). Th en, 60 ng of purified, digested DNA was religated with 2000 units of concentrated T4 DNA ligase (NEB labs) in a final volume of 20 l for 40 minutes at room temp. The ligation was then transformed into DH5 cells using protocol outlined under section for StAR cloning. All promoter constructs were verified by sequencing and rest riction digested with DraI. Mutagenesis of Putative Trans cription Factor Binding Sites Several transcription factor binding elements in the 2.9 Kb promoter were mutated to a NotI restriction site. These were constructed using the QuikChange XL SiteDirected Mutagenesis Kit (Str atagene.) The protocol combined 125 ng of each relevant forward and reverse primer (T able 2-5), 10 ng of the 2.9 Kb StAR promoter construct, 1 l of a proprietary dNTP mix, and 2.5 units of Pfu Turbo DNA polymerase in a final volume of 50 l. PCR amplification conditions for all QuikChange reactions are listed in Table 2-6 and Table 2-7. Following PCR amplif ication to create mutagenized promoter constructs, the parent, unmutage nized strands were digested w ith 10 units of DpnI at 37 C for 1 hour. 1 l of the DpnI digested PCR product was transformed into DH5 cells using the protocol outlined under the section for StAR cloning. The transformation was plated on LB/AMP agar plates (100 g/ml AM P). Several colonies were minipreped for

PAGE 58

45 each QuikChange reaction and were screened for creation of the desired mutation by digesting 2 g of DNA with NotI for 3 hours. Culturing of Y-1 Cells Y-1 mouse adrenal cells (passage 1) we re purchased from ATCC (American Type Culture Company). The cells were cultured in media containing: Ham's F12K medium with 2 mM L-glutamine supplemented with 1.5 g/L sodium bicarbonate; 15% horse serum; 2.5% FBS; and 1% penicillin-strepto mycin mix. The cells were grown in T-75 flasks at 37 C for normal propagation with a media change every other day to retain the endogenous steroidogenic activity (following th e recommendation from ATCC). After 4 days in culture, the cel ls typically reached about 70 to 80% confluency and were then split 1:3 after trypsinization. Each T-75 flask r eceived 2 ml of trypsin for 4 minutes at 37 C followed by inactivation of trypsin with 4 ml of media with serum. Lastly, 2 ml of the trypsinized cells where then added to one of the three pre-equilibr ated flasks with 13 ml of media. To help alleviate clumping, cells were gently pipeted up and down with a 2 ml glass pipet about 10 times. Transfection Assays Cells were trypsinized as outlined in th e section above on culturing of Y-1 cells, however, after inactivation of the trypsin by medi a with serum, the cells were pelleted at 1500 rpm for 5 minutes and resuspended with 10 ml of fresh media. Clumping of the cells was alleviated by pipeting the cells up and down with a 2 ml pipet and a brief 1 second vortex. Cells were counted with a hemacytometer and 150,000 cells/well were plated in 500 l of media with serum and allowed to attach and equilibrate for 24 hours. Cells received fresh media immediately prior to transfection. Tr ansfection reactions consisted of 0.1995 g of the appropriate St AR promoter construct, 0.0005 g of the

PAGE 59

46 control renilla luciferase vect or, and Fugene6, where the rati o of g of DNA to l of Fugene6 was 6:1 (1.2 l Fugene6/ 0.2 g to tal DNA), suspended in 20 l media with no serum or antibiotic. The transfection mixt ure sat for 30 minutes at room temperature before adding it dropwise to th e cells. All transfections were done in 24 well plates from Corning CoStar. Doses were prepared in the same manner for all experiments. dbcAMP doses were made fresh for each experiment by diluting th e appropriate amount of powder into cell culture media. For TGFexposures, stock solutions of 1 g/ml were kept aliquoted and frozen at -80 0C until appropriate dilutions were made fresh for each experiment. GFP Quantitation To measure the transfection efficienc y, 0.1995 g of GFP (pEGFP, Clontech) was transfected in place of the LMB StAR promot er DNA. Cells were transfected as normal and subsequently trypsinized for G FP quantitation on a hemacytometer. Luciferase Measurements After exposures were completed, cells we re immediately washed once with 1X PBS (phosphate buffered saline; 0.144 g/L KH2PO4, 9 g/L NaCl, 0.795 g/L Na2HPO4, pH 7.4) and then lysed with 100 l of 1X passi ve lysis buffer (Promega) for at least 15 minutes at room temperature. Luciferase measurements were done using reagents from the Dual Luciferase Kit (Promega). Firefly luciferase was measured first by adding 20 l of cell lysate to 100 l of reagent that contains substrate for th e firefly luciferase and luminescence was immediately measured by luminometer. Reni lla luminescence was then quantitated by adding 100 l of Stop and Glo reagent, which contains reagents to quench the firefly reaction and substrate for renilla luciferase.

PAGE 60

47 Mouse StAR Real-Time PCR Assay A real-time PCR assay was developed to measure endogenous levels of mouse StAR in the Y-1 cells. Total RNA was extract ed from the Y-1 cells by lysing with 500 l of RNASTAT (Tel-Test) and then following th e same protocol as outlined for LMB. Primer sequences for mouse StAR had previ ously been designed and published (Fielden et al., 2002); forward primer 5 TGCT AAGGATCGGGAACTGT 3; reverse primer 5 TCTGGCCTTTTACAGAGGAGA 3. The PCR reactions for the mouse StAR were set up in the same manner as the LMB StAR with the exception of having no standard curve, all quantitations were relative. The relative calculations for the mous e StAR real-time PCR were done by subtracting the 18S rRNA Ct value (cycle thresh old) from the StAR Ct values. Ct values represent the initial detection in the increase of fluoresence signal associated with an exponential increase of PCR product during the log-linear phase. An average of the normalized Ct values is obtained for each treatment and then the value for the experimental group is subtract ed from that of the control group, which leaves a log number that can be calculat ed into a fold change. Statistics Students T-test was used for evalua tion of significance between control and experimental groups. Results were reported as significant if P < 0.05.

PAGE 61

48 Table 2-1. Primers for 5 and 3 RACE. Primer name 5 RACE 3 RACE 5TTTTCGGGTGCTGAG TGGACATCCCAG3 (for 1st round of 5 RACE) Original primer 5TTCCACTCCCCCATT TGCTCCATATTT3 (for 2nd round of 5 RACE) 5ATCGGCCAAGACAC AATGGTTACC3 5CTTGGCCGATCTTTT GAAGGATCT3' Nested primer 5CAGGCAACATCTTAC TCAGGACTTTGTC3 5AGCGGAGAATGGAC CTACCTGTAT3

PAGE 62

49 Table 2-2. Thermocycler condi tions for 5 and 3 RACE. Original RACE Cycle Parameters Nested RACE Cycle Parameters Cycle 5X: 94 C / 5 seconds 72 C / 2:30 minutes Cycle 5X: 94 C / 5 seconds 70 C /10 seconds 72 C / 2:30 minutes Cycle 27X: 94 C /5 seconds 68 C / 10 seconds 72 C / 2:30 minutes Cycle 25X: 94 C / 5 seconds 68 C / 10 seconds 72 C / 2:30 minutes

PAGE 63

50 Table 2-3. Primers used to fix nucleotide mist akes in full length StAR cDNA sequence. QuickChange Reaction Forward Primer Reverse Primer 1 5CATATGAGGAACATGACA GGTTTGAGGAAGAATGCAA TG3 5CATTGCATTCTTCCTCAA ACCTGTCATGTTCCTCATAT G3 2 5GCCATCAGCATCCTCAGC GACCAGGA C3 5GTCCTGGTCGCTGAGGAT GCTGATGGC3 3 5AGTAACTGGATCAACCAA ACCCGAGGAAGAAGCTCCC TCCTCAG3 5CTGAGGAGGGAGCTTCTT CCTCGGGTTTGGTTGATCC AGTTACT3 4 5GCAGAGGGGTGTTGTCAG AGCGGAGAATG3 5CATTCTCCGCTCTGACAA CACCCCTCTGC3 5 5CTAAATATAGATCTAAAG GGCTGGATCCCAAAGACAA TCATAAAC3 5GTTTATGATTGTCTTTGG GATCCAGCCCTTTAGATCT ATATTTAG3 6 5AGTAACTGGATCAACCAA ACCCGAGGAAGAAGCTCCC TCCTCAG3 5CTGAGGAGGGAGCTTCTT CCTCGGGTTTGGTTGATCC AGTTACT3 7 5GGCCATCAGCATCCTTAG CGACCAGGACGG3 5CCGTCCTGGTCGCTAAGG ATGCTGATGGCC3 8 5GTGGACTTTGCCAACCAC CTCCGGCAAAGG3 5CCTTTGCCGGAGGTGGTT GGCAAAGTCCAC3

PAGE 64

51 Table 2-4. Thermocycler conditions for LMB StAR promoter cloning. Primary PCR Conditions: Secondary PCR Conditions: 7 cycles: 94 C / 2 seconds 72 C / 3 minutes 5 cycles: 94 C / 2 seconds 72 C / 3 minutes 37 cycles: 94 C / 2 seconds 67 C / 3 minutes 24 cycles: 94 C / 2 seconds 67 C / 3 minutes 67 C / 4 minutes 67 C / 4 minutes

PAGE 65

52 Table 2-5. Thermocycler conditions for cloning of LMB SF-1. Cycle Parameters Number of Cycles Temperature Time 1 95 C 2 minutes 95 C 30 seconds 61.8 C 25 seconds 40 72 C 45 seconds 1 72 C 10 minutes

PAGE 66

53 Table 2-6. Primers for promoter mutagenesis. Site Mutated Forward Primer Reverse Primer ERE/2678 5'ATAGCGCCTTTCTAGTCTTTGC GGCCGCTCAAAGCGCTTTTACAT G3' 5'CATGTAAAAGCGCTTTGAGCGG CCGCAAAGACTAGAAAGGCGCT AT3' COUP-TF/2027 5'GAATTGCAGTTTTCCCCATGGC GGCCGCTCATTAAAACCTGAAAC TGC3' 5'GCAGTTTCAGGTTTTAATGAGC GGCCGCCATGGGGAAAACTGCA ATTC3' ROR/1969 5'TAGGGAGCCATTTGAAATAGG CGGCCGCCCTACTTTGGCTCTTT GAAAAAAG3' 5'CTTTTTTCAAAGAGCCAAAGTA GGGCGGCCGCCTATTTCAAATGG CTCCCTA3' GATA/AP-1/ERE /1882 5'CTGTGGCTGAGTAATGCGGCCG CTAGTACTAGGCCTGTGT3' 5'ACACAGGCCTAGTACTAGCGG CCGCATTACTCAGCCACAG3' COUP-TF/2304 5'GGTGATATTTG CGAAGGAGCG GCCGCCACAAACGTCCTTTCCTG AA3' 5'TTCAGGAAAGG ACGTTTGTGGC GGCCGCTCCTTCGCAAATATCAC C3'

PAGE 67

54 Table 2-7. Thermocycler conditions for pr omoter mutagenesis with QuikChange-XL protocol. Cycles Temperature Time 1 95 C 1 minute 95 C 50 seconds 60 C 50 seconds 18 68 C 8 minutes 1 68 C 7 minutes

PAGE 68

55 Figure 2-1. Rapid amplification of cDNA ends (RACE). (A) 5 RACE protocol and (B) 3 RACE protocol to extend the cDNA sequence for LMB StAR. Both 5 and 3 RACE required primers specific to the original 345 bp sequence obtained for LMB StAR.

PAGE 69

56 Figure 2-2. Sample standard curve for real-tim e PCR. A) A standard curve is generated by plotting Ct (cycle thres hold) values versus the l og of the copy numbers for minipreps of the StAR plasmid done in 10X serial dilutions. Two replicates were done for each standard. Unknown samples are then extrapolated to the standard curve. B) Dissociation curve for LMB StAR to check for primer specificity. The dissociation of fluorescence from the amplified, doublestranded DNA was detected with a melting curve.

PAGE 70

57 Figure 2-3. Map of pET-28b vector.The full coding sequence for StAR was cloned into the pET-28b vector (Novagen) for His-tagged protein expression in BL21(DE3) bacterial cells.

PAGE 71

58 Figure 2-4. Location of peptide used for an tibody development is indicated by a green box. A 21 amino acid peptide was desi gned to a conserved region for LMB StAR. Two different rabbits were i mmunized with the peptide for production of StAR antibodies. 10 20 30 40 50 60 | | | | | | brook trout ML P ATFKLCAG I SYRH T RNM T GLR KN A M V A I H H ELN ML A -GP NP S S WI S H V R R R SSLL S rainbow trout ML P ATFKLCAG I SYRH M RNM T GLR KN A M V A I H H ELN ML A -GP NP S S WI S H V R R R SSLL S LMB ML P ATFKLCAG I SYRH M RNM T GLR KN A M V A I H H ELN RL A -GP GP S N WI N Q TR G R SSLL S zebrafish ML P ATFKLCAG I SYRH M RNM T GLR KN A M I A I H H ELN KL S -GP GA S T WI N H I R R R SSLL S pig ML L ATFKLCAG S SYRH V RNM K GLR HQ A V L A L G Q ELN RR A LG GP TS G S WI N Q V R R R SSLL G horse ML L ATFKLCAG S SYRH V RNM K GLR HQ A A L A I G Q ELN WR A PG GP TQ S GWI N Q V R R Q SSLL G human ML L ATFKLCAG S SYRH M RNM K GLR QQ A V M A I S Q ELN RR A LG GP TP S T WI N Q V R R R SSLL G ** ******** **** ***.***::* :*: :*** : ** **.: :****. Prim.cons. MLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNRLALGGPTPSSWINQVRRRSSLLS 70 80 90 100 110 120 | | | | | | brook trout S R I E E EQG Y N E A E V S Y VK QG EE A L Q KSI S IL G D Q D GW TT E IIAA NG DK V L SK VL P D V GKV rainbow trout S R I E E EQG Y N EA E V S Y VK QG EE A L Q KSI S IL G D Q D GW TT E IIAA NG DK V L SK VL P D V GKV LMB S R I E E EEG Y S D E E M S Y VK QG ED A L Q KAI S IL S D Q D GW TT E IVAA NG DK V L SK ML P D I GKV zebrafish S P I A E ETY S E A D Q C Y VQ QG QE A L Q KSI S IL E D QD GW QT E IESI NG EK V M SK VL P G I GKV pig S Q L E D TFY S D Q D L A Y IQ QG EE A M Q RAL D IL S N Q E GW KK E SRQE NG DE V L SK VI P D V GKV horse S Q L E D TLY S D Q E L A Y IQ QG EE A M Q KAL G IL R N Q E GW KE E NQQA NG DK V L SK VV P D V GKV human SR L E E TLY S D Q E L A Y LQ QG EE A M Q KAL G IL S N Q E GW KK E SQQD NG DK V M SK VV P D V GKV : : *.: : .*::**::*:*:::.** :*:** **::*:**::*.:*** Prim.cons. SRIEEE2GYSD2EL2YVQQGEEALQKAISILSDQDGW2TEI22ANGDKVLSKVLPDVGKV 130 140 150 160 170 180 | | | | | | brook trout F K LEV L LD Q RSDN LY G ELV G N ME Q MG D WNP N VK EV K I LQKIG QE T M V THE VS G P T P GN V V rainbow trout F K LEV L LD Q RSDN LY V ELV G N ME Q MG D WNP N VKEV K I LQKIG QE T M V THE VS G P T P GN V V LMB F K LEV M LE Q RPDN LY K ELV G N ME Q MG E WNP N VK QV K I LQKIG QD T M V THE VS A E T P GN V V zebrafish F K LEV T LE Q QTGD LY D ELV D N ME Q MG E WNP N VK QV K I LQKIG QE T M I THE IS A E T P GN V V pig F R LEV V VD Q PMER LY EELV E R ME A MG E WNP S VK KI K I LQKIG KD T V I THE LA A E A A GN L V horse F R LEV E VD Q PMER LY E ELV E R ME A MG E WNP N VK EI K V LQKIG KD T V I THE LA A E S A GN L V human F R LEV V VD Q PMER LY E ELV E R ME A MG E WNP N VK EI K V LQKIG KD T F I THE LA A E A A GN L V *:*** ::* ** *** .** **:***.**::*:*****::*.:***::. :.**:* Prim.cons. FKLEV2LDQ2M22LYEELV2NMEQMGEWNPNVKEVKILQKIGQDTMITHE2SAETPGNVV 190 200 210 220 230 240 | | | | | | brook trout GPRDFV S V RC A KRRGS T C F LAGM S T Q H PT MPEQ R G V V RAE N GPTC I V MR P S A D D P N KTK F rainbow trout GPRDFV S V RC A KRRGS T C F LAGM S T Q H PT MPEQ R G V V RAE N GPTC I V MR P S A D D P N KTK F LMB GPRDFV S V RC A KRRGS T C F LAGM S T Q H PK MPEQ R G V V RAE N GPTC IV MK P C V E D P N KTK F zebrafish GPRDFV N V RH A KRRGS T C F LAGM S T Q H PG MPEQ K G F V RAE N GPTC I V MR P S A D D P N KTK F pig GPRDFV S V GC T KRRGS V C V LAGM A T D F GE MPEQ K G V I RAE H GPTC M V LH P L A G S P S KTK L horse GPRDFV S V RC A KRRGS T C V LAGM A T Q F EE MPEQ KG V I RAE H GPTC M V LH P L A G S P S KTK L human GPRDFV S V RC A KRRGS T C V LAGM A T D F GN MPEQ K G V I RAE H GPTC M V LH P L A G S P S KTK L ******.* :*****.*.****:*:. ****:*.:***:****:*::* .*.***: Prim.cons. GPRDFVSVRCAKRRGSTCFLAGMSTQHP2MPEQKGVVRAENGPTCIVM2P2A2DPNKTKF 250 260 270 280 | | | | brook trout TWLL S I DLKGW I PK TI IN K VLSQTQVDF A NHLR Q R M A D NSVSKEMAPA C rainbow trout TWLL S I DLKGW I PK TI IN K VLSQTQVDF A NHLR Q R M A D NSVSMEMAAA C LMB TWLL N I DLKGW I PK TI IN K VLSQTQVDF A NHLR Q R M A N N-VSMEMAHA C zebrafish TWLL S L DLKGW I PK TV IN R VLSQTQVDF V NHLR D R M A S G-GGIDAAIA C pig TWLL S I DLKGW L PK TI IN Q VLSQTQVDF A NHLR K R L E S R---PALEAR C horse TWLL S I DLKGW L PK TI IN Q VLSQTQVDF A NHLR K R L E S S---PAPEAR C human TWLL S I DLKGW L PK SI IN Q VLSQTQVDF A NHLR K R L E SH---PASEAR C ****.:*****:**::**:*********.****.*: Prim.cons. TWLLSIDLKGWIPKTIIN2VLSQTQVDFANHLR2RMASNSVSP2MAAAC

PAGE 72

59 Figure 2-5. Promoter cloning. 3 Kb of the LM B StAR promoter was cloned using the Genomewalker protocol (C lontech). One round of PCR is done using GSP1 (gene specific primer) and a primer against the adaptor, AP1. A second round of PCR is done for added specificity us ing a nested gene specific primer, GSP2, and nested adapator primer, AP2.

PAGE 73

60 Figure 2-6. Map of pGL3 basic vector (Promega). The 2.9 kb StAR promoter was cloned into the pGL3-basic vector for transf ection experiments in Y-1 mouse adrenal cells. The promoter DNA was inserted into the MCS (multiple cloning site).

PAGE 74

61 CHAPTER 3 REGULATION OF STAR IN LARGEMOUTH BASS OVARIAN FOLLICLE CULTURES Introduction A major discovery was made in the ster oidogenic pathway within the past decade upon the identification of the Steroidogenic Ac ute Regulatory Protein (StAR Protein). It has now been well characterized in mammalian species that StAR transports cholesterol across the mitochondrial membrane and contro ls the rate-limiting step for steroidogenesis (Stocco and Clark, 1996). Humans with mutatio ns in StAR cant synthesize steroids from cholesterol, causing lethal accumula tion of lipids in the adrenal glands and ultimately cell death, underscoring the importa nce of proper StAR function (Khoury et al, 2004). It is clear, however, that steroidogenesis can be negatively impacted without the presence of mutations in StAR DNA, impli cating instead steps in regulation of StAR mRNA synthesis or protein expression. TGF, a signaling molecule, and toxins, such as phthalates, are known to repress mamma lian StAR transcription, however, a link, between the pathways they modulate has not be en fully established (Brand et al., 2000; Barlow et al., 2003). The steroid synthesis pathways of seve ral fish species, including LMB, white sucker, zebrafish, and goldfish, have been s hown in various cell culture systems to be targeted by toxins such as dioxins, pesticides, plasticizers, and paper mill effluents (Sepulveda et al., 2001; McMa ster et al., 1995; Carvan et al., 2000; MacLatchy and Van

PAGE 75

62 Der Kraak, 1995). The negative impact of these toxins on fish steroid production implores the examination of how they specifically regulate StAR. To date, there is minimal information about StAR in fish beyond the existence in the NCBI database of DNA sequences for a few lower vertebrate species, which now includes brook trout, rainbow trout, eel, cod, and zebrafish. There are 7 exons and 6 introns in the StAR gene which are conserved from mammals to fish (Goetz et al., 2004), however, the regulation of fish StAR has not been examined besides the induction of StAR mRNA in eel 1.5 hours post-injecti on with ACTH (adrenal corticotrophic hormone) (Li et al., 2003). The ACTH expe riment did suggest a conservation in the cAMP inducibility of StAR across species. Using largemouth bass (LMB) as a model fish since they are known to be sensitive to environmental toxins, I sequenced the en tire coding region of StAR. The sequence was used to develop a real-time PCR a ssay for mRNA quantitation of StAR and for generation of a polyclonal antibody to detect protein changes in LMB ovarian follicle cultures. The ovarian follicle cultures were used to establish StAR mRNA expression patterns by two potent signaling molecules, cAMP and TGF. It is known that cAMP can stimulate mammalian StAR, partially th rough activation of SF-1, and that TGFcan downregulate mammalian StAR transcription, ho wever, these studies have never been done in fish prior to this study. My main hypothesis was that LMB StAR mRNA expression is upregulated by cA MP and downregulated by TGFin ovarian follicle cultures. Since a direct link between -sitosterol exposure and TGFupregulation in prostate cancer cells has been shown (Kallen et al., 2000), this implies that toxins such as paper mill effluent repress steroid pr oduction through this signaling pathway.

PAGE 76

63 Results Cloning of StAR Protein A partial, 345 base pair sequence for LM B was amplified using degenerate primer based PCR (Figure 3-1). The partial sequen ce was then used to obtain the full length coding and untranslated regions with 5 and 3 RACE. Sequence alignment of LMB StAR with other species, in cluding brook and rainbow trout zebrafish, pig, horse, and human, shows 52% similarity between mamma lian and fish and 72% similarity amongst fish species (Figure 3-2). Based on the sequence comparison of StAR w ith other species, there appears to be conservation of a couple important re sidues, including an important PKA phosphorylation site at nucleotides 193196. The ScanProsite web program also putatively identified 4 PKC sites at nucle otides 5 7, 13 15, 60-62, and 187 189, as indicated by the number in the alignment (F igure 3-2). An important glutamic acid residue at nucleotide 170 which may bind to the hydroxyl group of cholesterol is also conserved amongst the species. The glutamic acid residue is within the START domain, the hydrophobic region for cholesterol binding, whic h spans from nucleotides 67 to 286. Seasonal Expression Changes in temporal expression of LMB StAR mRNA was quantitated from ovarian tissue samples previously collected at approximately two week intervals from October to April. RNA from seven fish at 11 time points during the year were analyzed and showed StAR mRNA levels do change in correlation with steroid production during the reproductive year (Figure 3-3). StAR levels peaked between February and March and began declining by April, therefore, suggesting a shor t window for maximal steroid

PAGE 77

64 expression. This information was useful in understanding when LMB are steroidogenic and which months are optimal fo r culturing ovarian follicles for in vitro studies. Regulation of StAR mRNA Expressi on in LMB Ovarian Cultures Alamar blue viability assay showed that the ovarian follicles remained viable after an 18 hour equilibration in the incubator. The follicles successfully reduced the components in the alamar blue reagent as detected with A570 and A600 spectrophotometry readings. The follicles remained viable and responsive to dbcAMP in either the absence or presence of charcoal-stripped serum, ther efore, serum was not added to culture media for experiments. Additionally, viability of th e follicles was also tested by inducing with dbcAMP after a couple of equilibration time points, showing that basal and induction levels werent impacted whether exposed fo r 6, 12, or 24 hours post-equilibration. cAMP Induction of LMB StAR About 20-30 mg pieces of ovarian tissue we re cultured and exposed to increasing doses of dbcAMP from 0 to 1 mM to char acterize the regulation of LMB StAR. Exposures of the ovarian tissue cultures to dbcAMP resulted in upregulation of StAR mRNA levels as quantitated by real-time P CR. A dose response of cultured ovarian slices to dbcAMP showed a significant 3.5 fo ld induction from a mean of 13,529 copies of StAR mRNA/g total RNA for controls to an averag e of 47,408 copies of StAR mRNA/g total RNA after exposure to 1mM dbcAMP for 4 hours (Figure 3-4). Additionally, more controlled experiments were done where number and stage of follicles cultured was monitored. Vitellogenic fo llicles with a specific diameter range of 0.68 mm to 0.76 mm were induced 5.9 fold from 18,054 copies of StAR mRNA/g total RNA for controls to 105,686 c opies of StAR mRNA/g tota l RNA after 4 hour exposure

PAGE 78

65 to 1mM dbcAMP (Figure 3-5). This suggests that more mature LMB follicles express more StAR, which fits the mammalian model for oocyte maturation (Logan et al., 2002). -sitosterol Exposures -sitosterol is known to downregulate ster oid production in goldfish (MacLatchy and Van Der Kraak, 1995), however, no consiste nt effects on LMB StAR mRNA could be seen at any given dose, timepoint, or stag e of follicle growth. Attempts were made with two different batches of fish after some initially promising results in the spring of 2003 suggested a decrease in LMB StAR mRNA levels by -sitosterol. The 2003 results, however, were never able to be reproduc ibly substantiated, su ggesting a suboptimal delivery of -sitosterol to the steroidogenic cells. TGFExposures TGF, a known repressor of steroid synthe sis in mammals (Brand et al., 2000; Gautier et al., 1997; Liakos, 2003), was tested in cultured follic les with diameters between 0.8 to 0.9 mm and 1 to 1.1 mm. The LMB follicle cultures showed a downregulation of StAR mRNA by about 2.3 fold after a 14 hour exposure to 1 ng/ml TGF(Figure 3-6). Some interesting preliminary results, howev er, were obtained from mature follicles between 1.2 and 1.3 mm. Data suggests that 1 ng/ml and 10 ng/ml TGFstimulates LMB StAR mRNA expression by about 2 fold after a 14 hour exposure, which could indicate that the various f actors which mediate the signa ling response may vary with follicle development (Figure 3-6).

PAGE 79

66 Antibody Development Currently, no polyclonal antibody exists fo r StAR that cross reacts with fish species, therefore, an antigenic peptide was designed and injected into two different rabbits to produce an immunogenic response against LMB StAR. The first step in testing and optimizing th e antiserum was to create purified protein for a positive control. A protein expressi on vector containing the full coding region for LMB StAR was created by PCR amplification (F igure 3-7) with subs equent cloning into the pET-28b expression vector containing a 6Xhistidine ta g (Novagen). The construct was verified by sequencing at the University of Florida DNA Sequencing Core. Bacterial cultures were induced by 1 mM or 3 mM IPTG for 1-4 hours and were analyzed on a Coomassie stained gel alongside negative contro ls of either empty vector or uninduced cultures. A band around 40 kD began to be ove rexpressed compared to controls after just one hour incubation with either 1 mM or 3 mM IPTG (Figure 3-8). Expression of StAR continued through 4 hours and appeared to be maximally induced by that timepoint. The expressed protein was successfully purified fr om the total protein extract using Ni-NTA spin columns (Qiagen) with a concen tration of 1.1 g/l (Figure 3-9). To confirm that the bacterially expresse d protein was StAR, the band was excised and in-gel digested with trypsin for analys is with two different mass spectrometers, QSTAR and LCQ. In-silico digest of the sequence matched several peaks seen by QSTAR, including peaks at 722.3833, 939.4723, 1404.7140, 1509.8019, 1601.8558, 2329.1177, and 2395.1682 (Figure 3-10). With the Q-STAR, we used a MALDI (matrix assisted laser desorption ioniza tion) based technique for sample analysis. Additionally, at least 5 tryptic peptides were identifie d by LCQ mass spec using an electrospray

PAGE 80

67 ionization technique (Figure 311). For LCQ, the peptides were separated by reversephase chromatography. Both the mass spec techni ques are complementary to each other. The rabbit antisera from rabbit UF408 a nd UF409 were tested by ELISA against the purified protein for presence of LMB St AR antibodies. After 30 minutes of exposure in an ELISA assay, 7 g/ml of purified prot ein had an absorbance at 450 nm of 0.3 with the pre-bleed versus about 2.8 for the test bleed at a 1:1600 dilution. ELISA results showed strong reactivity of the protein with antiserum dilu tions down to 1: 51,000, suggesting the presence of very strong and sp ecific LMB StAR antibodies (Figure 3-12). The ELISA results also helped to establish the dilution ranges of antiserum to test by western blot. To determine whether the anti bodies could also bind StAR in a western blot, I used the recombinant protein with ECL (chemilumi nesence) detection. The purified protein was successfully detected by the LMB StAR antibodies at several dilutions of primary antiserum as well as at severa l concentrations of protein. A 1:500 antiserum dilution was too concentrated for western blot and resulted in an overexposure on the film, even after a 1 second exposure, and a similar result was stil l seen with up to a 1:5000 dilution when a 1:20,000 secondary was used. The most optimal conditions to detect the recombinant StAR was at a 1:15,000 antiserum concentr ation with a 1:40,000 secondary dilution (Figure 3-13). Western Blot Detection of E ndogenous LMB StAR Protein Preliminary western blot analysis wa s done on LMB ovarian tissue exposed to either 0 or 1 mM dbcAMP for 24 hours. The western blot shows detection of StAR in both the control and 1 mM dbcAMP samples (F igure 3-14). The band detected for the dbcAMP sample appeared more intense compared to the control, however, the presence

PAGE 81

68 of non-specific bands suggests the StAR antibody needs to be purified from the anti-sera to obtain optimal results. The non-specific bands are of about the same intensity, indicating equivalent protein was loaded into each lane. Discussion Steroid hormones are key regulators of many cellular pathways and steroid synthesis can be regulated at the level of cholesterol transport with the StAR Protein. The StAR Protein has been cloned from severa l different species, ranging from higher mammals to lower vertebrates like fish, however, the similarities and differences in their function and regulation have never been inve stigated in any depth (Stocco and Clark, 1996; Goetz et al, 2004). A main goal of this project was to clone StAR from LMB and examine the conservation of key amino aci ds and whether the sequence similarity corresponds with conservation of function and activity. The ovarian tissue and follicle cultures we re excellent systems for comprehensive study of LMB StAR mRNA and protein regulation by e ndogenous signaling molecules like cAMP and TGF. Exposure of the ovarian cultu res to dbcAMP showed no blatant differences in magnitude of induction betw een species or the timepoints at which induction can be seen. There was a 6 fo ld induction of LMB StAR after a 4 hour exposure to dbcAMP, which compares with the 4 fold induction seen in MA10 mouse leydig cells (Clark et al., 1995.) The cAMP da ta establishes that th e overall re gulation of transcription is very similar across species and that key response elements in the promoter which mediate the response to cAMP, including those for SF-1, must be conserved. The exact molecular pathways are still being elucidated for regulation of StAR, therefore, it is important to detect changes in protein as well as mRNA levels since

PAGE 82

69 compounds could regulate either the transcription or transl ation of StAR. Previously, however, there was no antibody th at cross-reacted with vert ebrate species to examine protein alterations in StAR. We successf ully generated a polyclonal antibody that can detect LMB StAR protein by ELISA or western blot. Th e antibody proved to be very potent with purified protein and works optimally at very dilute con centrations of around 1:15,000. The antibody is being purified from th e anti-sera for optimal detection of StAR in complex protein samples, however, prelim inary results suggests protein levels are induced by cAMP. The ovarian follicle cultures were also used to establish whether LMB StAR mRNA is regulated by TGF, a potent signaling molecule like cAMP. The cultures showed about a 57% reduction in StAR mRNA levels after a 14 hour exposure to 1 ng/ml TGFfor two different diameter sizes of follicles. Follicles of the same size studied for the TGFexposures, such as the 0.8 0.9 mm, were inducible by dbcAMP. Interestingly, the stage at which follicles we re cultured may play a key role in the regulation since StAR mRNA levels we re activated by 1 or 10 ng/ml TGFin more mature follicles of 1.2 to 1.3 mm. One possibl e explanation for this may be that SMAD2 and SMAD3 proteins, the key signaling molecules of TGF, may not be present at sufficient levels in the more developed LMB follicles. It has been reported that both SMAD2 and SMAD3 levels are very low or non-existent in the la rge antral or preovulatory follicles of rats (Xu et al., 2002). SMAD3 has been shown to mediate the TGFrepression of mammalian St AR (Brand et al., 1998), TGF, unlike cAMP, significantly represse s mammalian StAR mRNA expression. The repression can range from 40% in unstim ulated cells treated with 2 ng/ml TGFfor

PAGE 83

70 12 hours to greater than 60% dow nregulation when induced w ith cAMP in H295 cells, a human adrenocortical cell line (Brand et al ., 1998). The LMB ovarian follicle data suggests that, in addition to the cAMP pathway, TGFsignaling is not only conserved between mammals and lower vertebrates but that the percent of inhi bition for developing oocytes by TGFis also relatively compar able. Preservation of TGFsignaling between species underscores the importance of this pathway and the chemicals that modulate it. Interestingly, a study showed TGFprotein levels are upreg ulated by at least 50% in prostate stromal cells after a 6 day exposure to -sitosterol, one major chemical found in paper mill contaminants (Kassen et al., 2000). Fish exposed to paper mill toxins, and more specifically, -sitosterol, exhibit de creased steroidogenic activity (MacLatchy and Van Der Kraak, 1995). The ovarian follicle cultures were used to determine if sitosterol exposure mimics TGFdownregulation of LMB St AR mRNA levels, thereby elucidating a possible signali ng pathway activated by environmental toxins. Overall, direct -sitosterol exposures at se veral doses and timepoints in the follicle cultures did not significantly impact StAR mRNA expre ssion, contradicting my original hypothesis that -sitosterol represses steroidoge nesis by downregulating StAR. A possible explanation for the -sitosterol results might be that ethanol was used as the solvent. Ethanol was chosen based on several published in vivo and in vitro experiments (Kassen et al, 2000; MacL atchy and Van Der Kraak, 1995). When administered in vivo -sitosterol is able to enter the bloodstream and be packaged into lipoproteins for delivery to st eroidogenic cells (Carter a nd Karpen, 2001). Although it is not definitively known which signa ling pathways are modulated by -sitosterol, it is

PAGE 84

71 possible that downstream signa ling requires activation throu gh the lipoprotein receptors. Using ethanol as a solvent might have bypa ssed the use of lipoprotein receptors or cellular lipid carri ers and could explain the lack of significant respons e I saw on StAR mRNA expression by -sitosterol. A recent publication reported that 15 g/ml cholesterol delivered via etha nol to Y-1 mouse adrenal cells did not significantly impact StAR protein levels (King et al., 2004), howev er, cholesterol incorporated in either HDL or LDL does increase mammalian StAR protei n production in Y-1 ce lls (Reyland et al., 2000). The ethanol study with cholesterol fu rther supported the potential importance of using the lipoprotein receptors as a r oute of entry for StAR regulation. Overall, the sequence, mRNA, and protein data suggests a similarity in signaling pathways and regulation across species for StAR. Induction of mRNA and protein levels by cAMP suggests that LMB StAR is subj ect to both transcriptional and posttranscriptional regulation by critical signa ling molecules such as cAMP. The combined mRNA and protein results can provide valu able insight into the mechanism by which environmental toxins, such as -sitosterol, can disrupt the normal regulation of StAR.

PAGE 85

72 Figure 3-1. PCR amplification of LM B StAR. A 345 bp partial cDNA sequence for LMB StAR was PCR amplified and run out on a 1% ag arose gel. (A) PCR marker (Promega) (B and C) PCR amplifications from gonadal RNA of two different fish. (D) negative co ntrol with no reverse transcriptase added. The amplified band for StAR is indicated by arrow.

PAGE 86

Figure 3-2. Alignment of LMB StAR cDNA with other species. Alignment of the cDNA sequence for LMB StAR with other sp ecies shows about a 53% similarity across all species and 73% similarity between fish and lower vertebrates. Several important regulatory sites were putatively identified in the LMB StAR cDNA, including; 4 PKC sites at amino acid positions (as indicated on alignment), 5 7 (TFK), 13 15 (SYR), 60-62 (SSR), and 187 189 (SVR). There is one conserved PKA site at nucleotides 193 196 (RRGS). Additionally, an important glutamic aci d residue at nucleotide 170, which may bind to the hydroxyl group of choleste rol, is conserved within the START domain (amino acids 67-286).

PAGE 87

74 10 20 30 40 50 60 | | | | | | brook trout ML P ATFKLCAG I SYRH T RNM T GLR KN A M V A I H H ELN ML A -GP NP S S WI S H V R R R SSLL S rainbow trout ML P ATFKLCAG I SYRH M RNM T GLR KN A M V A I H H ELN ML A -GP NP S S WI S H V R R R SSLL S LMB ML P ATFKLCAG I SYRH M RNM T GLR KN A M V A I H H ELN RL A -GP GP S N WIN Q T R G R SSLL S zebrafish ML P ATFKLCAG I SYRH M RNM T GLR KN A M I A I H H ELN KL S -GP GA S T WI N H I R R R SSLL S pig ML L ATFKLCAG S SYRH V RNM K GLR HQ A V L A L G Q ELN RR A LG GP TS G S WI N Q V R R R SSLL G horse ML L ATFKLCAG S SYRH V RNM K GLR HQ A A L A I G Q ELN WRA PG GP TQ S G WI N Q V R R Q SSLL G human ML L ATFKLCAG S SYRH M RNM K GLR QQ A V M A I S Q ELN RR A LG GP TP S T WI N Q V R R R SSLL G ** ******** **** ***.***::* :*: :*** : ** **.: :****. Prim.cons. MLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNRLALGGPTPSSWINQVRRRSSLLS 70 80 90 100 110 120 | | | | | | brook trout S R I E E EQG Y N E A E V S Y VK QG EE A L Q KSI S IL G D Q D GW TT E IIAA NG DK V L SK VL P D V GKV rainbow trout S R I E E EQG Y N E A E V S Y VK QG EE A L Q KSI S IL G D Q D GW TT E IIAA NG DK V L SK VL P D V GKV LMB S R I E E EEG Y S D E E M S Y VK QG ED A L Q KAI S IL S D Q D GW TT E IVAA NG DK V L SK ML P D I GKV zebrafish S P I A E ETY S E A D Q C YVQ QG QE A L Q KSI S IL E D Q D GW QT E IESI NG EK V M SK VL P G I GKV pig S Q L E D TFY S D Q D L A Y IQ QG EE A M Q RAL D IL S N Q E GW KK E SRQE NG DE V L SK VI P D V GKV horse S Q L E D TLY S D Q E L A Y IQ QG EE A M Q KAL G IL R N Q E GW KEE NQQA NG DK V L SK VV P D V GKV human S R L E E TLY S D Q E L A Y LQ QG EE A M Q KAL G IL S N Q E GW KK E SQQD NG DK V M SK VV P D V GKV : : *.: : .*::**::*:*:::.** :*:** **::*:**::*.:*** Prim.cons. SRIEEE2GYSD2EL2YVQQGEEALQKAISILSDQDGW2TEI22ANGDKVLSKVLPDVGKV 130 140 150 160 170 180 | | | | | | brook trout F K LEV L LD Q RSDN LY G ELV G N ME Q MG D WNP N VK EV K I LQKIG QE T M V THE VS G P T P GN V V rainbow troutF K LEV L LD Q RSDN LY V ELV G N ME Q MG D WNP N VK EV K I LQKIG QE T M V THE VS G P T P GN V V LMB F K LEV M LE Q RPDN LY K ELV G N ME Q MG E WNP N VK QV K I LQKIG QD T M V THE VS A E T P GN V V zebrafish F K LEV T LE Q QTGD LY D ELV D N ME Q MG E WNP N VK QV K I LQKIG QE TM I THE IS A E T P GN V V pig F R LEV V VD Q PMER LY E ELV E R ME A MG E WNP S VK KI K I LQKIG KD T V I THE LA A E A A GN L V horse F R LEV E VD Q PMER LY E ELV E R ME A MG E WNP N VK EI K V LQKIG KD T V I THE LA A E S A GN L V human F R LEV V VD Q PMER LY E ELV E RME A MG E WNP N VK EI K V LQKIG KD T F I THE LA A E A A GN L V *:*** ::* ** *** .** **:***.**::*:*****::*.:***::. :.**:* Prim.cons. FKLEV2LDQ2M22LYEELV2NMEQMGEWNPNVKEVKILQKIGQDTMITHE2SAETPGNVV 190 200 210 220 230 240 | | | | | | brook trout GPRDFV S V RC A KRRGS T C F LAGM S T Q H PT MPEQ R G V V RAE N GPTC I V MR P S A D D P N KTK F rainbow trout GPRDFV S V RC A KRRGS T C F LAGM S T Q H PT MPEQ R G V V RAE N GPTC I V MR P S A D D P NKTK F LMB GPRDFV S V RC A KRRGS T C F LAGM S T Q H PK MPEQ R G V V RAE N GPTC I V MK P C V E D P N KTK F zebrafish GPRDFV N V RH A KRRGS T C F LAGM S T Q H PG MPEQ K G F V RAE N GPTC I V MR P S A D D P N KTK F pig GPRDFV S V GC T KRRGS V C V LAGM A T D F GE MPEQ K G V I RAE H GPTCM V LH P L A G S P S KTK L horse GPRDFV S V RC A KRRGS T C V LAGM A T Q F EE MPEQ K G V I RAE H GPTC M V LH P L A G S P S KTK L human GPRDFV S V RC A KRRGS T C V LAGM A T D F GN MPEQ K G V I RAE H GPTC M V LH P L A G S P S KTK L ******.* :*****.*.****:*:. ****:*.:***:****:*::* .*.***: Prim.cons. GPRDFVSVRCAKRRGSTCFLAGMSTQHP2MPEQKGVVRAENGPTCIVM2P2A2DPNKTKF 250 260 270 280 | | | | brook trout TWLL S I DLKGW I PKTI IN K VLSQTQVDF A NHLR Q R M A D NSVSKEMAPA C rainbow trout TWLL S I DLKGW I PK TI IN K VLSQTQVDF A NHLR Q R M A D NSVSMEMAAA C LMB TWLL N I DLKGW I PK TI IN K VLSQTQVDF A NHLR Q R M A N N-VSMEMAHA C zebrafish TWLL S L DLKGW I PK TV IN R VLSQTQVDF V NHLR D R M A S G-GGIDAAIA C pig TWLL S I DLKGW L PK TI IN Q VLSQTQVDF A NHLR K R L E S R---PALEAR C horse TWLL SI DLKGW L PK TI IN Q VLSQTQVDF A NHLR K R L E S S---PAPEAR C human TWLL S I DLKGW L PK SI IN Q VLSQTQVDF A NHLR K R L E S H---PASEAR C ****.:*****:**::**:*********.****.*: Prim.cons. TWLLSIDLKGWIPKTIIN2VLSQTQVDFANHLR2RMASNSVSP2MAAAC

PAGE 88

75 Figure 3-3. Seasonal expression of LMB StAR. RNA was isolated from ovarian tissue of largemouth bass previously collected ev ery few weeks for most of a year throughout a year. Typical seasonal expression of StAR mRNA was determined by real-time PCR. RNA fr om seven fish at 11 time points during the year were analyzed. T-test show s a p< 0.05 between timepoints marked with red versus black stars.

PAGE 89

76 Figure 3-4. Dose response of LMB ovarian tis sue cultures to dbcAMP. Cultures of 20-30 mg pieces of ovarian tissue were expose d to a dose response of dbcAMP from 0 mM to 1mM for 4 hours. RNA was then isolated from the tissues and reverse transcribed to cDNA for realtime PCR analysis. T-test shows a p< 0.05 as indicated with red star. Results ar e from 2 different fish with triplicate assays done for each fish.

PAGE 90

77 Figure 3-5. cAMP induction of ovarian follicle s. Thirty follic les between 0.4 and 0.88 mm were cultured in 24 well culture pl ates and exposed to 0 or 1 mM dbcAMP for 4 hours. RNA was isolated from the follicles and reverse transcribed for analysis by real-time P CR. This experiment represents data obtained from one fish.

PAGE 91

78 Figure 3-6. Dose response exposur e of ovarian follicles to TGF. Follicles with diameter ranges between 0.8 0.9 mm, 1.0 1.1 mm, and 1.2 1.3 mm were exposed to 0 ng/ml or 1 ng/ml TGFfor 14 hours. Ten follicles were cultured in each well of a 24-well culture plate. RNA was isolated from the follicles and reverse transcribed for an alysis of StAR mRNA expression by real-time PCR. T-test shows a p< 0.05 indicated with red star.

PAGE 92

79 Figure 3-7. PCR amplification of entire LMB St AR cDNA. Specific primers were used to amplify 858 nucleotides of LMB StAR coding region. A) Low DNA mass ladder (Invitrogen). B and C) BamHI digest of two separate clones to confirm ligation of LMB StAR into the pET-28b vector.

PAGE 93

80 Figure 3-8. Bacterial expre ssion of LMB StAR. The en tire cDNA sequence for LMB StAR was cloned into the pET-28b vector (Novagen). Bacter ial expression of StAR protein was induced by 1 mM a nd 3 mM IPTG from 1 to 4 hours in BL21(DE3) bacterial cells. Total prot ein for each exposure was run out on a 4-12% Bis-Tris NuPAGE gel (Novex) a nd stained with Colloidal Coomassie Blue. Induction of StAR is indicated by an arrow.

PAGE 94

81 Figure 3-9. Purification of St AR. His-tagaged StAR was purified from the bacterial expression using Ni-NTA spin columns (Q iagen). Lane 1 contains 2 g of purified protein and lane 2 contains See Blue Plus 2 marker run out on a 412% Bis-tris gel (Novex) and stained w ith colloidal comassie blue stain. The band for purified StAR is indicated by an arrow.

PAGE 95

82 Figure 3-10. Identification of bacteria lly expressed StAR with Q-STAR mass spectrometry. The trypsin-digested StAR was analyzed with Q-STAR mass spectrometry for identification of bacterial LMB StAR expression. The coomassie blue stained band was excised a nd in-gel trypsin digested prior to a MALDI based mass spec analysis. The black arrows indicate which peaks were specific for predicted LMB StAR peptides.

PAGE 96

83 Figure 3-11. Identification of bacterially expressed StAR by LCQ mass spectrometry. The coomassie blue stained band for b acterially expressed LMB StAR was cut out and in-gel trypsin digested for analysis by LCQ mass spec. Predicted tryptic fragments were identified by Pros pector and the red arrows indicate which peptides were seen by LCQ.

PAGE 97

84 Figure 3-12. ELISA with LMB StAR antiserum and purified StAR protein. The antiserum from two differ ent rabbits injected with the StAR peptide were tested by ELISA with 7 g/ml purified protein.

PAGE 98

85 Figure 3-13. Western blot detection with LM B StAR antibody and purified StAR protein. 13 ug of purified protein was run out on a 4-12% bis-tris gel, transferred to a nitrocellulose membrane. The membrane was divided into strips for probing with several combinations of prim ary antibody (LMB StAR antiserum) and secondary (mouse anti-rabbit IgG) conjuga ted to horseradish peroxidase). Primary antibody incubations were done for 3 hours and secondary incubations done for 1 hour followed by chemiluminescence detection on kodak film.

PAGE 99

86 Figure 3-14. Western blot de tection of StAR in dbcAMP exposed LMB tissue cultures. 60 mg pieces of LMB ovarian tissue were cultured and exposed to 0 or 1 mM dbcAMP for 24 hours. Total protein wa s extracted and run on a 4-12% bistris for detection of StAR with a 1: 5,000 dilution of th e polyclonal antibody and 1: 40,000 secondary antibody.

PAGE 100

87 CHAPTER 4 TRANSCRIPTIONAL REGULATION OF THE LMB STAR PROMOTER Introduction Several transcription factor binding sites have been identified in the mammalian StAR promoter, including SF-1, AP-1, GATA4, C/EBP, involved in the activation of transcription, and DAX-1, a re pressor of mRNA synthesis (S ugawara et al., 2004; Jo and Stocco, 2004; Manna et al., 2004; Silverman et al., 2004). An alignment of the first 200 base pairs of mammalian promoters for mous e, rabbit, sheep, pig, and horse shows the locations are conserved for 2 SF-1, 1 GATA, and 2 C/EBP response elements. One of the SF-1 sites, in fact, is only about 12 base pairs upstream from the TATA box. The promoter alignments indicate how important those binding sites ar e in mediating the transcriptional activity of St AR, however, more studies are necessary before the function or even the identification of many of th e response elements is determined. It is well characterized in the mammalia n system that dbcAM P upregulates StAR transcription via PKA phosphoryl ation of SF-1 (Aesoy et al., 2002). The magnitude of activation of StAR by SF-1 is partially c ontingent on surrounding response elements and the proteins that they bi nd. C/EBP interaction with SF-1 can amplify the cAMP induction of StAR (Reinhart et al., 1999) however, when DAX-1 and SF-1 were cotransfected in HTB9 cells, a bladder carci noma cell line devoid of endogenous steroid production, SF-1 could no longer stimulate StAR in response to cAMP (Sandhoff and McLean, 1999). The SF-1 data underscores the importance and complexity of proteinprotein and protein-DNA inte ractions in StAR transc riptional regulation.

PAGE 101

88 StAR transcription can be modulated by both endogenous and exogenous factors, which includes environmental toxins. The Ah R element in the StAR promoter is a ligand for dioxins, a very toxic chemical found in water supplies and other locations. AhR cotransfection in Y-1 cells w ith 1.3 Kb of the human StAR promoter increased its transcriptional activity (Sugawara et al., 2001) Additionally, when the Y-1 cells were treated with -napthoflavone, a ligand for AhR, St AR promoter activity was also increased (Sugawara et al., 2001). Inducti on of the promoter by cAMP, however, was not changed by co-transfection with th e AhR construct. Although the AhR study suggested a pathway for StAR regulation th at is cAMP/PKA independent and could be targeted by toxins, it is importa nt to also characterize pa thways which use alternative signaling pathways and response elements. The mammalian StAR promoters were re-e xamined using the professional based web program and sites for COUP-TF, ER, RA R, and ROR were put atively identified. Their specific roles in StAR regulation were previously uncharacterized. COUP-TF can repress steroidogenesis and antagonize the induc tion of steroids by retinoic acids through competitive binding with the RAR/RXR re sponse element (Barger and Kelly, 1997; Butler and Parker, 1995), a possible new model to be examined in StAR downregulation. The ROR is a relatively novel cla ss of proteins related to th e RAR and their interactions with COUP-TF or ER are unclear and open for investigation (Jarvis et al., 2002). Estrogen receptors are significan t transcriptional regulators pa rtly because they can bind not only to their own ERE but also to AP-1 sites (Bjornstrom and Sjoberg, 2005), which are present in the StAR promoter.

PAGE 102

89 A major goal of this project was to clone the promoter for LMB StAR and develop a transfection assay with Y-1 cells for in-depth transcriptional analysis. The transfections were used to examine how deletions in promot er length or site-directed mutations in key response elements impact StAR transcri ption. To expand on the cAMP and TGFovarian follicle exposure data in chapter 3, the role of thos e same signaling molecules in modulating transcriptional regulation of LM B StAR was examined. My main hypothesis was that cAMP activates LMB StAR transc ription through sites independent of SF-1, such as the ROR. Additionally, there are cri tical regulatory sites fo r direct regulation of the StAR promoter by TGFsince LMB StAR mRNA is m odulated by the pathway. Results Cloning of the StAR Promoter A 2.9 Kb portion of the LMB StAR pr omoter was cloned to examine its transcriptional regulati on in transfection assays. The genomic DNA isolated for promoter sequencing was intact and of good quality with minimal if any protein contamination as evidenced by digestion with EcoRV, StuI, P vuII, and DraI compar ed to an undigested control (Figure 4-1). The digested genomic DNA was used to PCR amplify 2.9 Kb of the LMB StAR promoter with gene specific primer s starting from the coding region that was already sequenced. PCR products greater than 2 Kb were obtained from all four restriction libraries, as well as a shorter piece around 400 nucleotides from the EcoRV library. The amplified product from the PvuII library using promoter jk1 and promoter jk2 primers was ligated into a TOPO cloning vector. Ligation was c onfirmed by a double digest with EcoRV and DraI (Figure 4-2) and sent for sequencing at the DNA Sequencing Core at University of

PAGE 103

90 Florida. This PCR product was subsequen tly cloned into the pGL3 vector for transfection into Y-1 cells. Identification of the Transcriptional Start Site 5 RACE of the transcript was used to locate where the LMB StAR 5UTR ends, which is followed immediately by the transcri ptional start site and the beginning of the promoter (Figure 4-3). The length of the 142 base pair LMB StAR 5UTR was similar to sequences reported for brook trout (180 bp), rainbow trout (160 bp) and zebrafish (40 bp) sequence in the NCBI database. Additiona lly, the transcriptional start site identified from 5 RACE matched the site predic ted by a web-based program called Neural Network Promoter Prediction. A TATA box is located about 23 base pairs upstream from the start site. Identifying Transcriptional Response Elements Sequences upstream from the transcriptiona l site were analyzed by 3 different web search engines, MatInspect or V2.2, Professional-MatInspector V7.3, and TFSearch, for identification of putatively important respons e elements (Figure 4-4). Many important sites appeared to be conserved in the pr omoter between mammals and fish, although the specific position in the sequen ce didnt always correspond. Sites were only considered putatively f unctional if the same response element was identified by at least 2 of the 3 search program s, and if the matrix similarity score for the sequence was greater that 0.8 using the Profe ssional-MatInspector. A perfect match for a response element gets a matrix similarity score of 1.00, where each nucleotide in the sequence being analyzed ma tches the most conserved sequence known for a given response element. The matrix similarity scor e can be decreased by mismatches in highly conserved positions of the binding site. The professional web-based program identified

PAGE 104

91 several response elements in the LMB StAR promoter; 2 RAR, 1 ROR, 4 COUP-TF, 2 SF-1, 6 CREB, 8 GATA-1, 1 AhR/ARNT co mplex, 1 Smad3, and 1 full ERE site. Additional SF-1 sites were also identified by comparing mammalian EMSA data with LMB sequence information. Cloning of LMB Steroidogenic Factor -1 (SF-1) SF-1 has been extensively shown in mammalian species to upregulate StAR, therefore, a 280 bp sequence of LMB SF-1 was obtained to examine the sequence similarity across species. LMB SF-1 was am plified using specific primers designed to the ligand binding domain (Figure 4-5). Alignm ent of SF-1 with other species, including rainbow trout, sheep, and human, revealed great er than 40% similar ity within the region amplified. Optimization of Transfection Assays The StAR promoter was cloned into th e basic pGL3 luciferase vector and transfected into the Y-1 mouse adrenal cell line to examine its regulation by cAMP and TGF. Y-1 cells were chosen since they have endogenous steroidogenic activity and therefore, likely produce the cr itical transcription factors necessary for StAR regulation. The transfections were optimized with 3 differe nt ratios of volume of transfection reagent to mass of promoter DNA (l/g), 3:1, 3: 2, and 3:2, under control and 1 mM dbcAMP stimulated conditions. Under basal condi tions, the greatest amount of DNA was transfected using the 6:1 rati o and with 1 mM dbcAMP stimulation resulted in a 2.2 fold induction. Since the fold stimulation with 1 mM dbcAMP using the 6:1 ratio was about the same as the 3:1 ratio, and slightly more than the 1.9 fold stimulation obtained with the 3:2 ratio, the 6:1 ratio was used for all future experiments (Figure 4-6).

PAGE 105

92 Transfection time was also optimized. Y-1 cells were transfected for either 6 or 24 hours followed by exposure to either 0 or 1 mM dbcAMP for 20 hours (Figure 4-7). Both timepoints produced a similar fold induction by 1mM dbcAMP, however, the basal luciferase levels were higher at 24 hours and that timepoint was used for all subsequent transfections. The efficiency and percent of transf ected cells was quantitated by GFP cotransfection into the Y-1 cells (Figure 4-8). Transfection efficiency, using trypsinized cells on a hemacytometer, was calculated to be about 18%. dbcAMP Exposures Y-1 cells were transfected with the LMB St AR promoter and exposed to a range of dbcAMP between 0 mM and 2 mM for 20 hours. cAMP is known to induce mammalian StAR transcription, however, previously onl y SF-1 sites in the promoter were well characterized to mediate the response of St AR through this pathway (Brand et al., 2000). The 2.9 Kb LMB promoter was maximally induced between 0.75 mM and 1 mM dbcAMP by an average of about 2.4 fold (Figur e 4-9). The transfections were done in both the presence and absence of 17.5% serum to determine whether factors present in the serum impact transcripti onal activity. No impact wa s observed on the induction of StAR, therefore, all further transfections were done completely in the presence of serum for maximal viability of the cells. The Y-1 cells and other steroidogenic cells undergo a well characterized physical change in response to dbcAMP by rounding up. The exposures must be carried out in the presence of serum sin ce the cells appear to round up and eventually detach from the plate much mo re quickly without serum. It is possible that the sources for modulation of the tr anscription factors in volved in the dbcAMP signaling pathway, such as SF-1, are depleted more rapidly in the absence of serum.

PAGE 106

93 Also, since dbcAMP can be br oken down by phosphodiesterases, a phosphodiesterase inhibitor, IBMX, was adde d, to determine its impact on StAR induction. IBMX did not significantly impact the upregulation of StAR transcription, either at 0.1 mM or 0.5 mM IBMX, however, all experiments were carried out in the presence of IBMX to minimize dbcAMP breakdown during the 20 hour exposures. Promoter Deletion Experiments A deletion of the promoter was made to ex amine regulation of the shorter promoter by cAMP or TGF. Deletion of the 2.9 Kb promot er to 1.86 Kb was accomplished by using EcoRV and BstEII restriction sites alread y present in the sequence (Figure 4-10). The 1.86 Kb promoter deletion construct was exposed to dbcAMP for comparison in activity to the 2.9 Kb construct. The de letion appeared to significantly diminish the dbcAMP activation of LMB StAR by 70-80%, suggesting an important activation site was eliminated (Figure 4-11). The impact of TGFexposure on LMB StAR transcriptional activity was examined using the transfection assays since TGFis known to decrease steroid synthesis. Surprisingly, the 2.9 Kb constr uct was less susceptible to repression by 20 ng/ml TGFthan the 1.86 Kb construct after a 40 hour exposure (Figure 4-11). The 2.9 Kb promoter was downregulated about 20% versus a 40% repression for the 1.86 Kb construct, suggesting there are elements within that region which mediate TGFsignaling. Site-Directed Mutagenesis Experiments The role of individual tran scription factors in regula ting the StAR promoter was studied by mutating five different sites. Pote ntially important response elements involved in StAR regulation were identified us ing web based search programs, including

PAGE 107

94 Professional-MatInspector V7.3 and TFSearch. Mutation to a NotI restriction site was made for the following putative sites (location for all mutation sites are noted in reference to the LMB StAR transcripti onal start site): ERE at nuc leotide 2678; COUP-TF site at nucleotide 2027; ROR at nucleotide 1969; a combined GATA/AP-1/ERE site at nucleotide 1882; and another COUP-TF site at nucleotide 2304. The web-based programs predicted no transcription factors would bind to the mu tated sites. All mutations were confirmed by digestion with NotI (Figure 4-10). All of the site-mutagenesis constructs were exposed to 1 mM dbcAMP, however, none of the mutated promoters were as i nducible as the unmutated 2.9 Kb construct (Figure 4-12). Altering the ERE/2678 site re sulted in 30% loss of response to cAMP, however, the data was not significant according to T-test with a P value greater than 0.05. However, mutation of the COUP-TF/2027, combined GATA/AP-1/ERE/1882, or ROR/1969 sites reduced the cAMP induction by 80%. The site-directed mutations suggest that the COUP-TF, ROR, and GAT A/AP1/ERE sites are critical for dbcAMP regulation. Another goal of the site-directed mutagenesi s experiments was to evaluate the role of critical response elements specifically between the 1.86 and 2.9 Kb region, in TGFregulation of the StAR promoter. All of the site-mutagenesis constructs were exposed to 20 ng/ml TGFfor 40 hours (Figure 4-13). Muta tion of the ERE/2678 and Coup/2027 sites diminished the repression by about 16% suggesting these sites could mediate the inhibitory response to TGF. Mutation of COUP-TF/2304, GATA/AP-1/ERE/1882, or ROR/1969 resulted in no si gnificant change in TGFregulation, suggesting these elements are not involved in TGFsignaling.

PAGE 108

95 TGFRegulation of Y-1 Cell Endogenous Mouse StAR mRNA Y-1 mouse adrenal cells used for transf ections have endogenous steroidogenic activity, therefore, TGFimpact on mRNA expression of mouse StAR in Y-1 cells was examined by real-time PCR. A 40 hour exposure to 10 ng/ml TGFresulted in a 60% reduction of mouse StAR mRNA compared to controls (Figure 414). This set of experiments served as a reference point since TGFregulation of the entire mouse StAR gene was examined as opposed to either a pa rtial 2.9 Kb or 1.86 Kb promoter piece in the transfection studies. Discussion Mammalian StAR is regulated by a divers e array of proteins including SF-1, Dax1, and AP-1 (Stocco et al., 2001; Buholzer et al., 2005; Sandho ff and McLean, 1999; Shea-Eaton et al., 2002). A few of the re sponse elements putatively identified in mammalian StAR have been examined in mo re depth by transfection experiments or EMSA for functional capabilities. The bindi ng sites for SF-1 in the StAR promoter are probably, to date, the most well researched of all the sites identified (Sandhoff et al., 1998; Sugawara et al., 1997). Although SF-1 is ve ry critical to the upregulation of StAR, there are other potential response elements that have not even begun to be analyzed, such as the ERE, COUP-TF, and ROR. A main goal of this project was to develop assays to examine the LMB StAR promoter and to make inferences on factors which might modulate the response to dbcAMP and TGF, two critical and separate signaling pathways. dbcAMP upregulates LMB StAR transcrip tional activity in a similar manner as published for mammalian species. In chapte r 3, real-time PCR with ovarian follicle

PAGE 109

96 cultures showed a dose responsive induction of LMB StAR mRNA with dbcAMP. The mRNA data provided a benchmark for how th e promoter should re spond in transfection assays if functional and in a compatible ce ll line. The amount of induction of the LMB StAR promoter by 1mM dbcAMP was comparable to that of mammalian species. 1.3 Kb of human StAR promoter is upregulated about 2 fold after 24 hours in Y-1 cells (Sugawara et al., 2000), which parallels with the LMB resu lts. The cAMP transfection data implies that critical transcription f actors and their corresp onding protein sequences are conserved across species. This was a dditionally confirmed by the cloning of LMB SF-1. The amplified ligand binding domain for LMB SF-1 was about 42% similar with mammals and greater than 90% homologous to ot her fish. A conserved alanine at residue 288 on the alignment was published to be cri tical for transcripti onal activity of SF-1 (Wang et al., 2005), suggesting SF-1 is regula ted in a similar fashion for different species. Deletion of 1000 base pairs from the 2.9 Kb construct, leaving a 1.86 Kb construct, diminished the inducing effect of cAMP treatm ent. One explanation for the difference in response of the two constructs may be the removal of critical elements involved in StAR activation. There are AP-1, SF-1, RAR, ROR, as well as other sites within this region which could be crucial for inducti on of steroid synthesis. Directed mutations of several putative re sponse elements provided valuable insight into the upregulation of StAR by the cAMP pathway. Mutation of either the COUPTF/2027 or ROR/1969 diminished the cAMP induc tion compared to the control. It is known that COUP-TF is an inhibitor of steroid synthesis (Shibata et al., 2004; Tran et al., 1992), however, this study is th e first to implicate a putativ e role for COUP-TF in StAR

PAGE 110

97 transcriptional activity. The LMB StAR cAMP data with the various mutations suggests that COUP-TF does not repress StAR or steroi d synthesis by binding to its own response element but rather by competitively binding to other transcription sites. The mutation data suggests the response element for COUP-T F actually participates in the activation of StAR. The antagonistic effect of mutations in the C OUP-TF binding region on cAMP induction of the LMB promoter could be explai ned by its protein inte racting partners or the response elements it can bind. COUP-T F is known to homodimerize with itself or heterodimerize with RAR or ROR (Tran et al ., 1992; Schrader et al., 1996; Berrodin et al., 1992). COUP-TF can also bind to an ERE, even an ERE half-site (Klinge et al., 1997). More specifically, COUP-TF is known to downregulate steroid production through competitive binding w ith the RARE as opposed to binding to the response element for COUP-TF itself, thereby prev enting RAR from activating transcription (Butler and Parker, 1995; Tran et al., 1992) Additionally, COUP-TF can also bind response elements for ROR, further indicati ng the arbitrary response elements it binds (Schrader et al., 1996). Most nuclear receptors, unlike COUP -TF, have clear and defined binding sites, and the arbitrary sites COUP-T F binds leads to seve ral potential avenues for downregulation of the StAR promoter. This is the first study to show that the response element for COUP-TF may be involve d in the upregulation of steroidogenesis via StAR since mutation experiments rendere d the promoter significantly less responsive to cAMP activation. Little is known about the role of ER a nd ROR in StAR regula tion. The interesting interactions between COUP-TF and RAR, RA RE, ROR, or ERE further warranted the examination of the ROR and ERE function in StAR. We found RAR and ROR sites in

PAGE 111

98 both the mammalian and LMB promoters us ing public and professional web-based servers and examined the function of the ROR more closely. Th e transfection data suggests the ROR plays a critic al role in the induction of StAR transcription and it modulates the activity through a cAMP de pendent pathway. Interestingly, recent published results show that cholesterol is a ligand for the previ ously orphaned ROR (Kallen et al., 2002), and the LMB transfecti on data suggests this site could be one mechanism for feedback control of choleste rol metabolism. Mutating the ERE/2678 site, however, had no significant impact on cAMP activation. Deletion and mutagenesis experiments al so suggested putative transcription elements which mediate the TGFresponse exists between the 2.9 and 1.86 Kb region. The 1.86 Kb construct was more repressed by 20 ng/ml TGFcompared to the 2.9 KB promoter. The site-specific analysis showed that the ERE/2678, COUP-TF/2027, and relieved the 20% repression seen with the 2.9 Kb promoter by about 16%, suggesting that these sites might bind proteins involved in the TGFpathway. It has been published that both COUP-TF and ER can interact with the TGFsignaling pathway by binding to Smad complexes (Calonge et al., 2004). Fu rthermore, ER has been shown to bind to Smad3, the primary Smad found to mediate th e inhibitory respons e of StAR to TGF(Matsuda et al., 2001). It is possible there are other site s within the 1.8 Kb to 2.9 Kb region or upstream from this which mediat e the inhibitory response since none of the mutations restored the level of the repression to that seen with the 1.86 Kb length promoter. Overall, the transfection results suggest th at response elements far upstream in the promoter, at least 5 of the 1.86 region, are ju st as important or potentially even more

PAGE 112

99 important than sites present within the first 1000 to 1500 base pairs. Previous studies with mammalian StAR have focused on relativ ely short pieces of the StAR promoter, however, the LMB data implies that critical regulatory sites a nd mechanisms may be resolved by examining much longer constructs.

PAGE 113

100 Figure 4-1. Digested LMB ovarian genomic D NA for promoter cloning. The quality of genomic DNA isolated from LMB ovaria n tissue was tested before use in promoter cloning. 2.5 g of the DNA was digested for 20 hours with one of 4 restrictions enzymes, (B) DraI, (C) EcoRV, (D) PVUII, and (E) StuI and 5 ls was run out on a 0.5% agarose gel al ong with a DNA marker (A). (F) An undigested control was also run to co nfirm DNA integrity. Arrow indicates where undigested DNA would run. The gel was stained with ethidium bromide for visualization of bands.

PAGE 114

101 Figure 4-2. Cloning of LMB StAR promoter. The GenomeWalk er protocol was used to amplify the promoter for LMB StAR The 2.9 Kb length promoter was sequenced and ligated into the pCR 2.1-TOPO cloning vect or (Invitrogen). Creation of the promoter construct wa s confirmed with a double digest using EcoRV and DraI restriction enzymes. A) Low DNA mass ladder (Promega). B) EcoRV/ DraI double dige st of the LMB StAR promoter ligated into the TOPO vector.

PAGE 115

102 Figure 4-3. 5 and 3 untran slated region (UTR) for LMB StAR and identification of transcription start site. The overall structure for the LMB StAR gene is outlined and the DNA sequence for the 5 and 3 UTRs are shown. Both the 5 and 3 UTRs were obtained using RACE. 5 RACE was also used to identify the transcription start site, which is the first nucleotide following the end of the 5UTR.

PAGE 116

103 5CTGAGCTCACAGTGTTGCACAGAGTGAGGACTG AGTTTTCCCCCCATAATTTACATTTAAATTTCTTATT TCTTTTTAGTGAACAGGAGGAACATTTCAAATGGGC ATGTTTTGTTTTATGATAGACTTTTCCCTCAGAGC ACCTATTTTGGGAGTCACTGTACTTCATTACTTCAT GTAAATGCTCCGTACTTGTATAGCGCCTT TCTAGTC TTTTTGACCACT CAAAGCGCTTTTACATGTACATCTGCCTTCA CCAGTCACACACATTCATACACTGAGCC TAAGTGCTCAAACGGAAACTAACATTCACACTCAC TCGTACACTGGCGGAATAGCCCCCAGGGGCAATTC GGGGTTCA GTATCTTGCCCA AGGACACTTCGACCAGGGGGAGCCAGGGATTGAACCGCCGACCTTCCGA TTAACGGCCAACCTGCTCTACCTCCTGAGCCACAGC CGCCCTATCATATTGCAAATGAATGCCATCAGTG CTTGACTCAGGCAGAATTGTGGGATAAAGGGCCT GCTTCAAGGAATCTGAAAAAAATGGTCTGAAACCA GAGGTGACAGGGTGATATTTGCGAA GGAGCTTTGGACACA AACGTCCTTTCCTGAATCGCTTTTATCAAC CTTTATTGCACCTTTAATGTAATGTATGTTAC AGGTAAAAGAAGATCATTTT AACAAGCTCCTGGGTCCTG GTGTCATCATCAAACCCAACATGCCTGTTTGGCTC GTACATCTACAATACACAATCTTTCAGTAAGATTCT TACTTTTCAGATTTAGTTTTTTTAT AGGAGAGACTCTTTAACCTTCAGGTTCTCA CACCCTTG CTAGAAAA AAGAATTGCAGTTTTCCC CATGGCCAATGCTCA TTAAAACCTGAAACTGCTCTTGACAAATAGGGAGCCA TTTG AAATAGGCATATGACCTACTTTG GCTCTTTGAAAAAAGAGCTTTACGGC AGAAGAGACAT GGGTGT CTGGTTTGTCCACTGTGGCTGAGTAAT GGG TCAGATAGT ACTAG GCCTGTGTGTTCCCATATTAAATG AG AGAGGTCACCCTGCAGAAG GCCAGAAGAACTGATGGATAATT CCCTCCATCCAGCCAGAGGGACGCAAG GTTGCAGGTTAAGGTGACTCCGATTTTTCTTTGGGATT AATGACTGTTCCTGTTTTCAATGTGCACATCCCT GTTCACCAATCAGATGGCCTGCTTCTCTCTTGACAGGTTTCTATTCCTCTAGTCATCATGTGGACTGTTGA CTCTGAGTGCATGTTTGACGTGAGACAATATTGCT GTTAGATAGTTTGGATTTTGCATGGACTGAGTAAGC AAGGTTGTCATGAGTACCCTGATAAAACGTGTCATT AACAGAATATGACGTG CCATTTGTAAGCCTGTTG GAAAACTTGTGATTCAATCTCA CTGAAGAACCCGAGGCAGAGTC AGTTTGGATGTCAGTGTTA TGAACTG TGATGTGT TTCACAAGGTCCATAAACGCCA TGGAGAAAGTGACAGGCGCTGCTTTCATTAATTTCGAACC AAATGAAATAATTTGTCATGCTTATTCACAGATACTG GCTTTTTTCATTTTTGTG CCAGAGCATTTGATTTA TTGATTTCTGCTGGGATGTAAAGAGACTTTTCAACAACTC TGACAAAAATGCTTCT GAAATAAATTGACT AAACCCCAGATTTAAGTAGATTCTTACCTAAAAAATGAC TTCATAGTGTCCATTAACAGGCTTCTTTCCAG CCAAA ATGTCCTTG AACAAAACATGTTACTACCACCTGC TCCTTCACAATAAACTGCCCAAAGTATGGAC TTTTCAATTACACTGTCATGTAATTGCTGTGACCT CATGGTTACATTTCGTTG CTGTAATTCAGTTTTTGGA ATTTTTTTTTTTTGGGACGTTCTTCAAA CCCACAACGAAGAAAATTACT GTTAAAAGACCTGTTTATTCCT CCCTTTGAAACAAGCTGGACTCATGCATTTGCTGAAGGT GATACATTCTGGAAATGA CATGAATAATAAA CCAAATATGAAAATATTTCATGTTT GAAACAAATATATACATAT ATATATTTATTTTG TTTATTTTTATTTT GTTTGCTATAATTTTGTTCACTGACGAAGACACTGT AAAGTATGTGGACT CCGTGTTAGACCCTTTTTGAA GTTTCCAAAAACAGAAATCCAAATAG ACTGCCAGCACCAATCACTGTACTTACACTAAGATTTTGGACTT TTTAACCATGTCATCGTTTGAAAATTA TATTTATTTTAAGTAACTGTCAC ATAATTAAGATAAATCATTGA GTTTCAAGGTGATACGTTTAGGTTTTGAGCTGTTAAAAA GGATATAGAGTCTCCAGG ACTCCAAACCGAA CATTAGGGTTAATAGCTGTCAAT CATAACAAGGAAGAAACAAACCA AAATGAATTGAAATATTTTGGCCT CAAATTCCAGTGAGTGATTAGACTGCAATGGACCA TGAAGGAATGGATGAGTCACTCAGGGTCTCCCCTA CTGACCTCTGTTTTGTGCACTCT CATCATTGAAGAGAGGCTGCTCTC TTGTGTTTCACCCACTGGTATGAG AATGTTTGTGAATTTGTGTGTGTGTG TGTGTGTGTGTGTGTG TGTGTGCCCGTGTGTG TGTGTATGTGTGT GAGTGAGCAATGCAGTGGGCCT GGGCACTGGACGTTATCTGACAC AGTGTTTGTGAGAGTCTGAGACATT GCTCGTCGTGTCCGCCACG CAACCTTG ACGACATGGCCATTGGAACAGTGAGGTGATGTGTGTGTGTGTG TGTTTATCTATGTGGTGATAACACGGCGACCTTGTGGGCAGTGCTCTTATATATACAGCACTTTCAAACGT CCG 3 Figure 4-4. Putative transcri ption response elements id entified in the LMB StAR promoter. Sequence for 2.9 Kb of the LMB StAR promoter was analyzed by a professional web-based program, MatI nspector, for putative transcription sites. Several potential sites identified are highlighted: = CREBP; = AP-1; = GATA; = SF-1; =AhRE; = ER; = SF-1; = ROR; = RAR; = COUP-TF 1 -427 -921 -1417 -1910 -2402 -2825

PAGE 117

104 190 200 210 220 230 240 | | | | | | LMB Clone 1 --------------------------------PY T S S P E S IMG Y -A Y V D A Y Q S G SP P SF LMB Clone 2 --------------------------------PY T S S P E S IMG Y -A Y V D A Y Q S G SP P SF Rainbow Trout SMAMPPHAGSLQGYQAAYGHFQSTRTIKSEYPD PY T S S P E S IMG Y NP Y M D A Y Q T G SP P SF sheep PMAVPSTHGPLAGY--LYPAFPG-RAIKSEYPE PY A S P P Q A GPP Y -G Y P E P F S G G -P GV human PMAVPGAHGPLAGY--LYPAFPG-RAIKSEYPE PY A S P P QP GLP Y -G Y P E P F S G G -P NV **:*.*:. * :.:. *.. Prim.cons. PMAVP33HGPLAGYQALYPAFPGTRAIKSEYPEPYTSSPESIMGYN2Y2DAYQ2GSPPSF 250 260 270 280 290 300 | | | | | | LMB Clone 1 P H LI VE LL K C EPDE P QV Q A K I L AY LQ Q EQA SR G K H EK LN T F G L M C K MADQT LF SIV E WAR LMB Clone 2 P H LI VE LL K C EPDE P QV Q A K I L AY LQ Q EQA SR G K H EK LN T F G L M C K MADQT LF SIV D WAR Rainbow Trout PH LI VE LL K C EPDE P QV Q A K I L TY LQ Q EQA SR G K H EK LN T F G L M C K MADQT LF SIV E --sheep P E LI LQ LL Q L EPDE D QV R A R I V GC LQ E P-AK G R P DQ PA P F S L L C R MADQT FI SIV D WAR human P E LI LQ LL Q L EPDE D QV R A R I L GC LQ E P-TK S R P DQ PA AF G L L C R MADQT FI SIV D WAR *.**::**: **** **:*:*: **: ::.: :: .*.*:*:*****::***: Prim.cons. PHLIVELLKCEPDEPQVQAKIL2YLQQEQASRGKHEKLNTFGLMCKMADQTLFSIVDWAR 310 320 330 340 350 360 | | | | | | LMB Clone 1 RCMVFNH----------------------------------------------------LMB Clone 2 RCMVFNH----------------------------------------------------Rainbow Trout -----------------------------------------------------------Sheep RCMVFKELEVADQMTLLQNCWSELLVFDHIYRQIQHGKESSILLVTGQEVELTTVAAQAG Human RCMVFKELEVADQMTLLQNCWSELLVFDHIYRQVQHGKEGSILLVTGQEVELTTVATQAG Prim.cons. RCMVF22LEVADQMTLLQNCWSELLVFDHIYRQ2QHGKE2SILLVTGQEVELTTVA2QAG Figure 4-5. Cloning of LMB SF1. A) A 246 base pair sequence for SF-1 was amplified from LMB ovarian tissue using specific primers and gel purified using spin columns from Qiagen (gel purified P CR product indicated by arrow). A DNA ladder was run to approximate the size of the amplified gene. B) Alignment of two different clones sequenced fo r LMB SF-1 with rainbow trout, sheep, and human revealed a 42% similarity with mammals and greater than 90% similarity with other fish for the fr agment cloned within the ligand binding domain.

PAGE 118

105 Figure 4-6. Optimization of ratio for transf ection reagent (Fugene6) and promoter DNA concentration. Y-1 cells were transfected for 6 hours with either a 3:1, 3:2, or 6:1 ratio of Fugene6 (l)/promoter DNA (g) and exposed to 0 or 1mM dbcAMP for 20 hours. All wells were norma lized to Renilla. Students t-test was used to determine significance and a indicates a P < 0.05.

PAGE 119

106 Figure 4-7. Optimization of transf ection timepoint. Y-1 cells were transfected for 6 or 24 hours prior to a 20 hour exposure of 0 or 1 mM dbcAMP. Students t-test was used to determine significance and a indicates a P < 0.05.

PAGE 120

107 Figure 4-8. Quantitation of DNA transtec tion with GFP. GFP (0.1995 g) was transfected into Y-1 cells to measur e the amount of DNA getting transfected into the cells using the Fugene6 prot ocol. A) GFP fluorescence in a 1 mm2 square for one well of a 24-well plat e. B) GFP flouresence in 1 mm2 square of a hemacytometer using cells that we re trypsinized and diluted 1/10. C) Picture of 1 mm2 hemacytometer square in figure B with no fluorescence. Cells that fluoresced in figure B are indica ted by arrows in figure C.

PAGE 121

108 Figure 4-9. Dose response expos ure of Y-1 cells to dbcAMP. Y-1 cells were transfected with the 2.9 Kb LMB StAR promoter and exposed to increasing doses of cAMP from 0 to 2 mM dbcAMP. Re presented is one experiment done in triplicates which has been repeated 4 times. All values are normalized to Renilla. Students t-test was used to determine significance and a indicates a P < 0.05.

PAGE 122

109 Figure 4-10. Creation of promoter deletion a nd site-mutagenesis constructs. A deletion of the 2.9 Kb LMB StAR promoter to 1.86 Kb was created by digestion with EcoRV and StuI to 1.86 Kb. The dele tion was verified by digestion with DraI. A and F) DNA mass ladder B) unc ut 2.9 Kb C) DraI cut 2.9 Kb D) uncut 1.86 Kb E) DraI cut 1.86 Kb. S ite-directed mutati ons of potentially important transcription elements were made using the Stratagene QuikChange system. Primers were designed to mu tate 5 different putative response elements to a NotI restriction site. Cr eation of the constructs was verified by restriction digest with NotI; H) ERE/2678 construct I) COUP-TF/2027 construct J) GATA/AP-1/ERE/1882 cons truct K) COUP-TF/2304 construct L) ROR/1969 construct.

PAGE 123

110 Figure 4-11. Promoter deletion analysis. The 2.9 Kb and 1.86 Kb constructs were exposed to cAMP for 20 hours and TGFfor 40 hours to examine differences in transcriptional activity between the two promoter lengths.

PAGE 124

111 Figure 4-12. Exposures of promoter site-mutag enesis constructs to dbcAMP. Mutation of five putative transcri ptional elements between the 1.86 and 2.9 Kb region were made and individually evalua ted for induction by 1 mM dbcAMP after 20 hours via transfection in Y-1 cells. A ll values were normalized to renilla luciferase. All experiments were done in triplicates and repeated at least 3 times. Students test was used to test for significance and a indicated a P < 0.05.

PAGE 125

112 Figure 4-13. StAR promoter mutation analysis with TGFregulation. Various mutations of putative response elemen ts in the 2.9 Kb promoter were transfected into Y-1 cells and the regulation by 20 ng/ml TGFwas compared with the 1.86 Kb construct. The regulation of the endogenous mouse StAR mRNA in the Y-1 cells was also tested to help interpret the transfection results. All wells were normalized to renilla.

PAGE 126

113 Figure 4-14. Endogenous mRNA regulation of StAR in Y-1 cells by TGF. Y-1 cells were cultured in 24well plates and exposed to 10 ng/ml TGFfor 40 hours. RNA was extracted from the cells an d reverse transcribed to cDNA for analysis by real-time PCR with primers that probe for mouse StAR. All samples were normalized to 18S rRNA. Students test was used to test for significance and a indicated a P < 0.05.

PAGE 127

114 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Regulation of the StAR Prot ein is not fully understood for any species, primarily since it was only identified in th e last decade. StAR is involved in the critical step of cholesterol metabolism (Clark and Sto cco, 1996). Homeostasis of cholesterol metabolism is applicable to so many diffe rent diseases, includ ing heart disease, hypertension, LCAH (lipoid congenital adre nal hyperplasia), among others (Khoury et al., 2004; Moghadasian and Frohlic h, 1999). Understanding the re gulation of StAR at the level of transcription, transl ation, or post-translation are all crucial to pinpointing differences in StAR function under normal ve rsus atypical conditions. Additionally, the regulatory elements can be modulated by endogenous molecules or by exogenous ligands such as environmental toxins (Walsh et al., 2000; Barlow et al., 2003). This project primarily examines the transcriptional contro l of StAR using ovarian follicle cultures and transfection assays. The promoter for StAR is very complex with many transcriptional elements for which the function is still unknown. SF-1, DAX-1, GATA, AP-1, AhR, and CRE elements are consistently found in the mamm alian StAR promoters (Stocco et al., 2001; Sugawara et al., 2001), however, professiona l web-based programs identify many other potentially critical elements such as ER COUP-TF, ROR, RAR, GR, TTF1, or SMAD sites. Although the genome project is now complete for many species, the length of many of the StAR promoters used in past publications is typica lly around 1000 base pairs

PAGE 128

115 or less, leaving many of the sites upstream from this either unidentified or uncharacterized. A main objective of this di ssertation was to clone a large portion of the StAR promoter and to study its regulation by power ful signaling molecules such as cAMP and TGF. It is important to characterize signali ng pathways that activ ate or repress StAR since aberrant regulation on e ither side of this spectrum could have severe impact on steroidogenesis. In fact, publications show steroids are downregulated through modulation of StAR by environmental toxins such as insecticides and herbicides, however, the signaling pathways triggered ar e unknown (Walsh et al., 2000; Barlow et al., 2003). LMB was the model used for all studies in this project since their reproductive capabilities are severely repressed by downregulation of steroids upon paper mill contaminant exposure (Sepulveda, 2001). A dditionally, there are no published reports of transcriptional regulation of StAR in lowe r vertebrates, therefore, conservation or deviation from transcriptional elements in the mammalian promoters could provide valuable information. A large portion of the LMB StAR promot er, 2.9 Kb, was cloned and analyzed. Web based analysis showed numerous putat ive response elements, many of which are conserved with those published for mamma lian species, including SF-1, AP-1, AhR, and GATA sites. Additionally, RAR, ROR, and full ERE sites were found in both the LMB and mammalian promoters, however, nothing had been previously published about either their identification or functi on in StAR regulation.

PAGE 129

116 The presence of SF-1, AP-1, and GATA sites suggested that the LMB StAR promoter is regulated by cAMP in spite of differences in the DNA sequence. Transfection of the 2.9 Kb promoter confir med that LMB StAR is induced in a doseresponsive manner with dbcAMP, suggesting th at the function of StAR is maintained across species. Most of the transcriptional activation fo r mammalian StAR was reported to be in the first couple hundred base pairs. The transfections with LMB StAR deletion and mutation constructs suggests that regions further upstream from this region, between the 1.86 Kb and 2.9 Kb region, are extremely impor tant, at least for cAMP regulation. Mutation of no more than 5 nucleotides in the COUP-TF, ROR, or GATA/AP-1/ERE sites minimized the cAMP induction compar ed to unmutated controls. The GATA/AP1/ERE mutation served somewhat as a cont rol for the COUP-TF and ROR data since GATA and AP-1 are necessary for cAMP induc tion of mammalian StAR (Manna et al., 2004; Stocco et al., 2001). The transfection data actually sugg ests a novel mechanism for COUP-TF functioning. Previous publica tions show COUP-TF is involved in the downregulation of steroid synthesis via competitive binding to other sites such as RAR (Zhang and Pfahl, 1993). The LMB data shows that mutation of the COUP-TF site diminishes cAMP induction, suggesting that the response element for COUP-TF binds a protein which activates transcription. Th e only study that analyzed StAR regulation by COUP-TF involved overexpression of COUP-TF in a cel l line and subsequent observation of StAR transcriptional activity, howeve r, the function of the actua l response element has never been examined prior to this study (Buholzer et al., 2005).

PAGE 130

117 The same mutations in the LMB promot er were tested for regulation by TGFsince a major goal of this project was to el ucidate a signaling pathway for repression of StAR by environmental toxins. TGFhas been shown to dow nregulate steroidogenesis and also StAR itself in mammalian species, however, the mechanism is unclear (Brand et al., 2000). Mutation of COUP-TF/2304, GAT A/AP-1/ERE/1882, or ROR/1969 sites did not significantly change the regulation of LMB StAR promoter transcription by TGF, suggesting these response elements exam ined are modulated primarily by cAMP dependent signaling. The ERE/2678 and Coup/ 2027 sites might be responsive to TGF, however, their exact role is still unclear. LMB ovarian follicle cultures were very useful to study the effects of cAMP and TGFon expression of the endogenous LMB StAR mRNA and to correl ate that with the transfection data. The follicl e data showed that cAMP upregulates LMB StAR in a doseresponsive manner, matching the transfection results. When follicles between 0.8 and 1.1 mm were exposed to TGF, the mRNA expression of StAR was significantly repressed, substantiating the result s seen with the 1.86 Kb StAR promoter. The combination of ovarian follicle, pr omoter deletion, and promoter mutation data implies some very intere sting regulation by cAMP and TGFin the 1.86 Kb to 2.9 Kb region which needs to be fully examined. To date, almost all of the studies have focused on the first 1000 to 1500 base pairs of the StAR promoter, however, the LMB data shows that over 80% loss in transcriptional activity can be attributed to one site, including a COUP-TF, GATA/AP-1/ERE, or RO R site, upstream of 2000 base pairs of promoter.

PAGE 131

118 We identified critical response elements for cAMP induction of LMB that were not subjected to regulation by TGF. There are numerous transcriptional elements in the 1.86 Kb to 2.9 Kb region that could provide va luable information for the protein-protein or protein-DNA interactions which TGFmodulates. There are SMAD3 and FAST-1 sites in the StAR promoter which had previous ly not been identified or characterized and may help further elucidate the TGFpathway. The SMAD3 site located at about 1.9 Kb in the LMB StAR promoter may not have been previously identified for any other species since it is somewhat far upstream. Additionally there are at least 6 of the FAST-1 sites, an activin regulated SMAD interacting partne r, within the 2.9 Kb LMB promoter. Coincidentally, several of the FAST-1 sites are located within 70 nuc leotides of a COUPTF site for the LMB, brook trout and zebrafish StAR promoters, which could be another possible interaction to examine.

PAGE 132

119 REFERENCES Aesoy R, Mellgren G, Morohashi K, L und J. 2002. Activation of cAMP-dependent protein kinase increases th e protein level of steroi dogenic factor-1. Endocrinology. 143(1):295-303. Arakane F, King SR, Du Y, Kallen CB, Walsh LP, Watari H, Stocco DM, Strauss JF 3rd. 1997. Phosphorylation of Steroidogenic Acute Regulatory Protein (StAR) modulates its steroidogenic activ ity. J Biol Chem. 272(51):32656-62. Aringer L, Eneroth P, and Nordstrom L. 1979. Si de chain cleavage of 4-cholesten-3-one, 5-cholesten-3-ol, -sitosterol, and related steroids in endocrine tissues from rat and man. J Steroid Biochem. 11:1271-1285. Babin PJ. 1986. Effect of plasma lipoprotei ns in gonadotropin stimulation of 17estradiol production in the ovarian follicle of rainbow trout. Gen Comp Endocrinol. 64:456-467. Barger PM, Kelly DP. 1997. Identification of a retinoid/chicken ovalbumin upstream promoter transcription factor response element in the human retinoid X receptor gamma2 gene promoter. J Biol Chem. 272(5):2722-8. Barlow NJ, Phillips SL, Wallace DG, Sar M, Gaido KW, Foster PM. 2003. Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicol Sci. 73(2):431-41. Bastien J, Rochette-Egly C. 2004. Nuclear re tinoid receptors and the transcription of retinoid-target genes. Gene. 328:1-16. Bennett R, Heftmann E, Winter B. 1969. Conve rsion of sitosterol to progesterone by Digitalis Lanata. Naturw issenschaften. 56(9):463. Berrodin TJ, Marks MS, Ozato K, Linney E, Lazar MA. 1992. Heterodimerization among thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, chicken ovalbumin upstream promoter transcripti on factor, and an endogenous liver protein. Mol Endocrinol. 6(9):1468-78. Bertherat J. 1998. The nuclear receptor SF-1 (steroidogenic factor-1) is no longer an orphan. Eur J Endocrinol. 138:32-33.

PAGE 133

120 120 Bjornstrom L, Sjoberg M. 2005. Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on targ et genes. Mol En docrinol.19 (4):83342. Bose HS, Whittal RM, Baldwin MA, M iller WL. 1999. The active form of the Steroidogenic Acute Regulatory Protein, StAR, appears to be a molten globule. Proc Natl Acad Sci U S A. 96(13):7250-5. Boukhtouche F, Mariani J, Tedgui A. 2004. Th e "CholesteROR" protective pathway in the vascular system. Arterioscler Thromb Vasc Biol. 24(4):637-43. Bowman CJ, Kroll KJ, Gross TG, Denslow ND. 2002. Estradiol-induced gene expression in largemouth bass (Micropterus salmoi des). Mol Cell Endocrinol. 196(1-2):67-77. Brand C, Nury D, Chambaz EM, Feige JJ, Ba illy S. 2000. Transcrip tional regulation of the gene encoding the StAR protein in the human adrenocortical cell line, H295R by cAMP and TGFbeta1. Endocr Res. 26(4):1045-53. Brand C, Souchelnytskiy S, Chambaz EM, Fe ige JJ, Bailly S. 1998. Smad3 is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on StAR expression. Biochem Biophys Res Commun. 253(3):780-5. Buholzer CF, Arrighi JF, Abraham S, Pi guet V, Capponi AM, Casal AJ. 2005. Chicken ovalbumin upstream promoter-transcripti on factor is a negative regulator of steroidogenesis in bovine adrenal glomer ulosa cells. Mol Endocrinol. 19(1):65-75. Butler AJ, Parker MG. 1995. COUP-TF II homod imers are formed in preference to heterodimers with RXR alpha or TR beta in intact cells. Nucleic Acids Res. 23(20):4143-50. Callard GV, Tchoudakova AV, Kishida M, Wood E. 2001. Differential tissue distribution, developmental programmi ng, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. J Steroid Biochem Mol Biol. 79(15):305-14. Calonge MJ, Seoane J, Massague J. 2004. Opposite Smad and Chicken Ovalbumin Upstream Promoter Transcription Factor inputs in the regulation of the collagen VII gene promoter by Tran sforming Growth Factor. J Biol Chem. 279(22):23759-23765. Carter BA, Karpen SJ. 2001. Diet and disease: the "phyte" over intestinal cholesterol. Gastroenterology. 121(5):1255-6. Carvan MJ 3rd, Solis WA, Gedamu L, Nebe rt DW. 2000. Activation of transcription factors in zebrafish cell cultures by environmental po llutants. Arch Biochem Biophys. 376(2):320-7.

PAGE 134

121 121 Christenson L, Strauss J. 2000. Steroidogenic Acute Regulatory Protein (StAR) and the intramitochondrial transloca tion of cholesterol. Biochim Biophys Acta. 1529:175187. Clark BJ, Ranganathan V, Combs R. 2001. Steroidogenic Acute Regulatory Protein expression is dependent upon pos t-translational effects of cAMP-dependent protein kinase A. Mol Cell Endocrinol. 173(1-2):183-92. Clark BJ, Soo S, Caron K.1995. Hormonal and developmental regulation of the steroidogenic acute regulatory prot ein. Mol. Endocrinol. 9:1346-1355. Clark BJ, Stocco DM. 1996. StARA tissue specif ic acute mediator of steroidogenesis. TEM. 7(7):227-233. Clark BJ, Wells J, King SR, Stocco DM. 1994. The purification, cloning, and expression of a novel luteinizing ho rmone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of th e steroidogenic acute regulatory protein (StAR). J Biol Chem. 269(45):28314-22. Compassi S, Werder M, Weber FE, Bo ffelli D, Hauser H, Schulthess G. 1997. Comparison of cholesterol and sitoster ol uptake in different brush border membrane models. Biochemistry. 36(22):6643-52. de Caestecker M. 2004. The transforming grow th factor-beta superfamily of receptors. Cytokine Growth F actor Rev. 15(1):1-11. De Servi B, Hermani A, Medunjanin S, Maye r D. 2005. Impact of PKCdelta on estrogen receptor localization and activity in breas t cancer cells. Oncogene. [Epub ahead of print] Dohr O, Vogel C, Abel J. 1994. Modulat ion of growth factor expression by 2,3,7,8tetrachlorodibenzo-p-dioxin. Exp Clin Immunogenet. 11(2-3):142-8. Field FJ, Born E, Mathur SN. 1997. Effect of micellar -sitosterol on cholesterol metabolism in CaCo-2 cells. J Lipid Res. 38:348-360. Fielden MR, Halgren RG, Fong CJ, Staub C, Johnson L, Chou K, Zacharewski TR. 2002. Gestational and lactational exposure of ma le mice to diethylstilbestrol causes longterm effects on the testis, sperm fertilizi ng ability in vitro, a nd testicular gene expression. Endocrinology. 143(8):3044-59. Fleury A, Mathieu AP, Ducharme L, Ha les DB, LeHoux JG. 2004. Phosphorylation and function of the hamster adrenal steroidoge nic acute regulatory protein (StAR). J Steroid Biochem Mol Biol. 91(4-5):259-71. Gautier C, Levacher C, Saez JM, Habert R. 1997. Transforming growth factor beta1 inhibits steroidogenesis in dispersed fetal testicular cells in culture. Mol Cell Endocrinol. 131(1):21-30.

PAGE 135

122 122 Ginsberg HN. 1998. Lipoprotein physiology. Endocrinol Metab Clin North Am. 27(3):503-19. Goetz FW, Norberg B, McCauley LA, I liev DB. 2004. Characterization of the cod (Gadus morhua) steroidogenic acute regulat ory protein (StAR) sheds light on StAR gene structure in fish. Comp Bioche m Physiol B Biochem Mol Biol. 137(3):35162. Granot Z, Geiss-Friedlander R, MelamedBook N, Eimerl S, Timberg R, Weiss AM, Hales KH, Hales DB, Stocco DM, Orly J. 2003. Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins. Mol Endocrinol. 12:2461-76. Gutendorf B, Westendorf J. 2001. Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synt hetic estrogens, phytoestrogens and xenoes trogens. Toxicology. 166:79-89. Hammer G, Ingraham H. 1999. Steroidogenic Factor-1: Its role in endocrine organ development and differentiation. Fr ont Neuroendocrinol. 20:199-223. Hasegawa T, Zha L, Caron K, Majdic G, Suzuki T, Shizawa S, Sasano H, Parker K.2000.Developmental roles of the Steroi dogenic Acute Regulatory Protein as revealed by StAR knockout mice. Mol Endocrinol. 14(9):1462-71. Honda S, Morohashi K, Nomura M, Takeya H, Kitajima M, Omura T. 1993. Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily. J Biol Chem. 268(10):7494-502. Huang TJ, Shirley Li P. 2001. Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulator y protein in cultured rat preovulatory follicles. Biol Reprod. 64(1):163-70. Ito M, Achermann JC, Jameson JL. 2000. A natu rally occurring steroidogenic factor-1 mutation exhibits differential binding and activation of ta rget genes. J Biol Chem. 275(41):31708-14. Jarvis CI, Staels B, Brugg B, Lemaigre -Dubreuil Y, Tedgui A, Mariani J.2002. Agerelated phenotypes in the staggerer mouse expand the RORalpha nuclear receptor's role beyond the cerebellum. Mo l Cell Endocrinol. 186(1):1-5. Jones P, Howell T, MacDougall D, Feng J, Pars ons W. 1998. Shortterm administration of tall oil phytosterols improves plasma lipid profiles in subjects with different cholesterol levels. Metabolism 47(6):751-6. Jo Y, Stocco DM. 2004. Regulation of st eroidogenesis and Steroidogenic Acute Regulatory Protein in R2C cells by DAX-1 (dosage-sensit ive sex reversal, adrenal hypoplasia congenita, critical regi on on the X chromosome, gene-1). Endocrinology. 145(12): 5629-37.

PAGE 136

123 123 Kagawa H, Gen K, Okuzawa K, Tanaka H. 2003. Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream, Pagrus major. Biol Reprod. 68(5):1562-8. Kallen CB, Billheimer J, Summers S, Stayrook S, Lewis M, Strauss J. 1998. Steroidogenic Acute Regulatory Protein is a sterol transfer pr otein. J Biol Chem. 273(41):26285-26288. Kallen JA, Schlaeppi JM, Bitsch F, Geisse S, Geiser M, Delhon I, Fournier B. 2002. Xray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a chol esterol derivative is the natural ligand of RORalpha. Structure (Camb). 10(12):1697-707. Kassen A, Berges R, Senge T. 2000. Effect of beta-sitosterol on transforming growth factor-beta-1 expression and translocation protein kinase c alph a in human prostate stromal cells in vitro. Eur Urol. 735-741. Kazeto Y, Ijiri S, Place AR, Zohar Y, Trant JM. 2001. The 5'-flanking regions of CYP19A1 and CYP19A2 in zebrafis h. Biochem Biophys Res Commun. 288(3): 503-8. Khoury K, Ducharme L, LeHoux JG. 2004. Family of two patients wi th congenital lipoid adrenal hyperplasia due to StAR mutation. Endocr Res. 30(4):925-9. King SR, Matassa AA, White EK, Walsh LP, Jo Y, Rao RM, Stocco DM, Reyland ME. 2004. Oxysterols regulate expression of the Steroidogenic Acute Regulatory Protein. J Mol Endocrinol. 32(2):507-17. Klinge CM, Silver BF, Driscoll MD, Sathya G, Bambara RA, Hilf R. 1997. Chicken ovalbumin upstream promoter-transcription f actor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression. J Biol Chem. 272(50):31465-74. Kusakabe M, Nakamura I, Young G. 2003. 11beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid in rainbow trout: cl oning, sites of expression, and seasonal changes in gonads. Endocrinology. 144(6):2534-45. Lee PS, Chang C, Liu D, Derynck R.2003. Sumoylation of Smad4, the common Smad mediator of transforming growth facto r-beta family signaling. J Biol Chem. 278(30):27853-63. Lee HK, Yoo MS, Choi HS, Kwon HB, S oh J. 1999. Retinoic acids up-regulate steroidogenic acute regulat ory protein gene. Mol Ce ll Endocrinol. 148(1-2):1-10. Lehtinen K, Mattsson K, Tana J, Engstrom C, Lerche D, Hemming J. 1999. Effects of wood-related sterols on the reproduction, e gg survival, and offspring of brown trout. Ecotoxicol Environ Saf. 42(1):40-9.

PAGE 137

124 124 Liakos P, Lenz D, Bernhardt R, Feige JJ, Defaye G. 2003. Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression. J Endocrinol. 176(1):69-82. Li YY, Inoue K, Takei Y. 2003. Steroidogeni c Acute Regulatory Protein in eels: cDNA cloning and effects of ACTH and seaw ater transfer on its mRNA expression. Zoolog Sci. 20(2):211-9. Logan KA, Juengel JL, McNatty KP. 2002. On set of steroidogenic enzyme gene expression during ovarian follicular devel opment in sheep. Biol Reprod. 66(4):90616. Luo S, Yu H, Wu D, Peng C. 2002. Transf orming growth factor-beta1 inhibits steroidogenesis in human trophoblast cel ls. Mol Hum Reprod. 8(4):318-25. Lu K, Lee MH, Patel SB. 2001. Dietary choles terol absorption; more than just bile. Trends Endocrinol Metab. 12(7):314-20. MacLatchy DL, Peters L, Nickle J, Van Der Kraak GJ. 1997. Exposure to -sitosterol alters the endocrine status of goldfish differently than 17-estradiol. Environ Toxicol Chem. 16(9):1895-1904. MacLatchy DL, Van Der Kraak GJ. 1995. The phytoestrogen -sitosterol alters the reproductive endocrine status of goldfis h. Toxicol Appl Pharmacol. 134:305-312. Manabe N, Goto Y, Matsuda-Minehata F, I noue N, Maeda A, Sakamaki K, Miyano T. 2004. Regulation mechanism of selective atre sia in porcine folli cles: regulation of granulosa cell apoptosis during at resia. J Reprod Dev. 50(5):493-514. Manna PR, Dyson MT, Eubank DW, Clark BJ, Lalli E, Sassone-Corsi P, Zeleznik AJ, Stocco DM. 2002. Regulation of steroi dogenesis and the Steroidogenic Acute Regulatory Protein by a member of the cAMP response-element binding protein family. Mol Endocrinol. 16(1):184-99. Manna PR, Eubank DW, Stocco DM. 2004. Assessmen t of the role of activator protein-1 on transcription of the mouse Steroidoge nic Acute Regulatory Protein gene. Mol Endocrinol. 18(3):558-73. Marino M, Distefano E, Trentalance A, Smith CL. 2001. Estradiol-induced IP(3) mediates the estrogen receptor activit y expressed in human cells. Mol Cell Endocrinol. 182(1):19-26. Mathieu AP, Lavigne P, LeHoux JG. 2002. Mo lecular modeling and structure-based thermodynamic analysis of the StAR protein. Endocr Res. 28(4):419-23.

PAGE 138

125 125 Matsuda T, Yamamoto T, Muraguchi A, Saatcioglu F. 2001. Cross-talk between Transforming Growth Factor-B and estr ogen receptor signal ing through Smad3. J Biol Chem. 276(46):42908-42914. McMaster ME., Van Der Kraak GJ., Munk ittrick K.1995. Exposure to bleached kraft pulp mill effluent reduces the steroid bios ynthetic capacity of white sucker ovarian follicles. Comp Biochem Physiol. 112C(2):169-178. McMaster ME, Van Der Kraak GJ, Munk ittrick KR. 1996. An evaluation of the biochemical basis for steroid hormone depr essions in fish exposed to industrial wastes. J Great Lakes Res. 22:153-171. Moghadasian M, Frohlich J. 1999. Effect s of dietary phytosterols on cholesterol metabolism and atherosclerosis: clinical and experimental evidence. Am J Med. 107:588-594. Nagahama Y, Yoshikuni M, Yamashita M, Tokumoto T, Katsu Y. 1995. Regulation of oocyte growth and maturation in fish. Curr Top Dev Biol. 30:103-145. Nawata H, Yanase T, Oba K, Ichino I, Saito M, Goto K, Ikuyama S, Sakai H, Takayanagi R. 1999. Human Ad4BP/SF-1 and its related nuclear receptor. J Steroid Biochem Mol Biol. 69(1-6):323-8. Pang Y, Ge W. 2002. Gonadotropin and activ in enhance maturational competence of oocytes in the zebrafish (Danio rerio). Biol Reprod. 66(2):259-65. Petrescu AD, Gallegos AM, Okamura Y, Strauss JF 3rd, Schroeder F. 2001. Steroidogenic Acute Regulatory Prot ein binds cholesterol and modulates mitochondrial membrane sterol domai n dynamics. J Biol Chem. 276(40):36970-82. Petrino TR, Shuetz AW. 1986. Protein synthesi s and steroidogenesis in amphibian (rana pipiens) ovarian follicles: studies on the conversion of pregnenolone to progesterone. Gen Comp Endocrinol. 63:441-450. Pezzi V, Mathis JM, Rainey WE, Carr BR. 2003. Profiling transcript levels for steroidogenic enzymes in fetal tissues. J Steroid Biochem Mol Biol. 87(2-3):181-9. Pfahl M. 1993. Signal transduction by retinoid receptors. Skin Pharmacol. 6 Suppl 1:816. Pichler H, Riezman H. 2004. Where sterol s are required for endocytosis. Biochim Biophys Acta. 1666(1-2):51-61. Reinhart AJ, Williams S, Stocco DM. 1999. Tran scriptional regulation of the StAR gene. Mol Cell Endocrinol. 151:161-169.

PAGE 139

126 126 Reyland M, Evans R, White E. 2000. Li poproteins regulate expression of the Steroidogenic Acute Regulatory Protein in mouse adrenocortical cells. J Biol Chem. 275(47):36637-36644. Rosanoff A, Seelig MS. 2004. Comparison of mechanism and functional effects of magnesium and statin pharmaceutical s. J Am Coll Nutr. 23(5):501S-505S. Rust W, Stedronsky K, Tillmann G, Morley S, Walther N, Ivell R. 1998. The role of SF1/Ad4BP in the control of the bovine gene for the Steroidogenic Acute Regulatory Protein. J Mol Endocrinol. 21(2):189-200. Sakai N, Tanaka M, Takahashi M, Fukada S, Mason JI, Nagahama Y. 1994. Ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase of rainbow trout: its cDNA cloning and properties of the enzyme expr essed in a mammalian cell. FEBS Lett. 350(2-3):309-13. Salen G, Ahrens E, Grundy S. 1970. Metabolism of beta-sitosterol in man. J Clin Invest. 49:952-966. Sandhoff TW, Hales DB, Hales KH, McLean MP 1998. Transcriptional regulation of the rat steroidogenic acute regulatory prot ein gene by steroidogenic factor 1. Endocrinology. 139(12):4820-31. Sandhoff T, McLean M. 1999.Repression of the rat Steroidogenic Acute Regulatory (StAR) Protein gene by PGF2 is modulated by the negative tr anscription factor DAX-1. Endocrine 10(1):83-91. Schrader M, Danielsson C, Wiesenberg I, Carlberg C. 1996. Identification of natural monomeric response elements of the nucle ar receptor RZR/ROR. They also bind COUP-TF homodimers. J Biol Chem. 271(33):19732-6. Sepulveda MS, Quinn BP, Denslow ND, Holm SE, Gross TS. 2003. Effects of pulp and paper mill effluents on reproductive success of largemouth bass. Environ Toxicol Chem. 22(1):205-13. Sepulveda MS, Ruessler DS, Denslow ND, Holm SE, Schoeb TR, Gross TS.2001. Assessment of reproductive effects in la rgemouth bass (Micropterus salmoides) exposed to bleached/unbleached kraft m ill effluents.Arch. Environ. Contam Toxicol. 41(4):475-82. Shea-Eaton W, Sandhoff TW, Lopez D, Hales DB, McLean MP. 2002. Transcriptional repression of the rat Steroidogenic Acute Regulatory (StAR) Protein gene by the AP-1 family member c-Fos. Mo l Cell Endocrinol. 188(1-2):161-70. Shen WJ, Patel S, Natu V, Hong R, Wang J, Azhar S, Kraemer FB 2003. Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal. J Biol Chem. 278(44):43870-6.

PAGE 140

127 127 Shibata H, Kobayashi S, Kurihara I, Suda N, Yokota K, Murai A, Ikeda Y, Saito I, Rainey WE, Saruta T. 2004. COUP-TF and transcriptional co-re gulators in adrenal steroidogenesis. Endocr Res. 30(4):795-801. Shibata H, Nawaz Z, Tsai SY, O'Malley BW Tsai MJ. 1997. Gene silencing by chicken ovalbumin upstream promoter-transcripti on factor I (COUP-TFI) is mediated by transcriptional corepressors, nuclear r eceptor-corepressor (N-CoR) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT). Mol Endocrinol. 11(6):714-24. Shores EM, Picton H, Hunter M. 2000. Di fferential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serumfree culture. J Reprod Fertil. 118:211-219. Shultz VD, Phillips S, Sar M, Foster PM, Gaido KW. 2001. Altered gene profiles in fetal rat testes after in utero exposure to di(n -butyl) phthalate. T oxicol Sci. 64(2):233-42. Silverman E, Eimerl S, Orly J. 2004. CCA AT enhancer-binding protein beta and GATA4 binding regions within the promoter of the Steroidogenic Acute Regulatory Protein (StAR) gene are requi red for transcription in rat ovarian cells. J Biol Chem. 274(25):17987-96. Sirtori C, Manzoni C, Lovati M. 1991. M echanisms of lipid-lowering agents. Cardiology. 78:226-235. Skipper JK, Hamilton TH. 1977. Regulation by es trogen of the vitellogenin gene. Proc Natl Acad Sci U S A. 74(6):2384-8. Soccio RE, Breslow JL. 2004. Intracellular choles terol transport. Ar terioscler Thromb Vasc Biol. 24(7):1150-60. Spies RB, Rice DW.1988.The effects of orga nic contaminants on reproduction of the starry flounder Platichthys stellatus (Palla s) in San Francisco Bay. Mar Biol. 98: 191-200. Stocco DM, Clark BJ. 1996. Role of the Ster oidogenic Acute Regulat ory Protein (StAR) in steroidogenesis. Biochem. Pharmacol. 51:197-205. Stocco DM, Clark BJ. 1997. The role of the Steroidogenic Acute Re gulatory Protein in steroidogenesis. St eroids. 62:29-36. Stocco DM, Clark BJ, Reinhart AJ, Williams SC, Dyson M, Dassi B, Walsh LP, Manna PR, Wang XJ, Zeleznik AJ, Orly J. 2001. El ements involved in the regulation of the StAR gene. Mol Cell Endocrinol. 177(1-2):55-9. Strauss JF 3rd, Kishida T, Christenson LK Fujimoto T, Hiroi H. 2003. START domain proteins and the intracellula r trafficking of cholesterol in steroidogenic cells. Mol Cell Endocrinol. 202(1-2):59-65.

PAGE 141

128 Sugawara T, Kiriakidou M, McAllister J, Kallen C, Strauss J. 1997. Multiple Steroid Factor-1 binding elements in the huma n Steroidogenic Acut e Regulatory Protein gene 5-flanking region are required for maximal promoter activity and cyclic AMP responsiveness. Biochemistry. 36:7249-7255. Sugawara T, Nomura E, Sakuragi N, Fujimot o S. 2001. The effect of the arylhydrocarbon receptor on the human steroidogenic acute regulatory gene promoter activity. J Steroid Biochem Mol Biol. 78(3):253-60. Sugawara T, Nomura E, Nakajima A, Sa kuragi N. 2004. Characterization of binding between SF-1 and Sp1: predominant interaction of SF-1 with the N-terminal region of Sp1. J Endocrinol Invest. 27(2):133-41. Sugawara T, Saito M, Fujimoto S. 2000. Sp1 and SF-1 interact and cooperate in the regulation of human steroi dogenic acute regulatory pr otein gene expression. Endocrinology. 141(8):2895-903. Sugawara T, Shimizu H, Hoshi N, Nakajim a A, Fujimoto S. 2003. Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system. J Biol Chem. 278(43):42487-94. Tajima K, Babich S, Yoshida Y, Dantes A, Strauss JF 3rd, Amsterdam A. 2001. The proteasome inhibitor MG132 promotes accu mulation of the Steroidogenic Acute Regulatory Protein (StAR) and steroidogenesis. FE BS Lett. 490(1-2):59-64. Tchoudakova A, Kishida M, Wood E, Callard GV. 2001. Promoter characteristics of two cyp19 genes differentially expressed in th e brain and ovary of teleost fish. J. Steroid Biochem Mol Biol. 78(5):427-39. Thomson M. 1998. Molecular and cellular mech anisms used in the acute phase of stimulated steroidogenesis. Horm Metab Res. 30(1):16-28. Tran P, Zhang XK, Salbert G, Hermann T, Lehmann JM, Pfahl M. 1992. COUP orphan receptors are negative regulators of retin oic acid response pathways. Mol Cell Biol. 12(10):4666-76. Tsujishita Y, Hurley JH. 2000. Structure and lipid transport mechanism of a StAR-related domain. Nat Struct Biol. 7(5):408-14. Wallace R, Selman K. 1990. U ltrastructural aspects of ooge nesis and oocyte growth in fish and amphibians. J. Elect ron Microsc. Tech. 16:175-201. Walsh LP, McCormick C, Martin C, Sto cco DM. 2000. Roundup inhibits steroidogenesis by disrupting Steroidogenic Acute Regulator y (StAR) Protein expression. Environ Health Perspect. 108(8):769-75.

PAGE 142

129 Walsh LP, Webster DR, Stocco DM. 2000. Di methoate inhibits steroidogenesis by disrupting transcription of the steroi dogenic acute regulatory (StAR) gene. J Endocrinol. 167(2):253-63. Wang W, Zhang C, Marimuthu A, Krupka HI, Tabrizizad M, Shelloe R, Mehra U, Eng K, Nguyen H, Settachatgul C, Powell B, Milburn MV, West BL. 2005. The crystal structures of human steroidogenic fact or-1 and liver receptor homologue-1. Proc Natl Acad Sci U S A. 102(21):7505-10. Werbin H, Chaikoff I, Jones E. 1960. The metabolism of H3-B-sitosterol in the guinea pig: Its conversion to urinary cor tisol. J Biol Chem. 235(6):1629-1633. West LA, Horvat RD, Roess DA, Barisas BG, Juengel JL, Niswender GD. 2001. Steroidogenic Acute Regulatory Protei n and peripheral-type benzodiazepine receptor associate at the mitochondria l membrane. Endocrinology. 142(1):502-5. Wolff S, Harper PA, Wong JM, Mostert V, Wang Y, Abel J. 2001. Cell-specific regulation of human aryl hydrocarbon recep tor expression by transforming growth factor-beta(1). Mol Pharmacol. 59(4):716-24. Xu J, Oakley J, McGee EA. 2002. Stage-speci fic expression of Smad2 and Smad3 during folliculogenesis. Biol Reprod. 66(6):1571-8. Yaron Z, Gur G, Melamed P, Rosenfeld H, Elizur A, Levavi-Sivan B. 2003. Regulation of fish gonadotropins. In t Rev Cytol. 225:131-85. Young MJ, Clyne CD, Cole TJ, Funder JW. 20 01. Cardiac steroidogenesis in the normal and failing heart. J Clin Endocrinol Metab. 86(11):5121-6. Zhang XK, Pfahl M. 1993. Heteroand homodimeric receptors in thyroid hormone and vitamin A action. Receptor. 3(3):183-91.

PAGE 143

130 BIOGRAPHICAL SKETCH The author was born and raised primar ily in Indiana. After high school, she received her bachelors degr ee in chemistry from Indiana University in Bloomington. She continued her education by completing he r masters degree in biology at Purdue University in Indianapolis. Following her masters degree, she worked at Covance Centra l Laboratories in Indianapolis in the microbiology department. At Covance, she implemented tests for experimental antibiotics and medicines. She then moved to Florida and pursued her degree in the department of biochemistry. Following completion of her PhD degree, she will be doing a postdoctoral fellowship at Scripps Florida in the Drug Discovery department.


Permanent Link: http://ufdc.ufl.edu/UFE0010163/00001

Material Information

Title: Regulation of the Steroidogenic Acute Regulatory Protein (StAR) by cAMP and Transforming Growth Factor-Beta (TGF-Beta) Dependent Pathways
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0010163:00001

Permanent Link: http://ufdc.ufl.edu/UFE0010163/00001

Material Information

Title: Regulation of the Steroidogenic Acute Regulatory Protein (StAR) by cAMP and Transforming Growth Factor-Beta (TGF-Beta) Dependent Pathways
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0010163:00001


This item has the following downloads:


Full Text












REGULATION OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN
(StAR) BY cAMP AND TRANSFORMING GROWTH FACTOR-BETA
(TGF-BETA) DEPENDENT PATHWAYS














By

REBECCA JANNET KOCERHA


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


2005

































Copyright 2005

by

Rebecca Jannet Kocerha















ACKNOWLEDGMENTS

There are many people I would like to thank, but without my advisor, Dr. Nancy,

Denslow, I would not have had the opportunity to work on such an exciting and

interesting PhD project. I am so grateful and appreciative to Nancy for many things,

including her scientific creativity, mentoring skills, and friendship, none of which I will

forget.

I would also like to thank my committee, Dr. Flanegan, Dr. Purich, Dr. Bungert,

and Dr. James. Collectively, they really helped me to understand what it takes to have a

successful project and their advice is something I will take with me throughout the rest of

my career. I feel honored to have had such a talented group of scientists on my

committee.

The entire Denslow laboratory, Protein Core, Education Core, and Hybridoma Core

made my PhD experience such a pleasure. Kevin Kroll taught me so much about fish and

helped immensely to make the ovarian follicle cultures a success. Alfred Chung was

integral in the development of the LMB StAR antibody by synthesizing the peptides.

Dr. Stan Stephens and Dr. Andy Ottens were so helpful with the mass spec analysis. Dr.

Tara Sabo-Attwood was always there for me to bounce ideas through many lengthy

conversations and I feel very fortunate to have her as a very good friend. I would also

like to thank the newest member of the lab and my friend, Mindy Prucha, for doing such

a great job in continuing the StAR project and for all the enthusiasm and energy she









brings to the lab. Jason Blum, Natalia Reyero, Iris Knoebl, and Nicole Mullally were

also helpful resources throughout my time in the Denslow lab.

Last, but of course not least, I want to sincerely thank my parents and family for

their endless love and support. There truly are no words to describe the love, gratitude,

and appreciation that I have for them and always will.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES .................................................... ....... .. .............. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABBREVIATION S ......... .......................... .......... .......... ........... xi

ABSTRACT .............. ..................... .......... .............. xii

CHAPTER


1 IN TR OD U CTION ............................................... .. ......................... ..

L literature R review ............................................................................... .
Steroidogenic Acute Regulatory Protein (StAR) ..............................................1
Identification of the StAR protein.................................. ............. ...........1
M ode of action ............... .................................... ................ .. 2
Protein-protein interactions with StAR..................................4
M stations and associated diseases ...................................... ............... 5
Prom other ............................. .......... ..... .............. .. ............. 6
Endogenous regulators and signaling pathways................... ........... 9
Regulation by environmental contaminants .......... ..................................11
E ndocrine D isruption ........................................... ......... .................. ............ 11
Largem outh B ass as a M odel ........................................ ......................... 13
O v arian F o llicles ................................................. ................ 15
Cholesterol and Steroidogenesis.................... ...... .......................... 16
3-Sitosterol ........................................17
Transforming Growth Factor-Beta (TGF-) ................................................. 19
R e search O bjectiv es................................................................ ..................... .. 2 1

2 M ATERIALS AND M ETHODS ........................................ ......................... 27

A n im a ls ............................................................. ................ 2 7
LM B StAR mRNA Expression ............................................................................ 27
C loning of StA R ........................... .. .. .... .... .... ... .. ...... ..... ......... .... 27
Development of LMB Real-Time PCR Assay for mRNA Quantitation.............31


v









S e a so n a l S tu d y ............................................................................................... 3 3
LM B Ovarian Tissue Cultures....................................... .......................... 34
LM B Ovarian Follicle Cultures....................................... ......... ............... 34
LM B StAR Protein Quantitation ........................................ .......................... 35
Protein Expression Vector.................................................... 35
Bacterial Protein Induction........................................................... ..................... 36
Protein Purification........ .......................................... .. .. ..... ........ 38
D evelopm ent of StAR A ntibody ........................................ ...... ............... 38
Western Blots ................................................ 39
Transcriptional Regulation of LM B StAR ...................................... ............... 40
Cloning of the Promoter ........... .. ................................. 40
P rom other A analysis ..................... .. .. ...................... .. ...... ...............43
LM B SF-1 Cloning .......... ... ... ............. ....... .......... .............. 43
Prom other D election ............... ......................................... ....... ... ........ .... 43
Mutagenesis of Putative Transcription Factor Binding Sites..............................44
Culturing of Y -1 Cells .......................................... .. .... ..... ........ .... 45
Transfection Assays.................. .................................45
G F P Q uantitation ........ ................................................................ .... .... ... ... 46
Luciferase M easurem ents ......... ............... ................................. ............... 46
Mouse StAR Real-Time PCR Assay ...........................................47
S statistic s .................................................................................................... 4 7

3 REGULATION OF STAR IN LARGEMOUTH BASS OVARIAN FOLLICLE
CULTURES ................ ........ ........................ 61

Intro du action .................................................................................................... 6 1
Results ..... ...................... .. ........ ........ .. ............ ............ 63
Cloning of StAR Protein .................................. .....................................63
Seasonal Expression ...... .... ... ......... ........ .. .. .. .................... 63
Regulation of StAR mRNA Expression in LMB Ovarian Cultures ..................64
cAM P Induction of LM B StAR ........................................ ....... ............... 64
P-sitosterol Exposures ..................................... ............... ..... ..... 65
T G F -P E xposures........... .................................................. .......... ...... ..... .. 65
A ntibody D evelopm ent .....................................................................................66
Western Blot Detection of Endogenous LMB StAR Protein ...........................67
D iscu ssio n ............... ...... .. ......... .................................................................................. 6 8

4 TRANSCRIPTIONAL REGULATION OF THE LMB STAR PROMOTER.......... 87

Introdu action ............... ................. ......... ................. ................. 87
R esults.............................. .......... ........... ............................. 89
Cloning of the StAR Promoter ...................... .. ...................89
Identification of the Transcriptional Start Site .............................................. 90
Identifying Transcriptional Response Elements...............................................90
Cloning of LM B Steroidogenic Factor -1 (SF-1)..............................................91
Optimization of Transfection A says ...................................... ............... 91
dbcA M P E xposures .............................................. ....... .............................. 92









Prom other D election Experim ents ........................................ ....... ............... 93
Site-D directed M utagenesis Experim ents .................................... .....................93
TGF-P Regulation of Y-1 Cell Endogenous Mouse StAR mRNA ...................95
D isc u ssio n ............................................................................................................. 9 5

5 CONCLUSIONS AND FUTURE DIRECTIONS .............................114

R E F E R E N C E S ............. ................... ............ ..... ............................................ ..... 1 19

BIOGRAPHICAL SKETCH ............................................................. ............... 130
















LIST OF TABLES


Table p

2-1. Prim ers for 5' and 3' RA CE ................ ......... ............................... ............... 48

2-2. Thermocycler conditions for 5' and 3' RACE .............................. ................49

2-3. Primers used to fix nucleotide mistakes in full length StAR cDNA sequence........50

2-4. Thermocycler conditions for LMB StAR promoter cloning...............................51

2-5. Thermocycler conditions for cloning of LMB SF-1 ................ ...... .............52

2-6. Prim ers for prom other mutagenesis. ........................................ ....... ............... 53

2-7. Thermocycler conditions for promoter mutagenesis with QuikChange-XL
protocol ..................................... ................................... ......... 54
















LIST OF FIGURES

Figure pge

1-1. Alignment of mammalian StAR promoters.. ............. ......... ................ ........22

1-2. Fish ovarian follicle .............. .......... ...................................... ................ 23

1-3. General pathw ay for steroidogenesis.. ........................................ ............... 24

1-4. Structures of P-sitosterol and cholesterol. ................................. ...............25

1-5. TG F-P signaling pathw ay. ........................................................... .....................26

2-1. Rapid amplification ofcDNA ends (RACE). .................................. .................55

2-2. Sample standard curve for real-time PCR..................................... ............... 56

2-3. M ap of pE T -28b vector. ............................ ................................... .....................57

2-4. Location of peptide used for antibody development is indicated by a green box.
58

2-5. Promoter cloning. .................................... .. ........... ......... .... 59

2-6. Map of pGL3 basic vector (Promega)............... .................. ...............60

3-1. PCR amplification of LM B StAR. ........................................ ....................... 72

3-2. Alignment of LMB StAR cDNA with other species ...........................................73

3-3. Seasonal expression of LMB StAR................................ ...............75

3-4. Dose response of LMB ovarian tissue cultures to dbcAMP. ...............................76

3-5. cAM P induction of ovarian follicles. ............................................ .....................77

3-6. Dose response exposure of ovarian follicles to TGF- ........................................78

3-7. PCR amplification of entire LMB StAR cDNA............................................... 79

3-8. Bacterial expression of LM B StAR. ............................................. ............... 80









3 -9 P u rification of StA R ....................... ......... ...........................................................8 1

3-10. Identification of bacterially expressed StAR with Q-STAR mass spectrometry.....82

3-11. Identification of bacterially expressed StAR by LCQ mass spectrometry ..............83

3-12. ELISA with LMB StAR anti-serum and purified StAR protein ........................ 84

3-13. Western blot detection with LMB StAR antibody and purified StAR protein.. ......85

3-14. Western blot detection of StAR in dbcAMP exposed LMB tissue cultures.. ..........86

4-1. Digested LMB ovarian genomic DNA for promoter cloning.. ............................100

4-2. Cloning of LM B StAR prom oter.. ...................... ............................................. 101

4-3. 5' and 3' untranslated region (UTR) for LMB StAR and identification of
transcription start site.. ..................... .. ...................... .. .. ....... .... .......... 102

4-4. Putative transcription response elements identified in the LMB StAR promoter.. 103

4-5. Cloning ofLM B SF-1 .................................. .........................................104

4-6. Optimization of ratio for transfection reagent (Fugene6) and promoter DNA
concentration. ................................................................105

4-7. Optimization of transfection timepoint ........... .......................... ........ ........... 106

4-8. Quantitation of DNA transtection with GFP. 107

4-9. Dose response exposure of Y-1 cells to dbcAMP............ .................................108

4-10. Creation of promoter deletion and site-mutagenesis constructs.............................109

4-11. Prom other deletion analysis. ........... ................ ........... .......... ...............110

4-12. Exposures of promoter site-mutagenesis constructs to dbcAMP..........................111

4-13. StAR promoter mutation analysis with TGF-3 regulation....................................112

4-14. Endogenous mRNA regulation of StAR in Y-1 cells by TGF-P .........................113















ABBREVIATIONS


3-B-HSD = 3-beta-hydroxy-steroid-dehydrogenase
ACTH = adrenal corticotrophic hormone
AhR = aryl hydrocarbon receptor
bp = base pair
cAMP = cyclic adenosine monophosphate
COUP-TF = chicken ovalbumin upstream promoter transcription factor
CRE = cAMP response element
CREBP = cAMP response element binding protein
DAX-1 = dosage-sensitive sex reversal, adrenal hypoplasia critical region, on
chromosome X, gene 1
DHP = 17alpha, 20B-hydroxy-4-pregnen-3-one
ELISA = enzyme linked immunosorbent assay
EMSA = electromobility shift assay
ERE = estrogen response element
GFP = green fluorescence protein
GnRH = gonadotropin releasing hormone
Kb = kilobase
LCAH = lipoid congenital adrenal hyperplasia
LH = luteinizing hormone
LMB = Largemouth Bass
MALDI = matrix assisted laser desorption ionization
PBR = Peripheral Benzodiazepine Receptor
PCR = polymerase chain reaction
PKA = protein kinase A
PKC = protein kinase C
RACE = rapid amplification of cDNA ends
RAR = retinoic acid receptor
RARE = retinoic acid response element
ROR = retinoic acid related receptor
SBP = StAR binding protein
SF-1 = steroidogenic factor 1
StAR = Steroidogenic Acute Regulatory Protein
TGF-P = transforming growth factor beta















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

REGULATION OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN
(StAR) BY cAMP AND TRANSFORMING GROWTH FACTOR
(TGF-BETA) DEPENDENT PATHWAYS

By

Rebecca Jannet Kocerha

August 2005

Chair: Nancy Denslow
Major Department: Biochemistry and Molecular Biology

StAR is the rate-limiting step in steroid production and is transcriptionally down

regulated by toxin exposure. StAR transports cholesterol across the mitochondrial

membrane for metabolism into steroids. We cloned the entire coding region of

largemouth bass (LMB) StAR and used this sequence to develop a real-time PCR assay

to quantify StAR mRNA levels in LMB ovarian follicle cultures. Exposure to dbcAMP

and TGF-beta, two potent signaling molecules known to regulate mammalian

steroidogenesis, modulate LMB StAR. TGF-beta down regulates and dbcAMP

upregulates StAR mRNA. A polyclonal antibody specific to LMB StAR was developed

to measure protein levels by western blot. To further analyze the regulation of LMB

StAR, a 3 kb portion of the promoter was cloned. In silico analysis of this segment with

other StAR promoters available in the database showed potential conserved regulatory

sites that imply control by a wide range of transcription factors. The 3 kb promoter









segment was transfected into Y-1 cells, a mouse adrenalcortical cell line and tested with

dbcAMP and TGF beta. The 3 kb construct responded positively to dbcAMP but was not

significantly impacted by TGF-B exposure compared to the 1.8 kb length promoter.

Mutation of potential regulatory sites in the promoter, including ERE (estrogen response

elements), ROR (retinoic acid related receptor), and COUP-TF (chicken ovalbumin

upstream promoter) sites were tested for their role in cAMP and TGF-beta signaling.

Together, these data suggest that one way toxins may repress steroid synthesis, and more

specifically StAR, is through TGF-beta signaling.














CHAPTER 1
INTRODUCTION

The Steroidogenic Acute Regulatory Protein (StAR Protein) is the rate-limiting

step in steroidogenesis and can be regulated by endogenous and exogenous agents,

including environmental toxins (Walsh et al., 2000). Determining the regulation of StAR

is imperative since homeostasis of steroid production is vital for various cell signaling

and metabolic pathways. The aims of this project are to examine both the normal and

atypical regulation of StAR in largemouth bass, with a specific focus on delineating the

signaling cascades of cAMP and TGF-P, two potent molecules that can control steroid

production.

Literature Review

Steroidogenic Acute Regulatory Protein (StAR)

Extensive studies in mammalian models have confirmed StAR transports

cholesterol across the mitochondrial membrane. After transport, cholesterol is

metabolized by the side chain cleavage enzyme to pregnenolone and then ultimately to

steroid hormones (Stocco and Clark, 1996).

Identification of the StAR protein

Researchers had known for many years that de novo protein synthesis was required

for steroid synthesis; however, the identity of the protein involved in facilitating the

transport of cholesterol across the mitochondria eluded them until 1995. A 30 kDa

protein in rat adrenal cells stimulated by ACTH, now known as StAR, was first observed

when the [35S] methionine labeled proteins were electrophoresed through a 2D gel.









Cycloheximide, a protein synthesis inhibitor, blocked steroid production without

impairing the activity of the side chain cleavage or the delivery of cholesterol to the outer

mitochondrial membrane (Stocco and Clark, 1997). The 30 kDa protein was purified

from ACTH stimulated MA-10 mouse leydig cells using detergent solubilization

followed by separation of the proteins by ID and 2D gels. Bands at the 30 kDA location

were excised and digested with trypsin for microsequence analysis. Degenerate

oligonucleotides for PCR amplification of the 30 kDa protein were designed based on the

microsequencing results to obtain a 400-base pair partial product. The full coding

sequence for StAR from MA-10 cells was then screened from a cDNA library using the

400-base pair product as a probe (Clark et al., 1994). Since the original identification in

adrenal and leydig cells, StAR has also been located in the brain, kidney, and heart

(Young et al., 2001; Pezzi et al., 2003).

Mode of action

StAR is a mitochondrial protein synthesized as a 37 kDa precursor protein in the

cytosol of mammalian cells (Clark et al., 1995). Upon stimulation, the 37 kDa precursor

is targeted via its signal sequence to the mitochondria. As the precursor protein is

imported into the mitochondrial inner compartment, the protein's signal sequence is

removed by a matrix processing protease and contact sites are formed between the outer

and inner mitochondrial membranes. The precursor protein is further processed by the

mitochondrial intermediate processing peptide to remove the targeting sequence, forming

the mature 30 kDa protein (Stocco and Clark, 1997). The cytoplasmic 37 kDa protein

has a half-life of around 10-15 minutes; however, the 30 kDa inactive protein has a

longer half-life of up to several hours (Christenson and Strauss, 2000).









Two current models suggest StAR transports cholesterol by either acting on the

outside of the mitochondria as a molten globule (a protein with extensive secondary

structure but disorganized tertiary structure, potentially allowing for hydrophobic amino

acids to be exposed) or as an intermembrane shuttle. FRET (flouresence resonance

energy transfer) data indicate that StAR undergoes a conformational change to a molten

globule once it interacts with the outer mitochondrial membrane, enabling it to bind

cholesterol (Christenson et al., 2001). Additionally, recombinant mammalian StAR

protein lacking the first 62 amino acids is localized to the surface of the outer

mitochondria and is prevented from entering the intermembrane space. It remains active

and steroid production continues, suggesting that StAR functions by binding to the outer

membrane (Bose et al., 1999).

The second, but increasingly dubious, model shows StAR acting primarily to

shuttle cholesterol between the outer and inner mitochondria membranes, across the

intermembrane space (Mathieu et al., 2002). This theory was fueled by data showing

contact sites between the two mitochondrial membranes when cholesterol is bound

(Thomson, 1998). It is during the formation of these contact sites that cholesterol is

thought to be transported across the mitochondrial membranes to be metabolized.

Cholesterol is specifically encompassed or bound by the START (StAR-related

lipid-transfer) domain located towards the C-terminus of StAR. The START domain

contains about 210 amino acids with a hydrophobic core where cholesterol binds (Strauss

et al., 2003). Crystal structure of the START domain from the MLN64 protein, another

lipid transporter protein, suggests there would have to be a conformational change in the

hydrophobic core of StAR for a molecule of cholesterol to enter and bind under natively









folded conditions (Tsujishita and Hurley, 2000.) Furthermore, although the START

crystal structure shows room for one molecule of cholesterol, ligand binding assays with

recombinant StAR and increasing titrations of fluorescent cholesterol from 5-100 nM

gave a sigmoidal-shaped binding curve (Petrescu et al., 2001). The binding studies

suggest there are two cholesterol binding sites, necessitating a conformational change of

StAR for sterol binding and transfer, further negating the shuttle model.

Studies show that StAR protein can be degraded by proteasomes. In the presence

of a proteasome inhibitor, MG132, there is accumulation of the cytosolic, 37 kDa form of

StAR in human or rat granulosa cells (Tajima et al., 2001). There is even evidence

suggesting that StAR could be subjected to degradation by different proteases in a

biphasic manner, ensuring removal of residual protein that escaped the initial protease

(Granot et al., 2003). COS cells chased for 15 minutes with 35S methionine prior to

treatment with MG132 showed that degradation of StAR was prevented for the first 2

hours. The protein, however, began degrading after two hours of MG132 treatment,

suggesting that StAR is subjected to degradation by multiple proteases (Granot et al.,

2003).

Protein-protein interactions with StAR

Studies have indicated there are a couple of putative protein interacting partners for

StAR. FRET studies showed that PBR (peripheral-type benzodiazepine receptor)

associates with StAR at the mitochondrial membrane. A PBR-StAR association would,

in theory, make sense because PBR is necessary for cholesterol transport (West et al.,

2001).

Immunoprecipitation experiments showed that StAR also interacts, in vivo, with

HSL (hormone-sensitive lipase). HSL mediates the availability ofunesterified









cholesterol for steroid synthesis (Shen et al., 2003). Rats were injected with ACTH to

induce StAR expression and anti-HSL antibodies were used to immunoprecipitate HSL

from the adrenal glands. The HSL-immunoprecipitate complexes were separated on an

SDS-PAGE gel and the presence of StAR in the complex was detected with anti-StAR

antibodies.

Additionally, a recent study used a yeast two-hybrid system to identify another

protein that binds StAR, which they named SBP (StAR binding Protein). Binding of

StAR to SBP results in increased steroidogenesis (Sugawara et al., 2003). A recombinant

form of StAR (N-62 StAR) lacking the first 62 amino acids in which the mitochondrial

import signals are located was used as the bait for the yeast assay. Interaction of SBP

with N-62 StAR suggests the binding occurs in the cytoplasm or at the outer

mitochondrial membrane. Northern blot analysis suggests that SBP may be found in

several tissues, including the gonads, liver, lung, and kidney. It is possible that

identifying protein interacting partners will help to elucidate the mode of action for StAR

mediated cholesterol transport.

Mutations and associated diseases

StAR knockout mice and humans with LCAH lipoidd congenital adrenal

hyperplasia) disease exhibit similar symptoms of severely repressed steroid synthesis

(Hasegawa et al., 2000). Characteristic of LCAH patients is the presence of large adrenal

glands with high levels of cholesterol or cholesterol esters. Death can result in infancy if

patients are not treated with hormone replacement. A series of various nucleotide

insertions and deletions in StAR DNA are attributed to LCAH and these can vary among

the afflicted individuals. This disease just emphasizes the importance of StAR (Stocco

and Clark, 1996).









Promoter

Characterizing the response elements in the StAR promoter is important for

determining specific proteins and protein-DNA interactions used to regulate its

transcriptional activity. The promoter for StAR has been sequenced for several

mammalian species, including human, rat, mouse, pig, sheep and cow (Sugawara et al.,

1997; Reinhart et al., 1999; Rust et al., 1998). In these systems, the StAR promoter

contains a TATA box and several recognizable response elements for transcription

factors. Some of the most prevalent response elements identified in mammalian species

bind C/EBPs, GATA-4, SF-1, DAX-1, AP-1, and AhR (Manna et al., 2004; Sugawara et

al., 2001; Sandhoff and McLean, 1999) and are conserved across species (Figure 1-1).

Interestingly, although StAR is a cAMP-dependent regulated gene, a perfect consensus

site for CRE (cAMP response element) has not been recognized in any of the StAR

promoters sequenced. However, studies have shown that when wild type CREB (cAMP

response element binding protein) is transfected into several mammalian cell lines, there

is an increase in StAR promoter activity and mRNA expression (Stocco et al., 2001). It

is suggested that CREB may play a role in the absence of a consensus CRE by binding

indirectly to a non-consensus site. It is also possible that CREB regulates an activator of

StAR expression such as SF-1.

One of the most abundant response elements in the mammalian StAR promoter is

SF-1 (steroidogenic factor-1), a known inducer of steroidogenic transcriptional activity.

SF-1 is an orphan nuclear receptor transcription factor that has been sequenced from

many species, including mammalian and fish systems (Yaron et al., 2003). SF-1 also is

called AD4-BP (adrenal 4-binding protein) and is the mammalian homolog of the

Drosophila factor FTZ-F 1. FTZ-F 1 regulates transcription of the fushi tarazu homeobox









gene in fly embryos. Four transcripts are encoded by the SF-1 gene, including ELP1,

ELP2, ELP3, and SF-1, partly generated through alternate splicing (Nawata et al., 1999).

SF-1 is expressed in all steroidogenic tissues such as the adrenal, gonads, and the

placenta. SF-1 knockout mice implicate the importance of SF-1 in cellular functions.

The knockout mice lack adrenal glands and gonads, which leads to lethal adrenocortical

insufficiency (Hammer and Ingraham, 1999.)

It is not exactly known how ligands lead to the activation of SF-1; however, there

are a lot of studies showing post-translational modification of SF-1 occurs. SF-1 does

have a consensus site for protein kinase A (PKA) phosphorylation (Bertherat, 1998).

Further studies have shown that inhibition of mitogen activated protein kinase (MAPK)

can decrease SF-1 responsive genes (Hammer and Ingraham, 1999).

SF-1 binds as a monomer to its response element and has two zinc fingers which

helps it bind to its consensus site (Ito et al., 2000). Up to 6 SF-1 binding sites have been

identified in the mammalian StAR promoter, with the nearest element typically located

only about 40 base pairs away from the transcription start site. The first two SF-1 sites

closest to the start site appear to be conserved across species (Reinhart et al., 1999). SF-1

sites have been found in the promoter of aromatase for several fish species, including

goldfish, medaka, and zebrafish (Callard et al., 2001; Tchoudakova et al., 2001).

Aromatase is the final protein in the steroidogenic pathway involved in the conversion of

testosterone to estradiol (Callard, 2001). SF-1 sites in promoters of genes in fish studied

to date have the general consensus sequence of PyCAAGGPyPyPur, with the exception

that zebrafish have a purine instead of a pyrimidine for the first nucleotide (Kazeto et al.,

2001; Honda et al., 1993). It appears that one way in which SF-1 regulates StAR









transcriptional activity is by interacting with other transcription factors, including C/EBP,

AP-1, and SP1 (Reinhart et al., 1999; Shea-Eaton et al., 2002). Promoter regulation can,

therefore, be very complex and involve transcription factors acting individually or

cooperatively to exert their actions.

RAR and RXRs are part of the steroid-thyroid hormone receptor subfamily and

each are encoded by three genes, a, 0, and y. RAR mediates activation by

heterodimerizing with RXR and it is the heterodimer which binds RARE (retinoic acid

response element) (Pfahl, 1993). Typically, the RARE has a direct repeat of an

AGGTCA core motif which is separated by 2 or 5 nucleotides (Bastien and Rochette-

Egly, 2004).

ROR (retinoic acid receptor-related orphan receptor) is also a member of the

nuclear hormone receptor superfamily. ROR can bind to the RORE (ROR response

element) as a monomer or homodimer. If ROR binds as a monomer, it recognizes a 6

base pair A/T rich region followed by an AGGTCA motif. To bind as a homodimer, a

direct repeat of the RORE separated by 2 nucleotides is necessary (Boukhtouche et al.,

2004). ROR has been well characterized to activate gene transcription in the absence of

a ligand; however, a recent study shows that cholesterol is an ROR ligand (Kallen, 2002).

ROR alpha transcriptional activity is repressed in U20S osteosarcoma cells depleted of

cholesterol with stations, a family of drugs that inhibit cholesterol synthesis (Boukhtouche

et al., 2004). Although RORs are known to be involved in tissue development or

differentiation like some other nuclear receptors, there is still much to be known about

the genes that it regulates (Jarvis et al., 2002).









To date, only DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical

region, on chromosome X, gene 1) is well documented to repress StAR transcription (Jo

and Stocco, 2004). When Y-1 mouse adrenal cells were co-transfected with 2 Kb of the

rat StAR promoter and a vector encoding DAX-1, basal and ImM dbcAMP stimulated

luciferase activity were repressed by at least 40% (Sandhoff and McLean, 1999).

Although the exact mechanism is still being elucidated, it has been shown in mammalian

systems that DAX-1 binds to a hairpin structure in the promoter rather than at a

consensus site (Stocco et al., 2001).

COUP-TF (chicken ovalbumin upstream promoter-transcription factor) is also

known to downregulate steroidogenesis; however, its role in the regulation of StAR is

just beginning to be investigated (Buholzer et al., 2005; Shibata et al., 2004). A recent

study showed that mRNA induction of StAR by angiotensin II was completely

suppressed by overexpression of COUP-TF in bovine adrenal glomerulasa cells

(Buholzer et al., 2005). COUP-TF are nuclear orphan receptors that can form

homodimers and bind to response elements with variations of an AGGTCA core motif,

which includes the RARE (Tran et al., 1992). COUP-TF can also silence the activity of

other transcription factors like RXR by heterodimerizing with them and thereby limiting

their availability for other binding partners (Berrodin et al., 1992). COUP-TF can also

interact synergistically with corepressors like N-CoR (nuclear receptor corepressor) and

SMRT (silencing mediator of retinoid and thyroid hormone receptor) (Shibata et al.,

1997).

Endogenous regulators and signaling pathways

StAR has been shown to be upregulated by cAMP, forskolin, GnRH, ACTH, and

cholesterol containing lipoproteins, including both LDL and HDL (Clark and Stocco,









1996; Reyland et al., 2000). The signaling cascade triggered by cAMP and other related

inducers is complex and encompasses many different proteins and crosstalk between

various pathways.

It has been confirmed that cAMP modulates StAR through a PKA (protein kinase

A) mediated event. PKA activation can regulate StAR directly and indirectly. Indirectly,

PKA can induce transcriptional activation by phosphorylating SF-1, a transcription factor

commonly known to regulate StAR (Aesoy et al., 2002). There are multiple SF-1 sites

throughout the mammalian StAR promoter (Sandhoff et al., 1998).

Additionally, there are PKA sites within the protein sequence of StAR. In vitro

studies in mammalian cells showed specifically that mutation of a serine in a protein

kinase A (PKA) site at amino acid position 195 resulted in 40% less steroidogenic

capacity (Arakane et al., 1997). Studies with a normal and PKA mutant cell line showed

that phosphorylation of StAR most likely stabilizes the protein and therefore results in a

dose response increase to dbcAMP as seen by western blot (Clark et al., 2001).

[35S]methionine incorporation into StAR was shown in the PKA mutant cell line (Kin-8),

suggesting that PKA can act post-translationally.

Less is known about endogenous downregulators of StAR. Some studies have

shown PGF-2 (prostaglandin factor), TGF-3 (transforming growth factor beta), and

glucocorticoids can repress StAR transcription (Sandhoff and McLean, 1999; Brand et

al., 2000; Huang and Shirley, 2001). When dexamethasone, a glucocorticoid, was added

to follicles cultured from LH luteinizingg hormone) treated rats, StAR activity was

impaired but side chain cleavage was not (Huang and Shirley, 2001). This is potentially









one example of how steroids can regulate themselves and that StAR may be subject to

feedback inhibition by downstream steroids.

Regulation by environmental contaminants

Several recent studies have shown that StAR expression can be downregulated by

environmental contaminants, including phthalates, which are plasticizers (Shultz et al.,

2001; Barlow et al., 2003), the pesticide Roundup, the herbicide Lindane (Walsh et al.,

2000), and the insecticide Dimethoate (Walsh et al., 2000). Studies have shown that

these toxins could impact transcription or post-transcription activity. Experiments using

MA-10, mouse leydig cells, showed Lindane and Dimethoate inhibit both mRNA and

protein expression while not repressing overall protein synthesis or inhibiting PKA

activity (Walsh et al., 2000). Also, both microarray and real-time PCR analysis showed

di-butyl phthalate to repress StAR in fetal rat testes, further suggesting a negative

regulation of transcription by environmental contaminants (Shultz et al., 2001; Barlow et

al., 2003).

The regulation of StAR by paper mill contaminants, which is known to cause

repression of steroid levels, has not been investigated (McMaster et al., 1996). Several

studies have linked the altered steroid levels in fish to p-sitosterol (Lehtinin et al., 1999;

MacLatchy et al., 1995). Interestingly, StAR can transport p-sitosterol as efficiently as

cholesterol under in vitro conditions (Kallen et al., 1998). It is currently unknown how

these contaminants could specifically regulate the promoter, even though several of the

toxins listed above decreased StAR mRNA expression.

Endocrine Disruption

There is substantial evidence that humans and wildlife exposed to chemicals in the

environment can exhibit alterations in steroidogenic capacity, leading to changes in









secondary sex characteristics, gonad weight, and production and size of eggs (McMaster

et al., 1995; Sepulveda et al., 2001.). The consequences of steroid imbalance are so

significant and universal that the Environmental Protection Agency (EPA) Office of

Research and Development decided to incorporate endocrine disruption as one of its top

six research priorities. The EPA established several long-term goals, which included

providing a better understanding of the science underlying the effects, exposure,

assessment, and risk management of endocrine disruptors; determining the extent of the

impact on humans, wildlife, and the environment; and finally, supporting the EPA's

screening and testing program.

Environmental contaminants can impact reproduction and steroidogenesis by

mimicking the actions of endogenous androgens or estrogens, or by affecting their

synthesis or metabolism. Cellular signaling pathways can converge to mediate the

response to the endocrine disruptors, which includes endogenous signaling molecules like

cAMP and TGF-0 as well as critical transcription factors like SF-1, ER, COUP-TF, and

RAR.

Some environmental toxins that have been shown to impact steroidogenesis in fish

include PCB (Spies and Rice, 1988), polyaromatic hydrocarbons (PAH) (Spies and Rice,

1988), phthalates (Barlow et al., 2003), Roundup (Walsh et al., 2000), and paper mill

effluents (McMaster et al., 1996), among others. Extensive studies on several fish

species exposed to paper mill toxins, including the white sucker in Lake Superior and

largemouth bass (LMB) in Florida, show males have decreased testosterone and females

have decreased estrogen levels (McMaster et al., 1995; Sepulveda et al., 2001;

MacLatchy and Van Der Kraak, 1995). Several of the compounds present in paper mill









effluent have been identified, including resin acids, dioxins, abietic acid, and phytosterols

(MacLatchy and Van Der Kraak, 1995; McMaster et al., 1996; Sepulveda et al., 2001).

While paper mill effluent is a complex mixture of chemicals, attention has been focused

on P-sitosterol. P-sitosterol is a phytosterol released by paper mills into rivers and lakes.

Although published results suggest p-sitosterol acts through the estrogen receptor

(Gutendorf and Westendorf, 2001), other studies suggest p-sitosterol affects the transport

of cholesterol across the mitochondrial membrane (MacLatchy and Van Der Kraak, 1995;

MacLatchy et al., 1997). Studies show that goldfish injected with p-sitosterol exhibit

decreased steroid levels and expression of the P450 side chain cleavage enzyme, an

enzyme involved in the conversion of cholesterol to pregnenolone (MacLatchy et al.,

1997). However, when ovarian tissue cultures from p-sitosterol exposed fish are treated

with a membrane permeable form of cholesterol, steroid production is recovered

(MacLatchy et al., 1997). This study implicates StAR as a site of regulation by P-

sitosterol since it binds and transports cholesterol to the inner mitochondrial space for

steroid production.

Ovarian follicles were cultured from goldfish that were exposed in vivo to P-

sitosterol to test whether their steroidogenic ability was impaired by the toxic treatment

(MacLatchy and Van Der Kraak, 1995.) Culturing follicles has also been used to study

mammalian StAR function (Huang and Shirley, 2001) and it is a very relevant and

physiological assay to study regulation of steroidogenic proteins for many different types

of model species.

Largemouth Bass as a Model

Fish are often surrounded by many environmental contaminants in the water,

therefore, are a good model system to study the effects of toxins on reproduction in









vertebrates. Valuable information on the specific mode of cellular regulation by these

toxins can be gathered. In particular, LMB exposed to chemical contaminants discharged

from the Palatka paper mill into the St John's River have already been studied

extensively and have been shown to exhibit decreased steroid levels of 17-P- estradiol

(E2) and 11-ketotestosterone as well as other reproductive anomalies (Sepulveda et al.,

2001; Sepulveda et al., 2003). Additionally, the gonads were smaller in weight and

underdeveloped for fish closer to the source of contamination (Sepulveda et al., 2001).

LMB can bioaccumulate the environmental toxins in their tissues and organs since they

survive on a diet of other fish, crabs, frogs, snakes, mice, turtles, and birds

(http://www.go4bass.com/largemouth.html.) LMB are particularly useful for studying

exposure to environmental contaminants because they are a freshwater species found

across the country.


Their reproductive cycle extends for several months, usually from November

through April, which allows for an extended period for experiments. Because LMB are

annual spawners, their reproductive cycles are fairly synchronous making controlled

experiments more feasible. Some fish, such as zebrafish, spawn several times in a year

and do not have synchronized cycles. Our laboratory has preliminary data indicating

LMB exhibit seasonal changes in E2 and testosterone. Steroid levels were correlated with

the corresponding stage of ovarian follicle maturation during a one year time span. LMB

can release up to 100,000 eggs from the matured ovarian follicles per year

(http://www.go4bass.com/largemouth.html.)









Ovarian Follicles

Ovarian follicles provide an essential source of steroid biosynthesis in females.

The term follicle refers to the oocyte surrounded by an internal granulosa cell layer and

external thecal cell layer, which contains fibroblasts, collagen fibers, and thecal cells

(Figure 1-2). The thecal and granulosa layers are separated by a basal lamina (Babin,

1986) and both cell types are able to produce steroids. The thecal cells, in fish, are what

primarily have been found to form testosterone since biochemical and ultrastructural

studies show these cells contain 3-beta-hydroxy-steroid-dehydrogenase (3-P-HSD)

(Kusakabe et al., 2003). 3-P-HSD is an enzyme involved in the conversion of

pregnenolone to progesterone, and progesterone can then be metabolized to testosterone.

Granulosa cells contain the P450 aromatase enzyme, which converts testosterone to E2

(Nagahama et al., 1995). It has been shown in fish that synthesis of testosterone by

thecal cells increases during vitellogenesis (Nagahama et al., 1995).

Vitellogenesis is defined as the hepatic synthesis and secretion of vitellogenin

(VTG), an egg yolk precursor, followed by uptake of VTG into the oocyte from the

bloodstream by receptor-mediated endocytosis. After endocytosis, VTG is cleaved by

specific cathepsins to form yolk proteins (Wallace and Selman, 1990). The production of

VTG is stimulated by E2 (Skipper and Hamilton, 1977).

Ovarian follicles go through several stages of development which involves many

complex processes. At the most immature stage, oocyte cell cycles are arrested at

prophase of meiosis and are called primordial follicles (Wallace and Selman, 1990). The

phase during which the oocyte grows and granulosa cells proliferate is called the

primary-follicle stage. During this stage, the theca cells begin to differentiate and

continued follicle development becomes reliant on gonadotrophins, with FSH (follicle









stimulating hormone, also called GTHI in fish) levels being very elevated (Kagawa et

al., 2003).

Upon completion of follicle growth, LH luteinizingg hormone, also called GTHII in

fish) levels rise for the final maturation and ovulation of the oocyte. Gonadotropin

stimulates the production of a steroid, DHP (17cr, 20B-dihydroxy-4-pregnen-3-one),

which is involved in the maturation by binding directly to a receptor on the oocyte (Pang

and Ge, 2002). DHP is made in the granulosa cells from 17ea-hydroxyprogesterone,

which is made in the thecal cells, showing the interconnection between the two cells.

Following maturation, the oocyte is released and is either fertilized or undergoes atresia,

a process shown to involve apoptosis (Manabe et al., 2004). Completely developed

ovarian follicles in LMB are about 1.4 to 1.5 mm in diameter, but that diameter range can

vary across species.

Ovarian follicles have been cultured from various species, including mammals and

fish, to study the regulation of steroidogenic enzymes by endogenous and exogenous

substances (Petrino and Shuetz, 1986; Babin, 1986). Follicle diameter, stage of

maturation, and quantity of cultured follicles are all parameters that can be selected in

this assay. Another benefit of using cultured ovarian follicles is that the contact between

granulosa and thecal cells remains intact, which more closely resembles an in vivo system

(figurel-2). It has been shown that there is increased steroidogenesis in co-cultures of

granulosa and theca cells than when either cell type is cultured alone (Shores et al.,

2000).

Cholesterol and Steroidogenesis

Cholesterol is the backbone for steroid hormones and can be derived exogenously

from dietary sources or endogenously synthesized in the small intestine or liver.









Typically, hepatic or intestinal cholesterol production supplies 2 to 3 times the amount

that is absorbed from food (Lu et al., 2001). In addition to playing a vital role in

metabolic homeostasis, cholesterol is a major component of the plasma membrane,

helping to ensure the integrity of cellular structure. That same cholesterol can also be

extracted from the membranes for steroid production (Pichler and Riezman, 2004).

Multiple fates exist for cholesterol after being synthesized or absorbed, but it is

primarily packaged into lipoproteins for intracellular transport. Lipoproteins are

categorized as chylomicrons, LDL (low density lipoprotein), VLDL (very low density

lipoprotein), and HDL (high density lipoprotein) based on their roles in sterol transport.

Chylomicrons shuttle dietary cholesterol from the small intestine to the peripheral tissues;

VLDL and LDL transfer endogenously derived sterols from the liver to tissues. HDL

provides one of the only ways to clear cholesterol from the body by returning cholesterol

from the tissues back to the liver (Ginsberg, 1998). The liver metabolizes cholesterol into

bile acids that can either be further broken down by microorganisms in the large intestine

and excreted in urine, or re-used by the body to aid in fat digestion.

There are a couple of proteins which are crucial to maintaining appropriate levels

of cholesterol. HMG-CoA reductase is the rate-limiting enzyme in cholesterol

biosynthesis and is therefore a target of many pharmaceuticals to alleviate symptoms of

high blood pressure, atherosclerosis, as well as other diseases (Rosanoff and Seelig,

2004). The steroidogenic acute regulatory protein (StAR Protein) is the rate-limiting step

in cholesterol metabolism and it is just beginning to be investigated (Figurel-3).

p-Sitosterol

P-sitosterol is a phytosterol that shares structural similarity with cholesterol (Figure

1-4). It is discharged from paper mills into waterways upon processing of paper









products. p-sitosterol makes up about 65% of the phytosterols present in paper mill

effluent. The other phytosterols include campesterol, stigmasterol, and sitostanol

(MacLatchy and Van Der Kraak, 1995; McMaster et al., 1996; Sepulveda et al., 2001).

Unlike cholesterol, there is no endogenous production of the phytosterols in vertebrates

and they can only be obtained through dietary sources (Salen et al., 1970).

p-sitosterol is metabolized to pregnenolone and steroid hormones; however, studies

suggest p-sitosterol metabolism is less efficient than cholesterol (Bennett et al., 1969;

Aringer et al., 1979; Werbin et al., 1960). Absorbed phytosterols circulate in lipoprotein

particles but the rates of absorption for the different phytosterols vary. There is evidence

that phytosterols may accumulate in steroidogenic tissue, including the ovary, testis, and

adrenal gland of animals (Moghadasian and Frohlich, 1999). Absorption into

steroidogenic tissues suggests that phytosterols, like cholesterol, can serve as precursors

to steroid hormone synthesis.

p-sitosterol is used clinically to lower cholesterol. Studies have shown that plasma

cholesterol levels are significantly lowered by 10 days in humans fed a diet of 20[tg/g

body weight of phytosterols (Jones et al., 1998). Although only about 5% of phytosterols

are absorbed, studies have shown that p-sitosterol competes with cholesterol for uptake

into bile acid micelles (Compassi et al., 1997). In cells incubated with micelles

containing either P-sitosterol, cholesterol, or both, p-sitosterol was shown to decrease the

movement of cholesterol from the plasma membrane into the cell (Field et al., 1997). It

is thought that p-sitosterol displaces cholesterol from the bile acid micelles. Bile acids,

metabolites of cholesterol, are necessary for cholesterol absorption (Sirtori et al., 1991).

If cholesterol is displaced from the bile acid micelles by sitosterol, the absorbability of









cholesterol by cells is diminished (Field et al., 1997). Phytosterols lower overall plasma

cholesterol levels by inhibiting intestinal cholesterol absorption or by preventing

recirculation of bile acids.

Although p-sitosterol is used to treat patients with high cholesterol levels, high

plasma concentrations of phytosterols in animals may have deleterious effects on

reproductive organs. Rats injected with 0.5 to 5 mg/kg body weight per day of 0-

sitosterol exhibited a diminished sperm count and testes weight (Moghadasian and

Frohlich, 1999). In addition studies have shown that steroid levels are decreased in

goldfish when given a single injection of 5, 10, or 100 [tg/g p-sitosterol (MacLatchy and

Van der Kraak, 1995). Pregnenolone, a metabolite in the conversion of cholesterol to

steroid hormones, is also decreased with p-sitosterol exposure.

Transforming Growth Factor-Beta (TGF-P)

Although the exact mechanism of how p-sitosterol represses steroidogenesis is still

unknown, it has been shown that p-sitosterol upregulates TGF-P in prostate cells (Kassen

et al., 2000). There is also evidence that TGF-P may be regulated by dioxins, also found

in the environmental (Dohr et al., 1994), suggesting a possible signaling pathway used by

toxins to repress steroidogenesis. TGF-P, through SMAD proteins, has even been shown

to modulate the arylhydrocarbon receptor, a transcription factor which is regulated by

dioxins and for which a response element has been found in the human StAR promoter

(Wolff et al., 2001).

TGF-P is a powerful cytokine involved in cell signaling and has been shown to

inhibit steroidogenesis in adrenocortical, trophoblast, and testicular cells (Liakos et al,

2003; Luo et al., 2002; Gautier et al., 1997). Several recent studies have even shown the









StAR Protein to be a major target for TGF-P regulation. Transfection studies in a human

adrenocortical cell line, H295R, showed that 1.3 Kb of the human StAR promoter is

inhibited by about 25% when cells are treated with Ing/ml TGF-P for 24 hours (Brand et

al., 1998, 2000). The authors then tested several deletions of the 1.3 Kb promoter with

TGF-P and showed that all the deletions were downregulated except the 0.085 Kb

construct. The study suggests the regulation of StAR by TGF-P is mediated by elements

located between 0.085 Kb and 0.15 Kb upstream of the transcriptional start site in the

mammalian promoter. It was also shown that mutating the various SF-1 sites did not

alleviate the downregulation of the promoter, however, no other transcription factors

were examined.

There is substantial research outlining the specific signaling cascades that TGF-P

triggers. Two serine/threonine receptor tyrosine kinases, Type I and Type II, are

assembled and dimerized when TGF-P or other related ligands are bound. The

dimerization leads to Type I receptor activation by phosphorylation in a glycine/serine

rich domain. The active receptor can then phosphorylate and activate SMAD proteins

(Figure 1-5). SMAD proteins are the essential link in TGF-P signaling (de Caestecker,

2004).

Research has specifically shown SMAD3 involvement in TGF-P inhibition of

human StAR transcriptional activity in adrenocortical cells. Overexpression of a wild-

type SMAD3 protein in the cells potentiated the inhibitory action of TGF-P on StAR

mRNA levels, whereas, expression of a mutant SMAD3 alleviated some of that

repression (Brand et al., 1998). SMADS encompass a large and diverse family of

proteins that can either activate or repress gene expression. A primary way in which









TGF-P signaling can be terminated is by ubiquitinylation of SMADS in the nucleus,

which targets the proteins for proteosome-mediated degradation (Lee et al., 2003).

Research Objectives

The goals of this project were to examine cellular signaling mechanisms involved

in regulation of LMB StAR by environmental toxins. cAMP and TGF-P are two potent

signaling molecules known to regulate steroidogenesis in mammalian species, therefore,

their role in LMB StAR regulation was examined. The overall hypothesis of this project

was that LMB StAR transcription and post-translation activity are upregulated by cAMP

and downregulated by TGF-P.

The project was divided into three specific aims to meet the overall objective.

Specific aim 1 was cloning the LMB StAR cDNA and development of a specific

polyclonal antibody. Specific aim 2 was to develop LMB ovarian follicle cultures. The

follicle assays were used to measure changes in mRNA by real-time PCR and protein by

western blot detection with the anti-StAR antibody upon exposure to cAMP or TGF-P.

Specific aim 3 was to comprehensively examine the transcriptional regulation of LMB

StAR. For aim 3, the promoter was cloned and used in transfection assays in

conjunction with promoter deletion and site-directed mutagenesis experiments for

functional transcriptional response element analysis.












sheep
pig
human
rat


sheep
pig
human
rat


sheep
pig
human
rat


GGACC'AACTCCCTTOCCC3TTCGTG CTCGTCTOCfC O AAT -139
AGGCATCAG~CICOTTACT3VCTTCTIAACACTG=CCT C CCTCGCAAT -141
TGGCCCXGTCCCCCACICCCCCG iCCCC--CGC DCCAGC CCAAAC -133
GGTGACCA--CTGGTTTCCXCGC=GCCGATCTGGTCTAG CCca.CCACC-----T -130


* */IBP **


SF -1


*** P


sheep GCrAGAAACACCATCTGGC--- -1
pig GCAAGAACAA'--GTCCTGCCCAC -1
human GCAG-AACACCAGGTCC------ -1
rat AGAG-CACTTGC=TTGAGCCAGCT -1
** *





Figure 1-1. Alignment of mammalian StAR promoters. Line-up of the first hundred base
pairs for the sheep, pig, human, and rat showed conservation of several critical
transcriptional binding sites, including SF-1, GATA, and C/EBP.

















Epithelial membrane


Vitelline envelope




So 9 Theca and granulosa cells



Vitellogenin
nucleus








Figure 1-2. Fish ovarian follicle. The ovary of a fish is composed of many individual
follicles, where steroids are produced by the theca and granulosa cells, located
below the epithelial membrane. Vitellogenin, made in the liver, is the egg
yolk precursor for the growing follicle.















Cholesterol


Mitochondria



Pregnenolone in


Progesterone mm Mineralocorticosteroids


17-Hydroxyprogesterone mmm Glucocorticosteroids





Androstenedione ,, Steroid hormones:
Estrogens
Androgens





Figure 1-3. General pathway for steroidogenesis. Cholesterol is the backbone for all
steroid hormones. Cholesterol is metabolized in the mitochondria to
pregnenolone. The various steroids are then formed from pregnenolone.














Cholesterol




OH




Si p-sitosterol



OH











Figure 1-4. Structures of P-sitosterol and cholesterol. P-sitosterol is the plant equivalent
of cholesterol with the only structural difference being an additional ethyl
group (outlined in red) for B-sitosterol.



























3










Figure 1-5.TGF-0 signaling pathway. Binding of TGF-P to the Type II receptor kinase
induces dimerization of the Type II and Type I receptor. A cascade of
phosphorylation and signaling events mediated by SMAD proteins occurs
following the receptor dimerization, ultimately activating transcription of
many different genes.














CHAPTER 2
MATERIALS AND METHODS

The overall goals of this project were to characterize the expression of the

largemouth bass (LMB) Steroidogenic Acute Regulatory Protein (StAR) and then

specifically examine its regulation at the transcriptional level. Cloning the LMB StAR

coding region and a portion of the promoter region were the initial steps in achieving the

goals of this project. The sequence information was used for development of assays,

including real-time PCR, transfections, and western blots, to quantitate changes in LMB

StAR transcription and translation. StAR expression was examined in response to

various endogenous and exogenous chemicals, including cAMP and transforming growth

factor beta (TGF-P), central molecules involved in cell signaling pathways.

Animals

Largemouth bass (Micropterus Salmoides) were used for all tissue and ovarian

follicle cultures and were purchased from American Sport Fish Hatchery (Montgomery,

AL). All fish were housed at the Center for Environmental and Human Toxicology at

University of Florida in accordance with the National Institute for Health (NIH) Guide

for the Care and Use of Laboratory Animals.

LMB StAR mRNA Expression

Cloning of StAR

Total RNA was isolated from largemouth bass ovarian tissue using the RNeasy Kit

with spin columns (Qiagen). The quality of RNA was verified by examining 3-5 [g on a

formaldehyde based gel and looking for the presence of two bands, with the 28S rRNA









band being twice the intensity of the 18S rRNA band. 3 pg of RNA was reverse

transcribed into cDNA with 200 units of SuperscriptlI enzyme (Invitrogen). Basically,

the reverse transcription reaction involved heating the RNA and 150 ng random hexamers

at 70 o C for 10 minutes. A master mix of 10 mM DTT, 0.2 mM dNTP mix (stock has

2.5 mM each nucleotide), and 5X buffer [250 mM Tris-HCl (pH 8.3), 375 mM KC1, 15

mM MgCl2 ] was diluted to 1X in a 50 pl final reaction volume. After adding the master

mix, the reaction was heated at 42 o C for 2 minutes. The reaction was then stopped to

add 200 units of SuperscriptlI which was then followed by continued heating at 42 o C for

50 minutes. A final extension at 70 o C for 15 minutes completed the reaction.

LMB StAR was PCR amplified from the ovarian cDNA using partially

degenerate primers designed with the web program CODEHOP

(http://blocks.fhcrc.org/codehop.html) and an alignment of various mammalian sequences

in the database, including human, pig, horse, and cow. Sequences for the forward and

reverse primers were 5'TGGAGCAGATGGGCGANTGGAAYCC3' and

5'TTGATGATGGTCTTGGGCADCCANCCYTT3', respectively. The cDNA was

amplified using 10 picomoles (pmol) of each primer, 1 Unit of amplitaq, and 1.5 mM

MgC12, with 10X PCR buffer [500 mM KC1 and 100 mM Tris-HCl (pH 8.3)] diluted to

1X in a 20 pl final volume. The PCR reactions used a primer annealing temperature of

60.9 o C for 45 cycles in a Perkin Elmer 9600 model thermocycler. The PCR reaction

was run out on a 1% agarose gel and the predicted 350 base pair band was gel purified by

excising the ethidium bromide stained bands from two 20 pl loaded lanes under low UV

light and then using the gel purification kit and protocol from Qiagen.









The gel purified DNA was ligated into a pGEM-T cloning vector. The amount of

DNA to ligate into the vector was calculated by multiplying 50 ng of vector by the size of

the DNA to be inserted and then dividing that number by the size of the cloning vector,

which was 3 Kb for pGEM-T. The concentration of the gel purified product was

quantified using a DNA mass ladder. A molar ratio of both 1:1 and 3:1 for insert to

vector was used. Ligation was done at 16 o C overnight with a reaction containing the gel

purified product (see calculation above), 3 units T4 DNA ligase (Promega), 50 ng

pGEM-T vector (Promega), and 2X ligation buffer (Promega-proprietary) diluted to 1X

in a final volume of 10 il.

The ligation product (3 il) was transformed into 50 [il of DH5a cells (Invitrogen).

Basically, the transformation involved incubating the cells and ligation product on ice for

20 minutes, then heat shocking the cells for 45 seconds at 42 o C followed by immediate

cooling on ice for 2 minutes. The cells were then shaken in 950 [l of LB broth for 1 12

hours at 230 rpm before being plated on ampicillin (100 [g/il) LB/agar plates for

overnight incubation at 37 o C. Colonies were screened for ligation by PCR with M13

primers. Positive clones were minipreped using the Qiagen spin column kit and sent for

sequencing at the University of Florida ICBR DNA Sequencing Core.

RACE (rapid amplification of cDNA ends) were used to sequence the remaining

coding regions of LMB StAR using the SMART RACE protocol from Clontech/BD

Biosciences (Figure 2-1). Briefly, first-strand cDNA for 5' RACE was prepared by

reverse transcribing 1 [g of total RNA isolated from LMB ovary using 1 [l of

Powerscript Reverse Transcriptase (Clontech, proprietary), 1 ll of 5' RACE primer

(proprietary concentration; 5' (T)25VN3'), 1 ll SMART oligo









(5'AAGCAGTGGTATCAACGCAGAGTACGCGGG-3'), 1.3 mM DTT, and 2 il of 5X

First Strand Buffer (250 mM Tris-HC1, pH 8.3; 375 mM KC1; 30 mM MgC12) in a final

volume of 15 .il for a 1.5 hour incubation at 42 o C. The reverse transcriptase reaches the

5' end of the mRNA and adds several dC residues, which serves as a template on the

opposite strand for an oligo that contains 3 G residues (Smart II oligo) at its 3' end.

To make cDNA for 3' RACE, the same protocol was followed for that of 5'RACE,

however, the following primer [5'AAGCAGTGGTATCAACGCAGAGTAC(T)30VN3']

in proprietary concentrations was used and the SMART oligo was not added to the

reaction mix.

The 5'RACE and 3' RACE cDNA that was synthesized was then used in PCR

reactions with gene specific primers and primers to the Smart II oligo. The primers and

thermocycler conditions used for 5' and 3' RACE are listed in Table 2-1 and Table 2-2.

All gene specific primers, designed using the Oligo program, were between 23 and 28

nucleotides in length, 50-70% GC content, and a melting temperature of greater than 65 o

C. Nested gene specific primers were also designed to help eliminate non-specific PCR

amplification. All PCR reactions contained 1 pl of 50X Advantage 2 Polymerase Mix

(Invitrogen) and 0.2 mM dNTP stock mix in a final volume of 50 [l. Initial PCR

reactions were diluted 1:50 in Tricine-EDTA (10 mM Tricine-KOH-pH 8.5; 1 mM

EDTA) and 1 pl of the diluted DNA was used for nested/secondary PCR amplifications.

For 3'RACE, a gene specific primer and primer to the poly A adaptor oligo (Invitrogen)

that was attached during reverse transcription was used for PCR amplification. 5'RACE

amplification used a primer (Invitrogen) to the 5'cDNA adaptor oligo and gene specific

primers. Products were cloned into the pGEM-T vector and plated onto LB/AMP agar









plates as described in the above section. All sequence information was verified by at

least 3 different clones.

Development of LMB Real-Time PCR Assay for mRNA Quantitation

Quantitation of LMB StAR mRNA levels in ovarian tissue and follicle cultures was

done by real-time PCR using Taqman technology. RNA for all Taqman reactions was

extracted from about 5 mg (10 follicles) of bass ovarian follicles using RNA STAT-60

reagent (Tel-TEST). The ovarian follicles were homogenized in 500 ptl of RNA STAT-

60 using a polytron followed by addition of 150 ptl of chloroform. After centrifugation at

12,000 x g for 15 minutes, the upper aqueous RNA containing layer was removed for an

additional RNA STAT-60/chloroform extraction. The RNA was then precipitated with

250 ptl of 100% isopropanol overnight at -20 o C and then pelleted by a 30 minute

centrifugation at 12,000 x g at 4 o C. The RNA pellets were washed twice with 75%

ethanol (made with DEPC treated water) and then resuspended in 25 itl of RNAsecure, a

reagent that helps minimize RNase activity (Ambion). All of the RNA (all samples

contained less than 10 tg total RNA) was treated with 2 units of DNA-Free (Ambion) for

an hour to remove traces of contaminating chromosomal DNA. Quality of RNA was

checked on an agarose gel for the presence of 28S and 18S ribosomal bands.

3 [tg of each RNA sample was reverse-transcribed into cDNA using 25 units of

Stratascript enzyme (Stratagene), 1500 ng random primers, ImM of each dNTP (4 mM

total dNTP), along with 10X Stratascript buffer (Stratagene) diluted to be 1X in a final

reaction volume of 25 ptl. In short, RNA, random primers, and water were added first,

then all tubes were heated at 65 o C for 5 minutes followed by cooling at room

temperature. Then, 4 tl of a master mix of dNTPs, buffer, and Stratascript enzyme was









added to each reaction followed by incubation at 42 o C for 1 hour. The enzyme was

inactivated by heating the reactions at 90 o C for 5 minutes.

The primers for real-time PCR using Taqman technology were designed using the

Primer Express program (Applied Biosystems) based on the original sequence obtained

for LMB StAR. All PCR reactions used 10 pmoles of the forward primer,

5'ACCCCTCTGCTCAGGCATTT3', and 10 pmoles of the reverse primer,

5'GGGCTCCACCTGCTTCTTG3', to amplify 0.12 [g of reverse-transcribed RNA

using universal thermocycler parameters as recommended by Applied Biosystems. For

the real-time PCR assays, 2X Sybr Green reagent (Applied Biosystems) was used at a

concentration of 1X in the final reaction volume. Sybr Green is a fluorescent dye that

binds to double stranded DNA. Therefore, as more amplification occurs, more

fluorescence is detected. A dissociation curve was run with the LMB StAR Taqman

primers to check for amplification specificity after the PCR cycles are completed (Figure

2-2). A dissociation curve is generated by raising the temperature and obtaining a

specific melting point for separation of double stranded amplified StAR DNA, which is

represented by a steep loss of fluorescence signal.

A standard curve for real-time PCR quantitation was developed with known

amounts of the StAR plasmid. Standard curves typically were performed in 10X

dilutions from 8.1 X 106 to 8.1 X 101 copy numbers of plasmid and samples with

unknown amounts of StAR were quantitated by extrapolation to the standard curve. A

sample standard curve and amplification curve is shown in Figure 2-2. The slope for the

standard curve should be around -3.3, which indicates perfect doubling per cycle.









All Taqman reactions were normalized to 18S rRNA using universal thermocycler

conditions as recommended by Applied Biosystems. Each 25 gtl reaction contained 1.25

gl of 18S rRNA master mix containing proprietary amounts of primers and probes

(Applied Biosystems, catalog #4310893E), 0.12 gg of reverse transcribed RNA, and 2X

Taqman universal primer mix (Applied Biosystems, catalog # 4304437) diluted down to

1X in the final reaction volume.

All real-time PCR calculations were based on converting the concentration of the

the StAR plasmid used in standard curve to copy number. The conversion to copy

number was done since the concentration of the plasmid DNA includes the cloning vector

plus StAR DNA, which would not accurately reflect the concentrations for StAR.

Converting to copy number ensures that the plasmid is considered as one unit, including

both the vector and insert, and amplification is therefore a reflection of that unit. All

quantities from real-time PCR were measured as copy number of plasmids amplified.

The calculation for determining amount of copy number used to develop the standard

curve is :

6 X 1023 (copies/mol) X plasmid concentration (g/ul)
= copies / ul
molecular weight of the plasmid (g/mol)

Seasonal Study

Adult LMB between 2 and 3 years old were maintained in freshwater ponds at the

USGS facility in Gainesville, FL. Female fish were caught by electroshock bi-weekly

over a five month time span. Ovarian tissue was carefully removed and immediately flash

frozen with liquid nitrogen for long-term storage at -80 o C.









LMB Ovarian Tissue Cultures

Ovarian tissue cultures from LMB were cultured to detect changes in mRNA levels

after exposure to various chemicals. The ovarian tissue was carefully dissected into 20-

30 mg pieces, rinsed with culture media, and immediately placed in 1 ml of Dulbecco's

Modifed Eagle's Medium Nutrient Mixture (DMEM) with F-12 Ham containing L-

Glutamine and 15mM HEPES (Sigma) supplemented with 1.2 grams of sodium

bicarbonate and 1% antibiotic/antimycotic solution (ABAM). This media has the same

osmolality as LMB plasma, about 295 mOsmol/kg (Bowman thesis, 2001). Cultures

were equilibrated in a chilled incubator at 21-22 o C with 5% CO2 for 24 hours prior to

exposure with appropriate chemical to be studied. All experiments were carried out in 24

well culture plates, which were placed on a slow moving shaker during the exposures.

LMB Ovarian Follicle Cultures

Ovarian follicles were isolated from the ovary and cultured for further examination

of LMB StAR mRNA expression. Follicles were individually dissected and measured

with a micrometer to control size and stage of follicles incubated. Follicles were

incubated in 500 ptl of the DMEM culture media and equilibrated for 24 hours prior to

exposure. Ten follicles were incubated per well of a 24 well cell culture plate. Follicles

and culture were removed from the culture plate post exposure by using a BSA (bovine

serum albumin) coated wide boar 1 ml pipet tip and placed in a 1.5 ml microcentrifuge

tube. Follicles were gently pelleted to the bottom of the tube by centrifugation for 5

minutes at less than 3000 RPM. Follicles were washed once with IX PBS (phosphate

buffered saline) and then frozen at -80 o C until RNA isolation.

The viability of follicles was tested by adding 10% Alamar Blue reagent

(Biosource) to the cultures and looking for reduction of Resazurin (blue and









nonfluorescent) to resorufin (pink and highly fluorescent). Changes in the color from

blue to pink for metabolically active cells can be detected with spectrophotometry

readings at A570 and A600.

LMB StAR Protein Quantitation

Protein Expression Vector

There is no commercial antibody available for StAR that cross reacts with any fish

species, therefore, we developed a polyclonal antibody for western blot detection. First, a

protein expression vector was constructed by amplifying the entire coding region in one

piece using 10 pmol of ATGCTACCTGCAACCTTCAAACTGTG as the forward primer

and 10 pmol of TCAGCAGGCGTGAGCCATCTCCATA as the reverse primer. The

annealing temperature was 72 o C for 7 cycles followed by 67 o C for 42 cycles. The PCR

reaction contained the primers 1 [l of 10 mM dNTP mix, 2 [l of 25 mM Mg(OAc)2, 0.15

[g of LMB ovarian cDNA, 50X Advantage Genomic Polymerase Mix diluted to 1X

[Clontech: 5-6 units/pl Tth DNA polymerase, 0.5gg/[l TthStart antibody, 50% glycerol,

10 mM Tris-HCl (pH 7.5), 230 mM KC1], and 10X PCR reaction buffer [400 mM Tris-

HC1 (pH 9.3), 150 mM KOAc, 0.2% Triton X-100] diluted to IX in a final volume of 50

[l.

The entire coding region was ligated into the pET-28b vector (Novagen) (Figure 2-

3) and transformed into DH5a cells (Invitrogen) using similar procedures as outlined in

the section for cloning of StAR. Expression with the pET-28b vector produces proteins

with a 6 histidine tag on to N-terminus to ultimately allow for purification with an

appropriate affinity column.

Amplification of the full length StAR resulted in 8 amino acid mistakes that were

fixed using the QuikChange Kit (Stratagene). Primers to change the appropriate









nucleotides were designed using Stratagene's website program for site directed

mutagenesis (Table 2-3). The primers were designed to have the targeted mutation near

the middle, a minimum of 40% GC content, and a 3' end that terminates with one or

more G or C bases. Complementary reverse primers were designed against each forward

primer. The QuikChange protocol basically involved a PCR reaction using 125 ng of

each relevant forward and reverse primer, 38 ng of StAR plasmid template, 1 ll of a

proprietary dNTP mix, and 2.5 U/pl of Pfu Turbo DNA polymerase in a 50 pl final

volume. Reactions were amplified in a thermocycler for 12 cycles of 95 o C at 30

seconds, 55 o C for 1 minute, and 68 o C for 7 minutes. The parent strand is digested by

DpnI, leaving the corrected product to be transformed in DH5a cells. The transformed

constructs were minipreped with the Qiagen miniprep spin kit and DNA was sent for

sequencing to verify that the relevant nucleotides were fixed. Since pET-28b is a low-

copy number plasmid, concentration yields from minipreps were maximized by using 3

ml of bacterial culture and by eluting the purified DNA from the spin column with 70 o C

water. The final, completely corrected construct was transformed into BL21 (DE3) cells

(Novagen) for bacterial expression.

Bacterial Protein Induction

The LMB StAR expression construct was grown in 30 ml of LB broth with 24

[g/ml kanamycin for 4 hours at 37 o C with constant shaking at 280 rpm. The bacterial

cultures reached a desired density after the 4 hours with A600 spectrophotometry readings

between 0.6 to 0.8. StAR protein expression was then induced in the cultures with 1 mM

or 3 mM IPTG for an additional 4 hours with lml aliquots taken hourly. Bacterial cells

were pelleted by spinning down the 1 ml of culture at 10,000 x g for 1 minute, discarding

the supernatant. The pellet was resuspended in 100 .il of 1X phosphate buffered saline









(PBS) along with 1 pl of benzonase to lessen the viscosity. 100 tl of 4X SDS buffer

(250 mM Tris-HCl pH 6.8, 8% SDS, 10% 2-mercaptoethanol, 300 mM DTT, 40%

glycerol, and 0.02% bromophenol blue) was then added to the protein sample and heated

at 85 o C for 3 minutes for denaturation. The denatured protein samples were run on a 4-

12% Bis-Tris NuPAGE gel (Novex) with MES running buffer for 30 minutes. The gel

was stained with Colloidal Coomassie Blue Stain (Genomic Solutions) overnight

followed by de-staining for 2 hours.

The bands on the protein expression gel that were induced by IPTG compared to

the controls were excised and ultimately subjected to digestion with trypsin for definitive

identification of largemouth bass StAR peptide fragments in the gel. The in-gel trypsin

digestion protocol involved washing the gel pieces with 50% acetonitrile (ACN) 3 times

while vortexing for 15 minutes followed by dehydration of the gel with 100% ACN until

gel piece turns white. The gel was then rehydrated with 100 mM ammonium bicarbonate

(ABC) for 5 minutes. The proteins in the gel piece were reduced with 45 mM DTT for

30 minutes at 55 o C. DTT is a reducing agent that separates proteins which are linked

by disulfide bonds for more effective analysis by mass spectrometry. To prevent the

cysteine residues in the separated peptides from recombining, they are alkylated with 100

mM iodoacetatamide for 30 minutes in the dark at room temp. The gel piece was then

washed for 15 minutes 3 times with 50% ACN/50 mM ABC while vortexing. The gel

was completely dried in a speed vac prior to digestion with 12.5 ng/ipl Trypsin (Promega)

prepared in 50 mM ABC pH 8.4, 5 mM CaC12 on ice for 45 minutes. The enzyme

solution was then removed and replaced with just the buffer for incubation overnight at

37 o C prior to analysis by mass spectrometry.









Protein Purification

Purified LMB StAR was obtained by inducing 10 ml of bacterial culture for 4

hours with the StAR expression plasmid (see section on bacterial protein induction for

details). The bacteria were pelleted at 4000 x g for 15 minutes and stored at -80 o C until

ready for purification.

A nickel affinity column (Ni-NTA spin column kit, Qiagen) was used to purify

histidine-tagged StAR from the bacterial pellet under denatured conditions. The bacterial

cells were lysed by thawing the pellet for 15 minutes and then resuspending them in a

buffer of: 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HC1, pH 8.0. The cells were shaken

for 1 hour at room temperature. Cellular debris was removed from the lysate by

centrifuging at 10,000 X g for 20 minutes. Spin columns were equilibrated with 600 pl

of the pH 8 buffer followed by centrifugation at 700 x g for 2 minutes. Histidine tagged

proteins were bound to the nickel affinity column by flowing 600 tl of the bacterial

lysate through the equilibrated column at 700 x g for 2 minutes. The columns were

washed 3 times with 600 tl of a buffer containing: 8 M urea, 0.1 M NaH2PO4, 0.01 M

Tris-HC1, pH 6.3. Finally, the bound proteins were eluted from the spin column with 200

pl of the following buffer: 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HC1, pH 4.5.

The purified protein was quantitated by protein assay using Coomassie Plus

Reagent. Two micrograms of the purified protein was run out on a 4-12% Bis-Tris

NuPAGE gel (Novex) and stained overnight in Colloidal Coomassie Blue followed by a

2 hour de-stain.

Development of StAR Antibody

A polyclonal antibody was made against the largemouth bass StAR Protein. The

antibody was produced by injecting two different rabbits (Cocalico company) with a









synthetic peptide designed to a part of the coding sequence. The antigenic peptide used

for rabbit injections, CFLAGMSTQHPKMPEQRGVVR (Figure 2-4), was constructed in

an area of the coding region where prolines were present so the antibodies are able to

recognize relatively exposed areas of StAR. The peptide was conjugated to a carrier

protein, KLH (keyhole limpet hemocyanin), at the University of Florida Protein Core to

help ensure an immunogenic response to the StAR peptide.

For the rabbit injections, the following schedule was followed; Day 0 =

Prebleed/Initial Inoculation, Day 14 = Boost, Day 21 = Boost, Day 35 = Test Bleed, Day

49 = Boost, Day 56 = Test Bleed. Monthly boosts were subsequently continued for

about 12 year to obtain more antiserum.

Western Blots

The specificity and reactivity of the LMB StAR antibody was tested by western

blot using the purified StAR protein as a positive control. 1 gg of purified protein was run

on a 4-12% Bis-Tris NuPAGE gel followed by transfer to nitrocellulose membrane at 100

volts for 1 hour. The transfer buffer contained 20 mM Tris, 144 mM glycine, and 20 %

methanol. The nitrocellulose membrane was blocked for 3 hours, while shaking, with 5%

Carnation non-fat dry milk diluted in 1X TBST (25 mM Tris, 0.15 M NaC1, 0.05 %

Tween-20, pH 7.6) to help reduce non-specific antibody binding. The membranes were

then washed 4 times, at 15 minutes each, with 1X TBST.

Washed membranes were then exposed to the primary antibody for 3 hours with

vigorous shaking. Dilutions of 1:5000 and 1: 15000 primary antibody were tested.

Following the incubation, primary antibody was removed by washing 4 times with TBST

before incubation with concentrations of 1:20000 and 1:40000 secondary antibody for

one hour. The secondary antibody used was a mouse anti-rabbit IgG conjugated to









horseradish peroxidase (Pierce). Membranes were again thoroughly washed with TBST

before antibody binding was detected using chemiluminescence after exposing for 3-4

minutes to 4 ml of each chemiluminescent reagent (Super Signal West Pico

Chemiluminescence Kit, Pierce.) Presence of luminescence was captured on Kodak film.

Transcriptional Regulation of LMB StAR

Cloning of the Promoter

Most transcriptional regulation occurs in the promoter, therefore, the first goal for

this part of the project was to clone the promoter for LMB StAR using the

GenomeWalker Kit (Clontech) (Figure 2-5). The promoter cloning started with isolating

high quality genomic DNA that was phenol/chloroform purified twice from 20 mg of

LMB ovarian tissue using the Wizard Kit (Promega.). Basically, the genomic isolation

involved lysing the tissue with 600 ptl of a lysis solution (proprietary, Promega) for 20

minutes at 65 o C. Contaminating RNA was degraded by incubation of the lysate for 20

minutes at 37 o C with 3 ptl of an RNase solution (proprietary, Promega). The solution

was chilled on ice with 200 [il of a Protein Precipitation Solution (proprietary) and the

protein was removed by centrifuging at 14,000 X g for 4 minutes. The supernatant was

put into a new tube. Genomic DNA was precipitated from the remaining supernatant

with 600 ptl ofisopropanol and pelleted by centrifuging at 15000 X g for 1 minute. The

pellet was washed with 70% ethanol and then air dried for 12 minutes. The pellet was

resuspended in 100 [tl of TE buffer (10 mM Tris-HC1, pH7.3; 1 mM EDTA, pH 8.0) by

incubating at 65 o C for 1 hour. Residual protein was removed from the genomic DNA

with a phenol/chloroform extraction followed again by resuspension in TE buffer (10

mM Tris-HC1, 1 mM EDTA; pH 7.5).









Genomic DNA (2.5 Gg) was digested for 20 hours with 4 different restriction

enzymes; StuI, EcoRV, Dral, and PvuII to create digested libraries for PCR

amplification. Complete digestion of the genomic DNA was checked by running 5 pl on

a 0.5% agarose gel stained with ethidium bromide at 70 volts for one hour. The

restriction libraries were purified with 2 phenol/chloroform extractions. An adaptor oligo

(Clontech) was ligated onto the various digested pieces of genomic DNA by incubating

with 3 units of T4 DNA ligase for 20 hours at 16 o C.

PCR amplification of the LMB StAR promoter from the restriction libraries was

done using a primer to the ligated adaptor oligo and a gene specific primer. Two

different sets of gene specific primers were used, one set of primers started closer to the

5'end of the coding sequence than the other, which gave overlapping sequences and

provided for some sequence verification. One set of gene specific primers used was

5'CAGGCAACATCTTACTCAGGACTTTGTC3' (promoter jkl). It was followed by

5'TCACCTTGCTTCACATAAGACATCTCT3' (promoter jk2) for the nested reaction.

The second construct used 5'TTCCACTCCCCCATTTGCTCCATATTT3' (promoter

jk3) for the primary PCR reaction and

5'CAGGCAACATCTTACTCAGGACTTTGTC3' (promoter jkl) for the nested

reaction. All primers were designed using the Oligo Program and were between 26 and

30 nucleotides with a 40-60% GC content, with no more than 3 G and C's in the last 6

bases of the 3' end. Nested gene specific primers were used to help reduce non-specific

amplification. All secondary PCR reactions using the nested primers were done using a

1:50 dilution of the primary PCR product in a final volume of 50 il. Thermocycler

conditions for the promoter cloning are listed in Table 2-4. PCR products were gel









purified with spin columns and protocol from Qiagen and ligated into the pCR2.1-TOPO

vector (Invitrogen) using a similar protocol as outlined under the section for StAR

cloning. The sequence for the 2.9 Kb promoter fragment (named pSP1 plasmid) was

verified with two independent PCR reactions.

The promoter was cloned into the pGL3 basic vector (Promega) (Figure 2-6) for

transfections. The cloning of the promoter involved two phases, first, both the pGL3 and

pSP1 plasmids were double digested with Xhol and HindIII promega enzymes and Buffer

B (6 mM Tris-HC1, 6 mM MgC12, 50 mM NaC1, 1 mM DTT, pH 7.5) for 13 hour

digestion at 37 o C. The XhoI/HindIII fragment vector was ligated into the cut pGL3

vector by protocol outlined under StAR cloning section and the plasmid was named

pLUCStAR1. The pLUCStAR1 construct, however, still had the translational start site

from the LMB StAR sequence since the primers used for promoter cloning originated in

the coding region. Removing the ATG start site from the plasmid was essential since the

pGL3 plasmid contains its own ATG site for luciferase protein synthesis. The ATG start

site was removed from the pLUCStAR 1 with a double digest of HindIII (Stratagene) and

BlpI (NEB labs) in buffer #2 (NEB labs = 20 mM Tris-OAc, 50 mM KOAc,

10 mM Mg(OAc)2, 1 mM DTT; pH 7.90) @ 250C.

All promoter constructs were obtained using endotoxin-free maxiprep kits

(Qiagen). The maxiprep protocol basically involved growing up a single colony of the

plasmid in DH5a cells in a 3 ml LB/Ampicillin (100 [tg/ml AMP) starter culture for 8

hours. The starter culture was then added to 100 ml of LB/Ampicillin (100 tg/ml AMP)

for overnight growth at 37 o C. The plasmid was then purified under endo-toxin free

conditions from the DH5a cells by binding of the DNA to an anion-exchange resin. The









plasmid was eluted from the resin under high salt conditions (1.6 M NaC1; 50 mM

MOPS; 15% isopropanol). The DNA was then concentrated and rid of salt with

isopropanol precipitation. All maxipreps were suspended with 2 ml of TE buffer (10 mM

Tris-HC1, pH 8.0; 1 mM EDTA).

Promoter Analysis

The transcription start site for the LMB StAR promoter was identified by

sequencing to the end of the 5'UTR (untranslated region) using 5'RACE. The 5'UTR

information was then matched up with genomic sequence to determine where the UTR

ends and the promoter begins. Sequence upstream of the transcription start site was then

analyzed with three different web search engines, MatInspector V2.2, Professional-

MatInspector V7.3, and TFSearch, to identify putative consensus binding sites.

LMB SF-1 Cloning

A 246 base pair portion of LMB SF-1 was cloned from ovarian tissue by PCR with

a forward primer 5'CCAACCGCACCATCAAGTCNGARTAYCCNG 3' and reverse

primer 5'GAAGACCATGCAGCGGCKNGCCCANTC 3'. The PCR reactions consisted

of 10 pmol each primer, 1.5 mM MgC12, 0.2 mM dNTP mix, 1 unit of amplitaq (Perkin

Elmer/Applied Biosystems), 0.45 pg cDNA, and 10X PCR buffer [500 mM KC1 and 100

mM Tris-HCl (pH 8.3)] diluted to 1X in a 20 il final volume. PCR amplification

conditions using a Perkin Elmer 9600 thermocycler are listed in Table 2-5.

Promoter Deletion

One deletion of the 2.9 Kb LMB StAR promoter was made. The deletion was

made via a double digest with EcoRV and BstEII. The EcoRV restriction site is at the

5'end of the promoter and the BstEII is about 1000 bp from the 5' end, leaving a 1.86 Kb

promoter when digested. The digested promoter construct was analyzed on a 1% agarose









gel and the bands corresponding to the proper deletion pieces were purified using spin

columns from a Qiagen kit. Additionally, unlike EcoRV, BstEII is not a blunt-end

cutting enzyme, which left incompatible ends, requiring filling in the ends with Klenow

enzyme (NEB labs). For the Klenow reactions, 1 unit of Klenow was incubated with 1 lg

DNA and 0.2 il of a 10 mM dNTP mix in a final volume of 20 [l. The Klenow reactions

were stopped immediately after 15 minutes by flowing the DNA through spin columns

from a PCR purification kit (Qiagen). Then, 60 ng of purified, digested DNA was re-

ligated with 2000 units of concentrated T4 DNA ligase (NEB labs) in a final volume of

20 il for 40 minutes at room temp. The ligation was then transformed into DH5a cells

using protocol outlined under section for StAR cloning. All promoter constructs were

verified by sequencing and restriction digested with Dral.

Mutagenesis of Putative Transcription Factor Binding Sites

Several transcription factor binding elements in the 2.9 Kb promoter were mutated

to a NotI restriction site. These were constructed using the QuikChange XL Site-

Directed Mutagenesis Kit (Stratagene.) The protocol combined 125 ng of each relevant

forward and reverse primer (Table 2-5), 10 ng of the 2.9 Kb StAR promoter construct, 1

tl of a proprietary dNTP mix, and 2.5 units of Pfu Turbo DNA polymerase in a final

volume of 50 [il. PCR amplification conditions for all QuikChange reactions are listed in

Table 2-6 and Table 2-7. Following PCR amplification to create mutagenized promoter

constructs, the parent, unmutagenized strands were digested with 10 units of DpnI at 37 o

C for 1 hour. 1 pl of the DpnI digested PCR product was transformed into DH5a cells

using the protocol outlined under the section for StAR cloning. The transformation was

plated on LB/AMP agar plates (100 [g/ml AMP). Several colonies were minipreped for









each QuikChange reaction and were screened for creation of the desired mutation by

digesting 2 pg of DNA with NotI for 3 hours.

Culturing of Y-1 Cells

Y-1 mouse adrenal cells (passage 1) were purchased from ATCC (American Type

Culture Company). The cells were cultured in media containing: Ham's F12K medium

with 2 mM L-glutamine supplemented with 1.5 g/L sodium bicarbonate; 15% horse

serum; 2.5% FBS; and 1% penicillin-streptomycin mix. The cells were grown in T-75

flasks at 37 o C for normal propagation with a media change every other day to retain the

endogenous steroidogenic activity (following the recommendation from ATCC). After 4

days in culture, the cells typically reached about 70 to 80% confluency and were then

split 1:3 after trypsinization. Each T-75 flask received 2 ml of trypsin for 4 minutes at 37

o C followed by inactivation of trypsin with 4 ml of media with serum. Lastly, 2 ml of

the trypsinized cells where then added to one of the three pre-equilibrated flasks with 13

ml of media. To help alleviate clumping, cells were gently pipeted up and down with a 2

ml glass pipet about 10 times.

Transfection Assays

Cells were trypsinized as outlined in the section above on culturing of Y-1 cells,

however, after inactivation of the trypsin by media with serum, the cells were pelleted at

1500 rpm for 5 minutes and resuspended with 10 ml of fresh media. Clumping of the

cells was alleviated by pipeting the cells up and down with a 2 ml pipet and a brief 1

second vortex. Cells were counted with a hemacytometer and 150,000 cells/well were

plated in 500 pl of media with serum and allowed to attach and equilibrate for 24 hours.

Cells received fresh media immediately prior to transfection. Transfection reactions

consisted of 0.1995 [g of the appropriate StAR promoter construct, 0.0005 [g of the









control renilla luciferase vector, and Fugene6, where the ratio of tg of DNA to [il of

Fugene6 was 6:1 (1.2 pl Fugene6/ 0.2 tg total DNA), suspended in 20 pl media with no

serum or antibiotic. The transfection mixture sat for 30 minutes at room temperature

before adding it dropwise to the cells. All transfections were done in 24 well plates from

Corning CoStar.

Doses were prepared in the same manner for all experiments. dbcAMP doses were

made fresh for each experiment by diluting the appropriate amount of powder into cell

culture media. For TGF-P exposures, stock solutions of 1 tg/ml were kept aliquoted and

frozen at -80 C until appropriate dilutions were made fresh for each experiment.

GFP Quantitation

To measure the transfection efficiency, 0.1995 pg of GFP (pEGFP, Clontech) was

transfected in place of the LMB StAR promoter DNA. Cells were transfected as normal

and subsequently trypsinized for GFP quantitation on a hemacytometer.

Luciferase Measurements

After exposures were completed, cells were immediately washed once with 1X

PBS (phosphate buffered saline; 0.144 g/L KH2PO4, 9 g/L NaC1, 0.795 g/L Na2HPO4, pH

7.4) and then lysed with 100 il of IX passive lysis buffer (Promega) for at least 15

minutes at room temperature.

Luciferase measurements were done using reagents from the Dual Luciferase Kit

(Promega). Firefly luciferase was measured first by adding 20 pl of cell lysate to 100 [l

of reagent that contains substrate for the firefly luciferase and luminescence was

immediately measured by luminometer. Renilla luminescence was then quantitated by

adding 100 pl of Stop and Glo reagent, which contains reagents to quench the firefly

reaction and substrate for renilla luciferase.









Mouse StAR Real-Time PCR Assay

A real-time PCR assay was developed to measure endogenous levels of mouse

StAR in the Y-1 cells. Total RNA was extracted from the Y-1 cells by lysing with 500 il

of RNASTAT (Tel-Test) and then following the same protocol as outlined for LMB.

Primer sequences for mouse StAR had previously been designed and published (Fielden

et al., 2002); forward primer 5' TGCTAAGGATCGGGAACTGT 3'; reverse primer 5'

TCTGGCCTTTTACAGAGGAGA 3'. The PCR reactions for the mouse StAR were set

up in the same manner as the LMB StAR with the exception of having no standard curve,

all quantitations were relative.

The relative calculations for the mouse StAR real-time PCR were done by

subtracting the 18S rRNA Ct value (cycle threshold) from the StAR Ct values. Ct values

represent the initial detection in the increase of fluoresence signal associated with an

exponential increase of PCR product during the log-linear phase. An average of the

normalized Ct values is obtained for each treatment and then the value for the

experimental group is subtracted from that of the control group, which leaves a log

number that can be calculated into a fold change.

Statistics

Student's T-test was used for evaluation of significance between control and

experimental groups. Results were reported as significant ifP < 0.05.











Table 2-1. Primers for 5' and 3' RACE.


Primer name 5' RACE 3' RACE



5'TTTTCGGGTGCTGAG
TGGACATCCCAG3'
(for 1 st round of 5' RACE)
Original primer 5'ATCGGCCAAGACAC
AATGGTTACC3'
5'TTCCACTCCCCCATT
TGCTCCATATTT3'
(for 2nd round of 5'
RACE)


5'CTTGGCCGATCTTTT
GAAGGATCT3'

Nested primer 5'AGCGGAGAATGGAC
CTACCTGTAT3'
5'CAGGCAACATCTTAC
TCAGGACTTTGTC3'












Table 2-2. Thermocycler conditions for 5' and 3' RACE.


Original RACE Cycle Parameters Nested RACE Cycle Parameters


Cycle 5X:
94 C / 5 seconds
72 C / 2:30 minutes


Cycle 5X:
94 C / 5 seconds
70 o C/10 seconds
72 C / 2:30 minutes


Cycle 27X:
94 oC /5 seconds
68 C / 10 seconds
72 C / 2:30 minutes


Cycle 25X:
94 C / 5 seconds
68 o C /10 seconds
72 C / 2:30 minutes










Table 2-3. Primers used to fix nucleotide mistakes in full length StAR cDNA sequence.

QuickChange Forward Primer Reverse Primer
Reaction
5'CATATGAGGAACATGACA 5'CATTGCATTCTTCCTCAA
1 GGTTTGAGGAAGAATGCAA ACCTGTCATGTTCCTCATAT
TG3' G3'

5'GCCATCAGCATCCTCAGC 5'GTCCTGGTCGCTGAGGAT
2 GACCAGGA GCTGATGGC3'
C3'


5'AGTAACTGGATCAACCAA 5'CTGAGGAGGGAGCTTCTT
3 ACCCGAGGAAGAAGCTCCC CCTCGGGTTTGGTTGATCC
TCCTCAG3' AGTTACT3'

5'GCAGAGGGGTGTTGTCAG 5'CATTCTCCGCTCTGACAA
4 AGCGGAGAATG3' CACCCCTCTGC3'


5'CTAAATATAGATCTAAAG 5'GTTTATGATTGTCTTTGG
5 GGCTGGATCCCAAAGACAA GATCCAGCCCTTTAGATCT
TCATAAAC3' ATATTTAG3'

5'AGTAACTGGATCAACCAA 5'CTGAGGAGGGAGCTTCTT
6 ACCCGAGGAAGAAGCTCCC CCTCGGGTTTGGTTGATCC
TCCTCAG3' AGTTACT3'

5'GGCCATCAGCATCCTTAG 5'CCGTCCTGGTCGCTAAGG
7 CGACCAGGACGG3' ATGCTGATGGCC3'


5'GTGGACTTTGCCAACCAC 5'CCTTTGCCGGAGGTGGTT
8 CTCCGGCAAAGG3' GGCAAAGTCCAC3'











Table 2-4. Thermocycler conditions for LMB StAR promoter cloning.

Primary PCR Conditions: Secondary PCR Conditions:


7 cycles: 5 cycles:
94 C / 2 seconds 94 o C / 2 seconds
72 C / 3 minutes 72 o C / 3 minutes


37 cycles: 24 cycles:
94 C / 2 seconds 94 o C / 2 seconds
67 C / 3 minutes 67 o C / 3 minutes



67 o C / 4 minutes 67 o C / 4 minutes












Table 2-5. Thermocycler conditions for cloning ofLMB SF-1.


Number of Cycle Parameters
Cycles
Temperature Time


1 95 0 C 2 minutes



95 0 C 30 seconds


40
61.8 oC 25 seconds



72 o C 45 seconds



1 72 C 10 minutes










Table 2-6. Primers for promoter mutagenesis.

Site Mutated Forward Primer Reverse Primer

5'ATAGCGCCTTTCTAGTCTTTGC 5'CATGTAAAAGCGCTTTGAGCGG
ERE/2678 GGCCGCTCAAAGCGCTTTTACAT CCGCAAAGACTAGAAAGGCGCT
G3' AT3'


5'GAATTGCAGTTTTCCCCATGGC 5'GCAGTTTCAGGTTTTAATGAGC
COUP-TF/2027 GGCCGCTCATTAAAACCTGAAAC GGCCGCCATGGGGAAAACTGCA
TGC3' ATTC3'


5'TAGGGAGCCATTTGAAATAGG 5'CTTTTTTCAAAGAGCCAAAGTA
ROR/1969 CGGCCGCCCTACTTTGGCTCTTT GGGCGGCCGCCTATTTCAAATGG
GAAAAAAG3' CTCCCTA3'


5'CTGTGGCTGAGTAATGCGGCCG 5'ACACAGGCCTAGTACTAGCGG
GATA/AP-1/ERE CTAGTACTAGGCCTGTGT3' CCGCATTACTCAGCCACAG3'
/1882


5'GGTGATATTTGCGAAGGAGCG 5'TTCAGGAAAGGACGTTTGTGGC
COUP-TF/2304 GCCGCCACAAACGTCCTTTCCTG GGCCGCTCCTTCGCAAATATCAC
AA3' C3'











Table 2-7. Thermocycler conditions for promoter mutagenesis with QuikChange-XL
protocol.
Cycles Temperature Time


1 95 C 1 minute





95 0 C 50 seconds





18 60 C 50 seconds





68 0 C 8 minutes





1 68 0 C 7 minutes













5' AAAAAA 3' (RNA)
Adaptor
1
5' AAAAAA 3' (RNA)
3'ccc --- 5'

1
5' AAAAAA 3' (RNA)
Primer to qligo
3' .5'

1
Gene specific
primer
5' 1 3'


5' AAAAAA 3' (RNA)
I Adaptor

5' AAAAAA 3' (RNA)
3' --- 5'


5' AAAAAA 3' (RNA)
Gene specific
primer
3' | 5'


Primer to adaptor
5'- | 3'


3' 1 15'



Figure 2-1. Rapid amplification of cDNA ends (RACE). (A) 5' RACE protocol and (B)
3' RACE protocol to extend the cDNA sequence for LMB StAR. Both 5' and
3' RACE required primers specific to the original 345 bp sequence obtained
for LMB StAR.












A



















B.






D 5









Figure 2-2. Sample standard curve for real-time PCR. A) A standard curve is generated
by plotting Ct (cycle threshold) values versus the log of the copy numbers for
minipreps of the StAR plasmid done in 10X serial dilutions. Two replicates
were done for each standard. Unknown samples are then extrapolated to the
standard curve. B) Dissociation curve for LMB StAR to check for primer
specificity. The dissociation of fluorescence from the amplified, double-
stranded DNA was detected with a melting curve.






























T7 Promoter 370-386
T7 Transcription site 369
His-Tag coding sequence 270-287
Multiple cloning sites (BamHI to Xhol) 158-203
T7 terminator 26-72
Kanamycm coding sequence 3995-4807
fl origin 4903-5358


Xho 1(15s)
Not 1(166)
Eag 1(161)
Hind III(13)
Sal 11179)
Sac 1(1901
EcoR 1(1021
EjInH I. t';.
Bp1102 [80)o~ Nhe lI: ii
Dra 111(5127) No 1296)
-Xba 1035)
lf* "Bgl 11W01)
,.. f SgrA 1(442)
/\ Sphl(5Be)


PVU 1(4426) / '
Sgf 1(4426)
Sma ](4300) Mlu (1.123)
Sc -Bet 1 (1137)
Cla 1(4117) i
Nru 1(4os) n T-28b Bs1E11(1304:
,Ap(1334)
5368 bp
B ssH 111534)
Eco571(3772)\ EcoR V(1573]
Hpa 1(1629)
AlwN 1(3640)


BssS [(3397) ) PshA 1(1963)
BspLU11 I(3224) 1 Bg1(2187)
Sap 1(108) Fsp 1(2205)
Bstl 107 (295) -~Psp11(223)
Tthl 11 1(2969)'


Figure 2-3. Map of pET-28b vector.The full coding sequence for StAR was cloned into
the pET-28b vector (Novagen) for His-tagged protein expression in
BL21(DE3) bacterial cells.










58



10 20 30 40 50 60
I I I I I I
brook trout MLPATFKLCAGISYRHTRNMTGLRKNAMVAIHHELNMLA-- GPNPSSWI SVRRRSSLLS
rainbow troutMLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNMLA-- GPNPSSWISFHVRRRSSLLS
LMB MLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNRLA--GPGPSNWINQTRGRSSLLS
zebrafish MLPATFKLCAGISYRHMRNMTGLRKNAMIAIHHELNKLS--GPGASTWINHIRRRSSLLS
pig MLLATFKLCAGSSYRHVRNMKGLRHQAV LALGQELNRRALGGPTSGSWINQVRRRSSLLG
horse MLLATFKLCAGSSYRHVRNMKGLRHQAALAIGQELNWRAPGGPTQSGWINQVRRQSSLLG
human MLLATFKLCAGSSYRHMRNMKGLRQQAVVMAISQELNRRALGGPTPSTWINQVRRRSSLLG

Prim. cons. MLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNRLALGGPTPSSWINQVRRRSSLLS

70 80 90 100 110 120

brook trout SRIEEEQGYNEAEVSYVKQGEEALQKSISILGDQDGWTTEIIAANGDKVLSKVLPDVGKV
rainbow troutSRIEEEQGYNEAEVSYVKQGEEALQKSISILGDQDGWTTEIIAANGDKVLSKVLPDVGKV
LMB SRIEEEEGYSDEEMSYVKQGEDALQKAISILSDQDGWTTEIVAANGDKVLSKMLPDIGKV
zebrafish SPIAEET-YSEADQCYVQQGQEALQKSIS ILEDQDGWQTEIESINGEKVMSKVLPGIGKV
pig SQLEDTF-YSDQDLAYIQQGEEAMQRALDILSNQEGWKKESRQENGDEVLSKVIPDVGKV
horse SQLEDTL-YSDQELAYIQQGEEAMQKALGILRNQEGWKEENQQANGDKVLSKVVPDVGKV
human SRLEETL-YSDQELAYLQQGEEAMQKALGILSNQEGWKKESQQDNGDKVMSKVVPDVGKV

Prim.cons. SRIEEE2GYSD2EL2YVQQGEEALQKAISILSDQDGW2TEI22ANGDKVLSKVPDVGKV

130 140 150 160 170 180

brook trout FKLEVLLDQRSDNLYGELVGNMEQMGDWNPNEVEVKILQKIGQETMVTHEVSGPTPGNV
rainbow troutFKLEVLLDQRSDNLYVELVGNMEQMGDWNPNKVKEVQKQKIGQETMVTHEVSGPTPGNVV
LMB FKLEVMLEQRPDNLYKELVGNMEQMGEWNPNVKQVKILQKIGQDTMVTHEVSAETPGNVV
zebrafish FKLEVTLEQQTGDLYDELVDNMEQMGEWNPNVKQVKILQKIGQETMITHEISAETPGNVV
pig FRLEVVVDQPMERLYEELVERMEAMGEWNPSVKKIKILQKIGKDTVITHELAAEAAGNLV
horse FRLEVEVDQPMERLYEELVERMEAMGEWNPNVKEIKVLQKIGKDTVITHELAAESAGNLV
human FRLEVVVDQPMERLYEELVERMEAMGEWNPNVKEIKVLQKIGKDTFITHELAAEAAGNLV

Prim. cons. FKLEV2LDQ2M22LYEELV2NMEQMGEWNPNVKEVKILQKIGQDTMITHE2SAETPGNVV

190 200 210 220 230 240

brook trout GPRDFVSVRCAKRRGS' cu AEMiTBSIQHETIrm1FEQRVV ENGPTCIVMRPSADDPNKTKF
rainbow troutGPRDFVSVRCAKRRGS CFLAGMSTQHPTMPEQRGWEV ENGPTCIVMRPSADDPNKTKF
LMB GPRDFVSVRCAKRRGS CFLAGMSTQHPKMPEQRGWFV ENGPTCIVMKPCVEDPNKTKF
zebrafish GPRDFVNVRHAKRRGS CFLAGMSTQHPGMPEQKGFV 1ENGPTCIVMRPSADDPNKTKF
pig GPRDFVSVGCTKRRGS CVLAGMATDFGEMPEQKGVI FfEHGPTCMVLHPLAGSPSKTKL
horse GPRDFVSVRCAKRRGS CVLAGMATQFEEMPEQKGVI IEHGPTCMVLHPLAGSPSKTKL
human GPRDFVSVRCAKRRGS 1CJT pM A 'Tn T'T~Tl'1T EHGPTCMVLHPLAGSPSKTKL

Prim. cons. GPRDFVSVRCAKRRGSTCFLAGMSTQHP2MPEQKGWRAENGPTCIVM2P2A2DPNKTKF

250 260 270 280

brook trout TWLLSIDLKGWIPKTIINKVLSQTQVDFANHLRQRMADNSVSKEMAPAC
rainbow troutTWLLSIDLKGWIPKTIINKVLSQTQVDFANHLRQRMADNSVSMEMAAAC
LMB TWLLNIDLKGWIPKTIINKVLSQTQVDFANHLRQRMANN-VSMEMAHAC
zebrafish TWLLSLDLKGWIPKTVINRVLSQTQVDFVNHLRDRMASG-GGIDAAIAC
pig TWLLSIDLKGWLPKTIINQVLSQTQVDFANHLRKRLESR--- PALEARC
horse TWLLSIDLKGWLPKTIINQVLSQTQVDFANHLRKRLESS ---PAPEARC
human TWLLSIDLKGWLPKSIINQVLSQTQVDFANHLRKRLESH--- PASEARC

Prim. cons. TWLLSIDLKGWIPKTIIN2VLSQTQVDFANHLR2RMASNSVSP2MAAAC













Figure 2-4. Location of peptide used for antibody development is indicated by a green

box. A 21 amino acid peptide was designed to a conserved region for LMB

StAR. Two different rabbits were immunized with the peptide for production

of StAR antibodies.
































Genomic DNA
\ ---GSP2
-GSP1

AP2->
GSP1
AP Primary PCR amplification


GSP2
AP2"L Nested, secondary, PCR amplification




Run PCR product on agarose gel


Figure 2-5. Promoter cloning. 3 Kb of the LMB StAR promoter was cloned using the
Genomewalker protocol (Clontech). One round of PCR is done using GSP1
(gene specific primer) and a primer against the adaptor, AP1. A second round
of PCR is done for added specificity using a nested gene specific primer,
GSP2, and nested adapator primer, AP2.


Genomic DNA Restriction libraries:
Stul
Pvull
EcoRV
Dral

























pGL3 Basic Vector
4818 bp


Figure 2-6. Map of pGL3 basic vector (Promega). The 2.9 kb StAR promoter was cloned
into the pGL3-basic vector for transfection experiments in Y-1 mouse adrenal
cells. The promoter DNA was inserted into the MCS (multiple cloning
site).


Luciferase














CHAPTER 3
REGULATION OF STAR IN LARGEMOUTH BASS OVARIAN FOLLICLE
CULTURES

Introduction

A major discovery was made in the steroidogenic pathway within the past decade

upon the identification of the Steroidogenic Acute Regulatory Protein (StAR Protein). It

has now been well characterized in mammalian species that StAR transports cholesterol

across the mitochondrial membrane and controls the rate-limiting step for steroidogenesis

(Stocco and Clark, 1996). Humans with mutations in StAR can't synthesize steroids

from cholesterol, causing lethal accumulation of lipids in the adrenal glands and

ultimately cell death, underscoring the importance of proper StAR function (Khoury et al,

2004).

It is clear, however, that steroidogenesis can be negatively impacted without the

presence of mutations in StAR DNA, implicating instead steps in regulation of StAR

mRNA synthesis or protein expression. TGF-P, a signaling molecule, and toxins, such as

phthalates, are known to repress mammalian StAR transcription, however, a link,

between the pathways they modulate has not been fully established (Brand et al., 2000;

Barlow et al., 2003).

The steroid synthesis pathways of several fish species, including LMB, white

sucker, zebrafish, and goldfish, have been shown in various cell culture systems to be

targeted by toxins such as dioxins, pesticides, plasticizers, and paper mill effluents

(Sepulveda et al., 2001; McMaster et al., 1995; Carvan et al., 2000; MacLatchy and Van









Der Kraak, 1995). The negative impact of these toxins on fish steroid production

implores the examination of how they specifically regulate StAR.

To date, there is minimal information about StAR in fish beyond the existence in

the NCBI database of DNA sequences for a few lower vertebrate species, which now

includes brook trout, rainbow trout, eel, cod, and zebrafish. There are 7 exons and 6

introns in the StAR gene which are conserved from mammals to fish (Goetz et al., 2004),

however, the regulation of fish StAR has not been examined besides the induction of

StAR mRNA in eel 1.5 hours post-injection with ACTH (adrenal corticotrophic

hormone) (Li et al., 2003). The ACTH experiment did suggest a conservation in the

cAMP inducibility of StAR across species.

Using largemouth bass (LMB) as a model fish since they are known to be sensitive

to environmental toxins, I sequenced the entire coding region of StAR. The sequence

was used to develop a real-time PCR assay for mRNA quantitation of StAR and for

generation of a polyclonal antibody to detect protein changes in LMB ovarian follicle

cultures. The ovarian follicle cultures were used to establish StAR mRNA expression

patterns by two potent signaling molecules, cAMP and TGF-P. It is known that cAMP

can stimulate mammalian StAR, partially through activation of SF-1, and that TGF-P can

downregulate mammalian StAR transcription, however, these studies have never been

done in fish prior to this study. My main hypothesis was that LMB StAR mRNA

expression is upregulated by cAMP and downregulated by TGF-P in ovarian follicle

cultures. Since a direct link between p-sitosterol exposure and TGF-P upregulation in

prostate cancer cells has been shown (Kallen et al., 2000), this implies that toxins such as

paper mill effluent repress steroid production through this signaling pathway.









Results

Cloning of StAR Protein

A partial, 345 base pair sequence for LMB was amplified using degenerate primer

based PCR (Figure 3-1). The partial sequence was then used to obtain the full length

coding and untranslated regions with 5' and 3' RACE. Sequence alignment of LMB

StAR with other species, including brook and rainbow trout, zebrafish, pig, horse, and

human, shows 52% similarity between mammalian and fish and 72% similarity amongst

fish species (Figure 3-2).

Based on the sequence comparison of StAR with other species, there appears to be

conservation of a couple important residues, including an important PKA

phosphorylation site at nucleotides 193-196. The ScanProsite web program also

putatively identified 4 PKC sites at nucleotides 5 7, 13 15, 60-62, and 187 189, as

indicated by the number in the alignment (Figure 3-2). An important glutamic acid

residue at nucleotide 170 which may bind to the hydroxyl group of cholesterol is also

conserved amongst the species. The glutamic acid residue is within the START domain,

the hydrophobic region for cholesterol binding, which spans from nucleotides 67 to 286.

Seasonal Expression

Changes in temporal expression of LMB StAR mRNA was quantitated from

ovarian tissue samples previously collected at approximately two week intervals from

October to April. RNA from seven fish at 11 time points during the year were analyzed

and showed StAR mRNA levels do change in correlation with steroid production during

the reproductive year (Figure 3-3). StAR levels peaked between February and March

and began declining by April, therefore, suggesting a short window for maximal steroid









expression. This information was useful in understanding when LMB are steroidogenic

and which months are optimal for culturing ovarian follicles for in vitro studies.

Regulation of StAR mRNA Expression in LMB Ovarian Cultures

Alamar blue viability assay showed that the ovarian follicles remained viable after

an 18 hour equilibration in the incubator. The follicles successfully reduced the

components in the alamar blue reagent as detected with A570 and A600 spectrophotometry

readings. The follicles remained viable and responsive to dbcAMP in either the absence

or presence of charcoal-stripped serum, therefore, serum was not added to culture media

for experiments. Additionally, viability of the follicles was also tested by inducing with

dbcAMP after a couple of equilibration timepoints, showing that basal and induction

levels weren't impacted whether exposed for 6, 12, or 24 hours post-equilibration.

cAMP Induction of LMB StAR

About 20-30 mg pieces of ovarian tissue were cultured and exposed to increasing

doses of dbcAMP from 0 to 1 mM to characterize the regulation of LMB StAR.

Exposures of the ovarian tissue cultures to dbcAMP resulted in upregulation of StAR

mRNA levels as quantitated by real-time PCR. A dose response of cultured ovarian

slices to dbcAMP showed a significant 3.5 fold induction from a mean of 13,529 copies

of StAR mRNA/tg total RNA for controls to an average of 47,408 copies of StAR

mRNA/tg total RNA after exposure to ImM dbcAMP for 4 hours (Figure 3-4).

Additionally, more controlled experiments were done where number and stage of

follicles cultured was monitored. Vitellogenic follicles with a specific diameter range of

0.68 mm to 0.76 mm were induced 5.9 fold from 18,054 copies of StAR mRNA/tg total

RNA for controls to 105,686 copies of StAR mRNA/tg total RNA after 4 hour exposure









to ImM dbcAMP (Figure 3-5). This suggests that more mature LMB follicles express

more StAR, which fits the mammalian model for oocyte maturation (Logan et al., 2002).

p-sitosterol Exposures

p-sitosterol is known to downregulate steroid production in goldfish (MacLatchy

and Van Der Kraak, 1995), however, no consistent effects on LMB StAR mRNA could

be seen at any given dose, timepoint, or stage of follicle growth. Attempts were made

with two different batches of fish after some initially promising results in the spring of

2003 suggested a decrease in LMB StAR mRNA levels by P-sitosterol. The 2003 results,

however, were never able to be reproducibly substantiated, suggesting a suboptimal

delivery of P-sitosterol to the steroidogenic cells.

TGF-P Exposures

TGF-P, a known repressor of steroid synthesis in mammals (Brand et al., 2000;

Gautier et al., 1997; Liakos, 2003), was tested in cultured follicles with diameters

between 0.8 to 0.9 mm and 1 to 1.1 mm. The LMB follicle cultures showed a

downregulation of StAR mRNA by about 2.3 fold after a 14 hour exposure to 1 ng/ml

TGF-P (Figure 3-6).

Some interesting preliminary results, however, were obtained from mature follicles

between 1.2 and 1.3 mm. Data suggests that 1 ng/ml and 10 ng/ml TGF-P stimulates

LMB StAR mRNA expression by about 2 fold after a 14 hour exposure, which could

indicate that the various factors which mediate the signaling response may vary with

follicle development (Figure 3-6).









Antibody Development

Currently, no polyclonal antibody exists for StAR that cross reacts with fish

species, therefore, an antigenic peptide was designed and injected into two different

rabbits to produce an immunogenic response against LMB StAR.

The first step in testing and optimizing the antiserum was to create purified protein

for a positive control. A protein expression vector containing the full coding region for

LMB StAR was created by PCR amplification (Figure 3-7) with subsequent cloning into

the pET-28b expression vector containing a 6X- histidine tag (Novagen). The construct

was verified by sequencing at the University of Florida DNA Sequencing Core. Bacterial

cultures were induced by 1 mM or 3 mM IPTG for 1-4 hours and were analyzed on a

Coomassie stained gel alongside negative controls of either empty vector or uninduced

cultures. A band around 40 kD began to be overexpressed compared to controls after just

one hour incubation with either 1 mM or 3 mM IPTG (Figure 3-8). Expression of StAR

continued through 4 hours and appeared to be maximally induced by that timepoint. The

expressed protein was successfully purified from the total protein extract using Ni-NTA

spin columns (Qiagen) with a concentration of 1.1 [tg/ptl (Figure 3-9).

To confirm that the bacterially expressed protein was StAR, the band was excised

and in-gel digested with trypsin for analysis with two different mass spectrometers, Q-

STAR and LCQ. In-silico digest of the sequence matched several peaks seen by Q-

STAR, including peaks at 722.3833, 939.4723, 1404.7140, 1509.8019, 1601.8558,

2329.1177, and 2395.1682 (Figure 3-10). With the Q-STAR, we used a MALDI (matrix

assisted laser desorption ionization) based technique for sample analysis. Additionally, at

least 5 tryptic peptides were identified by LCQ mass spec using an electrospray









ionization technique (Figure 3-11). For LCQ, the peptides were separated by reverse-

phase chromatography. Both the mass spec techniques are complementary to each other.

The rabbit antisera from rabbit UF408 and UF409 were tested by ELISA against

the purified protein for presence of LMB StAR antibodies. After 30 minutes of exposure

in an ELISA assay, 7 tg/ml of purified protein had an absorbance at 450 nm of 0.3 with

the pre-bleed versus about 2.8 for the test bleed at a 1:1600 dilution. ELISA results

showed strong reactivity of the protein with antiserum dilutions down to 1: 51,000,

suggesting the presence of very strong and specific LMB StAR antibodies (Figure 3-12).

The ELISA results also helped to establish the dilution ranges of antiserum to test by

western blot.

To determine whether the antibodies could also bind StAR in a western blot, I used

the recombinant protein with ECL (chemiluminesence) detection. The purified protein

was successfully detected by the LMB StAR antibodies at several dilutions of primary

antiserum as well as at several concentrations of protein. A 1:500 antiserum dilution was

too concentrated for western blot and resulted in an overexposure on the film, even after a

1 second exposure, and a similar result was still seen with up to a 1:5000 dilution when a

1:20,000 secondary was used. The most optimal conditions to detect the recombinant

StAR was at a 1:15,000 antiserum concentration with a 1:40,000 secondary dilution

(Figure 3-13).

Western Blot Detection of Endogenous LMB StAR Protein

Preliminary western blot analysis was done on LMB ovarian tissue exposed to

either 0 or 1 mM dbcAMP for 24 hours. The western blot shows detection of StAR in

both the control and 1 mM dbcAMP samples (Figure 3-14). The band detected for the

dbcAMP sample appeared more intense compared to the control, however, the presence









of non-specific bands suggests the StAR antibody needs to be purified from the anti-sera

to obtain optimal results. The non-specific bands are of about the same intensity,

indicating equivalent protein was loaded into each lane.

Discussion

Steroid hormones are key regulators of many cellular pathways and steroid

synthesis can be regulated at the level of cholesterol transport with the StAR Protein.

The StAR Protein has been cloned from several different species, ranging from higher

mammals to lower vertebrates like fish, however, the similarities and differences in their

function and regulation have never been investigated in any depth (Stocco and Clark,

1996; Goetz et al, 2004). A main goal of this project was to clone StAR from LMB and

examine the conservation of key amino acids and whether the sequence similarity

corresponds with conservation of function and activity.

The ovarian tissue and follicle cultures were excellent systems for comprehensive

study of LMB StAR mRNA and protein regulation by endogenous signaling molecules

like cAMP and TGF-P. Exposure of the ovarian cultures to dbcAMP showed no blatant

differences in magnitude of induction between species or the timepoints at which

induction can be seen. There was a 6 fold induction of LMB StAR after a 4 hour

exposure to dbcAMP, which compares with the 4 fold induction seen in MA10 mouse

leydig cells (Clark et al., 1995.) The cAMP data establishes that the overall regulation of

transcription is very similar across species and that key response elements in the

promoter which mediate the response to cAMP, including those for SF-1, must be

conserved.

The exact molecular pathways are still being elucidated for regulation of StAR,

therefore, it is important to detect changes in protein as well as mRNA levels since









compounds could regulate either the transcription or translation of StAR. Previously,

however, there was no antibody that cross-reacted with vertebrate species to examine

protein alterations in StAR. We successfully generated a polyclonal antibody that can

detect LMB StAR protein by ELISA or western blot. The antibody proved to be very

potent with purified protein and works optimally at very dilute concentrations of around

1:15,000. The antibody is being purified from the anti-sera for optimal detection of StAR

in complex protein samples, however, preliminary results suggests protein levels are

induced by cAMP.

The ovarian follicle cultures were also used to establish whether LMB StAR

mRNA is regulated by TGF-P, a potent signaling molecule like cAMP. The cultures

showed about a 57% reduction in StAR mRNA levels after a 14 hour exposure to 1 ng/ml

TGF-P for two different diameter sizes of follicles. Follicles of the same size studied for

the TGF-P exposures, such as the 0.8 0.9 mm, were inducible by dbcAMP.

Interestingly, the stage at which follicles were cultured may play a key role in the

regulation since StAR mRNA levels were activated by 1 or 10 ng/ml TGF-P in more

mature follicles of 1.2 to 1.3 mm. One possible explanation for this may be that SMAD2

and SMAD3 proteins, the key signaling molecules of TGF-P, may not be present at

sufficient levels in the more developed LMB follicles. It has been reported that both

SMAD2 and SMAD3 levels are very low or non-existent in the large antral or pre-

ovulatory follicles of rats (Xu et al., 2002). SMAD3 has been shown to mediate the

TGF-P repression of mammalian StAR (Brand et al., 1998),

TGF-P, unlike cAMP, significantly represses mammalian StAR mRNA expression.

The repression can range from 40% in unstimulated cells treated with 2 ng/ml TGF-P for









12 hours to greater than 60% downregulation when induced with cAMP in H295 cells, a

human adrenocortical cell line (Brand et al., 1998). The LMB ovarian follicle data

suggests that, in addition to the cAMP pathway, TGF-P signaling is not only conserved

between mammals and lower vertebrates but that the percent of inhibition for developing

oocytes by TGF-P is also relatively comparable. Preservation of TGF-P signaling

between species underscores the importance of this pathway and the chemicals that

modulate it.

Interestingly, a study showed TGF-P protein levels are upregulated by at least 50%

in prostate stromal cells after a 6 day exposure to p-sitosterol, one major chemical found

in paper mill contaminants (Kassen et al., 2000). Fish exposed to paper mill toxins, and

more specifically, P-sitosterol, exhibit decreased steroidogenic activity (MacLatchy and

Van Der Kraak, 1995). The ovarian follicle cultures were used to determine if p-

sitosterol exposure mimics TGF-P downregulation of LMB StAR mRNA levels, thereby

elucidating a possible signaling pathway activated by environmental toxins. Overall,

direct P-sitosterol exposures at several doses and timepoints in the follicle cultures did

not significantly impact StAR mRNA expression, contradicting my original hypothesis

that p-sitosterol represses steroidogenesis by downregulating StAR.

A possible explanation for the P-sitosterol results might be that ethanol was used as

the solvent. Ethanol was chosen based on several published in vivo and in vitro

experiments (Kassen et al, 2000; MacLatchy and Van Der Kraak, 1995). When

administered in vivo, P-sitosterol is able to enter the bloodstream and be packaged into

lipoproteins for delivery to steroidogenic cells (Carter and Karpen, 2001). Although it is

not definitively known which signaling pathways are modulated by P-sitosterol, it is









possible that downstream signaling requires activation through the lipoprotein receptors.

Using ethanol as a solvent might have bypassed the use of lipoprotein receptors or

cellular lipid carriers and could explain the lack of significant response I saw on StAR

mRNA expression by P-sitosterol. A recent publication reported that 15 gg/ml

cholesterol delivered via ethanol to Y-1 mouse adrenal cells did not significantly impact

StAR protein levels (King et al., 2004), however, cholesterol incorporated in either HDL

or LDL does increase mammalian StAR protein production in Y-1 cells (Reyland et al.,

2000). The ethanol study with cholesterol further supported the potential importance of

using the lipoprotein receptors as a route of entry for StAR regulation.

Overall, the sequence, mRNA, and protein data suggests a similarity in signaling

pathways and regulation across species for StAR. Induction of mRNA and protein levels

by cAMP suggests that LMB StAR is subject to both transcriptional and post-

transcriptional regulation by critical signaling molecules such as cAMP. The combined

mRNA and protein results can provide valuable insight into the mechanism by which

environmental toxins, such as 3-sitosterol, can disrupt the normal regulation of StAR.










A B C D


1000 bp-
750 bp
500 bp
300 bp
150 bp-
50 bp


Figure 3-1. PCR amplification ofLMB StAR. A 345 bp partial cDNA sequence
for LMB StAR was PCR amplified and run out on a 1% agarose gel.
(A) PCR marker (Promega) (B and C) PCR amplifications from gonadal RNA
of two different fish. (D) negative control with no reverse transcriptase
added. The amplified band for StAR is indicated by arrow.









Figure 3-2. Alignment of LMB StAR cDNA with other species. Alignment of the cDNA
sequence for LMB StAR with other species shows about a 53% similarity
across all species and 73% similarity between fish and lower vertebrates.
Several important regulatory sites were putatively identified in the LMB StAR
cDNA, including; 4 PKC sites at amino acid positions (as indicated on
alignment), 5 7 (TFK), 13 15 (SYR), 60-62 (SSR), and 187 189 (SVR).
There is one conserved PKA site at nucleotides 193 196 (RRGS).
Additionally, an important glutamic acid residue at nucleotide 170, which may
bind to the hydroxyl group of cholesterol, is conserved within the START
domain (amino acids 67-286).









74






10 20 30 40 50 60

brook trout MLPATFKLCAGISYRHTRNMTGLRKNAMVAIHHELNMLA--GPNPSS
rainbow troutMLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNMLA--GPNPSWISHVRRR
LMB MLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNRLA--GPGPNN SLLS
zebrafish MLPATFKLCAGISYRHMRNMTGLRKNAMIAIHHELNKLS--GPGAST
pig MLLATFKLCAGSSYRHVRNMKGLRHQAVLALGQELNRRALGGPTSGS
horse MLLATFKLCAGSSYRHVRNMKGLRHQAALAIGQELNWRAPGGPTQSG
human MLLATFKLCAGSSYRHMRNMKGLRQQAVMAISQELNRRALGGPTPST

Prim.cons. MLPATFKLCAGISYRHMRNMTGLRKNAMVAIHHELNRLALGGPTPSWINQVRRR

70 80 90 100 110 120

brook trout SRIEEEQGYNEAEVSYVKQGEEALQKSISILGDQDGWTTEIIAANGDVLSVLPDVGV
rainbow troutSRIEEEQGYNEAEVSYVKQGEEALQKSISILGDQDGWTTEIIAANGDKVLSKVLPDVGKV
LMB SRIEEEEGYSDEEMSYVKQGEDALQKAISILSDQDGWTTEIVAANGG
zebrafish SPIAEET-YSEADQCYVQQGQEALQKSISILEDQDGWQTEIESINGE
pig SQLEDTF-YSDQDLAYIQQGEEAMQRALDILSNQEGWKKESRQENGDEVLSKVIPDVGKV
horse SQLEDTL-YSDQELAYIQQGEEAMQKALGILRNQEGWKEENQQANGDKVLSKVVPDVGKV
human SRLEETL-YSDQELAYLQQGEEAMQKALGILSNQEGWKKESQQDNGG

Prim.cons. SRIEEE2GYSD2EL2YVQQGEEALQKAISILSDQDGW2TEI22ANGDKVLSKVLDVGKV

130 140 150 160 170 180

brook trout FKLEVLLDQRSDNLYGELVGNMEQMGDWNPNVKEVKILQKIGQETMV
rainbow troutFKLEVLLDQRSDNLYVELVGNMEQMGDWNPNVEVKILQKIGQETMVN
LMB FKLEVMLEQRPDNLYKELVGNMEQMGEWNPNVKQVKILQKIGQDTMV
zebrafish FKLEVTLEQQTGDLYDELVDNMEQMGEWNPNVKQVKILQKIGQETMI
pig FRLEVVVDQPMERLYEELVERMEAMGEWNPSVKKIKILQKIGKDTVI
horse FRLEVEVDQPMERLYEELVERMEAMGEWNPNVKEIKVLQKIGKDTVI
human FRLEVVVDQPMERLYEELVERMEAMGEWNPNVKEIKVLQKIGKDTFI

Prim.cons. FKLEV2LDQ2M22LYEELV2NMEQMGEWNPNVKEVKILQKIGQDTMI

190 200 210 220 230 240

brook trout GPRDFVSVRCAKRRGSTCFLAGMSTQHPTMPEQRGVVRAENGPTCIV
rainbow troutGPRDFVSVRCAKRRGSTCFLAGMSTQHPTMPEQRGVVRAENGPTCIV
LMB GPRDFVSVRCAKRRGSTCFLAGMSTQHPKMPEQRGVVRAENGPTCIV
zebrafish GPRDFVNVRHAKRRGSTCFLAGMSTQHPGMPEQKGFVRAENGPTCIV
pig GPRDFVSVGCTKRRGSVCVLAGMATDFGEMPEQKGVIRAEHGPTCMV
horse GPRDFVSVRCAKRRGSTCVLAGMATQFEEMPEQKGVIRAEHGPTCMV
human GPRDFVSVRCAKRRGSTCVLAGMATDFGNMPEQKGVIRAEHGPTCMV

Prim.cons. GPRDFVSVRCAKRRGSTCFLAGMSTQHP2MPEQKGVVRAENGPTCIV

250 260 270 280

brook trout TWLLSIDLKGWIPKTIINKVLSQTQVDFANHLRQRMADNSVSKEMAA
rainbow troutTWLLSIDLKGWIPKTIINKVLSQTQVDFANHLRQRMADNSVSMEMAC
LMB TWLLNIDLKGWIPKTIINKVLSQTQVDFANHLRQRMANN-VSMEMAH
zebrafish TWLLSLDLKGWIPKTVINRVLSQTQVDFVNHLRDRMASG-GGIDAAI
pig TWLLSIDLKGWLPKTIINQVLSQTQVDFANHLRKRLESR---PALEARC
horse TWLLSIDLKGWLPKTIINQVLSQTQVDFANHLRKRLESS---PAPEA
human TWLLSIDLKGWLPKSIINQVLSQTQVDFANHLRKRLESH---PASEA

Prim.cons. TWLLSIDLKGWIPKTIIN2VLSQTQVDFANHLR2RMASNSVSP2MAC























*


I Ir n


10-26-99 11-2-99 11-19-9911-30-99 12-21-99 1-18-00 2-1-00 2-25-00 3-15-00 3-29-00 4-11-00
Date of sampling


Figure 3-3. Seasonal expression of LMB StAR. RNA was isolated from ovarian tissue of
largemouth bass previously collected every few weeks for most of a year
throughout a year. Typical seasonal expression of StAR mRNA was
determined by real-time PCR. RNA from seven fish at 11 time points during
the year were analyzed. T-test shows a p< 0.05 between timepoints marked
with red versus black stars.







76








I?
6
S 80

z

50 *


W T














Figure 3-4. Dose response of LMB ovarian tissue cultures to dbcAMP. Cultures of 20-30
< 20
4--
0


0
o 0mM 025mM 05 mM 075 mM 1 mM
dbcAMP



Figure 3-4. Dose response of LMB ovarian tissue cultures to dbcAMP. Cultures of 20-30
mg pieces of ovarian tissue were exposed to a dose response of dbcAMP from
0 mM to ImM for 4 hours. RNA was then isolated from the tissues and
reverse transcribed to cDNA for real-time PCR analysis. T-test shows a p<
0.05 as indicated with red star. Results are from 2 different fish with triplicate
assays done for each fish.












0) M

Ct
E














E
z
1,
A) 0




o 0)







o a
U)


control 1 mM dbcAMP



Figure 3-5. cAMP induction of ovarian follicles. Thirty follicles between 0.4 and 0.88
mm were cultured in 24 well culture plates and exposed to 0 or 1 mM
dbcAMP for 4 hours. RNA was isolated from the follicles and reverse
transcribed for analysis by real-time PCR. This experiment represents data
obtained from one fish.


0.4 0.6 mm


control 1 mM dbcAMP








0.68 0.76 mm







control 1 mM dbcAMP







0.8-0.88 mm












08-09mm


1-11mm


o

< 30-
Z
- 25
m
S20-


z
E 10-

< 5

0)
4--


o
0

0


12-125 mm


0 ng/ml 1 ng/ml 10 ng/ml

TGF-1


Figure 3-6. Dose response exposure of ovarian follicles to TGF-P. Follicles with
diameter ranges between 0.8 0.9 mm, 1.0 1.1 mm, and 1.2 1.3 mm were
exposed to 0 ng/ml or 1 ng/ml TGF-P for 14 hours. Ten follicles were
cultured in each well of a 24-well culture plate. RNA was isolated from the
follicles and reverse transcribed for analysis of StAR mRNA expression by
real-time PCR. T-test shows a p< 0.05 indicated with red star.


0 ng/ml 1 ng/ml 0 ng/ml 1 ng/ml

TGF-P













n f'


2000 bp-

1200 bp-

800 bp


400 bp-


Figure 3-7. PCR amplification of entire LMB StAR cDNA. Specific primers were used to
amplify 858 nucleotides of LMB StAR coding region. A) Low DNA mass
ladder (Invitrogen). B and C) BamHI digest of two separate clones to
confirm ligation of LMB StAR into the pET-28b vector.






80



1 hour 2 hours 3 hours 4 hours


rE E
0 c


00 00


SE E E E
O r crO O CO


49 kDa
'-38 kDa












Figure 3-8. Bacterial expression of LMB StAR. The entire cDNA sequence for LMB
StAR was cloned into the pET-28b vector (Novagen). Bacterial expression of
StAR protein was induced by 1 mM and 3 mM IPTG from 1 to 4 hours in
BL21(DE3) bacterial cells. Total protein for each exposure was run out on a
4-12% Bis-Tris NuPAGE gel (Novex) and stained with Colloidal Coomassie
Blue. Induction of StAR is indicated by an arrow.


0 0

0 -
OU -CO








1 2


I'


62 kDa
49 kDa


StAR I- 38 kDa


28 kDa


Figure 3-9. Purification of StAR. His-tagaged StAR was purified from the bacterial
expression using Ni-NTA spin columns (Qiagen). Lane 1 contains 2 [g of
purified protein and lane 2 contains See Blue Plus 2 marker run out on a 4-
12% Bis-tris gel (Novex) and stained with colloidal comassie blue stain. The
band for purified StAR is indicated by an arrow.










1249,5*5


894723
722.=3
41 1021-5457


I A M A22,4461
| M4
1 ^llul i j)


277
,-1131

LI
il,.L


1 08oo8g


S EE


\ 1420 4fl0
404r140


kLJ hL


14B.BB21


68.85547


1747

,, 171


L_ ,t11,


U 241 160DO
23M 682
23M.117 2A IM


1,61 ,I ,


Figure 3-10. Identification of bacterially expressed StAR with Q-STAR mass
spectrometry. The trypsin-digested StAR was analyzed with Q-STAR mass
spectrometry for identification of bacterial LMB StAR expression. The
coomassie blue stained band was excised and in-gel trypsin digested prior to a
MALDI based mass spec analysis. The black arrows indicate which peaks
were specific for predicted LMB StAR peptides.


54A440


I


I - w^^.16o


~~U~~-Y~- -ul-rurql'-g~U~y~~)~lY1l














286
32431.3
113.4
+11


I


IW


I


Res
1
B
16
19
25
236
38
52
54
61
712
85
107
111
118
121
135
152
155
159
182
18a
191
192
193
206
213
217
236
239
24A
253
259B
272
'74S


M-as Type: Mana
Mass rlang: 0-10000000
Sart By Position
Display: Mr
Add Nam*: -
Residue(s): -


_ I C I Retei
41.4
1 1 39.4
9.3
25.1
-2.5
38,0
40.6
1.9
27.5
2 42.1
18.7
57.1
23.2
36.5
24.4
1 54.9
50.2
9.5
27,6
45.9
32.4
1 8.7
1.0
1.0
1 35.3
17.9
20,4
2 42.6
0.5
69.4
30.8
25.6
456.7
2.3
1 40.4


Figure 3-11. Identification of bacterially expressed StAR by LCQ mass spectrometry.
The coomassie blue stained band for bacterially expressed LMB StAR was cut
out and in-gel trypsin digested for analysis by LCQ mass spec. Predicted
tryptic fragments were identified by Prospector and the red arrows indicate
which peptides were seen by LCQ.


Enymln: Trypsin
Cyst CAM


LangthI
MW;

Charge;


I


Mol Wt
806 .4
939.5
442.2
690.3
146.1
1403 7
1509 08
231.1L
748,4
1935.8
687.4
2303.1
445 ,3
772.4
392.2
1746.9
1973. 9
373 .2
500.3
2393 .2
721.-4
377.2
174 ,1
174.1
1620. 7
659 3
429,3
2158.0
247.2
1261.7
599. 3
587.4
1626.6
302.2
1464.4


*


..r.. ~ r I I --


Len
7r
e
3
6


14
2
7-


22
14
2



7,
16

322

4
7
3



14
17

23

3

1
1

15


5



14


13


S10 20
MLPATFK
LCAGISYR *
HMR i
UMTGLR
K :
NAWVAKIHELNA I
LAG PGPSNW I QTR
GR :
SSLLSSR
IEEeBGYSDEEI4SYVKr
QGEDALQK :
AISILDODQWTT EIVAANGDK
VLSK
MLPDIGK .
VFK
LEVMLm QRPDNLYK *
ELVGN1EOGEWN PNVK .4* l
QVK X

ZOODTMVTHEVSAETPGNVV CP U
DFVSVR
CAK *
R




AENGPTCIVMKPC'VEDPNK *
TK
FTWLLNIDLK .-
GWIPK i
TIINK
VLSFTQVDFANHLR
OR :
MANNVSMEMAHAC *







84







UF408 Rabbit


UF409 Rabbit





Lc_


-*- Pre-Bleed
--- Test-Bleed


1 : 1600 1: 3200 1:6400 1: 12,800 1: 2-5 00 1: 51 200
Ab Ab Ab Ab Ab Ab
-*-Pre-Bleed 0239 0.22 0.189 0199 016 0-142
---Test-Breed 2.691 2.162 1.581 0.771 0.558 0.346



Figure 3-12. ELISA with LMB StAR anti-serum and purified StAR protein. The
antiserum from two different rabbits injected with the StAR peptide were
tested by ELISA with 7 pg/ml purified protein.


3.
3-2.5 --a--


2-
2 -.-F Pre-Bleed
S --Test-Bleed
*S 1

< 0.5
0
- - --- ----

1:1600 1: 3200 1: 6400 1: 12,800 1: 25,600 1: 51,200
Ab Ab Ab Ab Ab Ab
- Pre-Bleed 0.347 0.306 0.199 0.174 0.18 0.173
-- Test-Bleed 2.842 2 842 2.586 2.078 1.3 0.762



















V-




cc


62 kDa-


49 kDa-


38 kDa-


28 kDa-


17 kDa-


00

0'


0'



0~
0~I


Figure 3-13. Western blot detection with LMB StAR antibody and purified StAR protein.
13 ug of purified protein was run out on a 4-12% bis-tris gel, transferred to a
nitrocellulose membrane. The membrane was divided into strips for probing
with several combinations of primary antibody (LMB StAR antiserum) and
secondary (mouse anti-rabbit IgG) conjugated to horseradish peroxidase).
Primary antibody incubations were done for 3 hours and secondary
incubations done for 1 hour followed by chemiluminescence detection on
kodak film.


0
rL

0?

5V
0^


0
0,













-- 62 kDa

S49 kDa

.- ,. 38 kDa

28 kDa

17 kDa


Control 1 mM dbcAMP


Figure 3-14. Western blot detection of StAR in dbcAMP exposed LMB tissue cultures.
60 mg pieces of LMB ovarian tissue were cultured and exposed to 0 or 1 mM
dbcAMP for 24 hours. Total protein was extracted and run on a 4-12% bis-
tris for detection of StAR with a 1: 5,000 dilution of the polyclonal antibody
and 1: 40,000 secondary antibody.














CHAPTER 4
TRANSCRIPTIONAL REGULATION OF THE LMB STAR PROMOTER

Introduction

Several transcription factor binding sites have been identified in the mammalian

StAR promoter, including SF-1, AP-1, GATA-4, C/EBP, involved in the activation of

transcription, and DAX-1, a repressor of mRNA synthesis (Sugawara et al., 2004; Jo and

Stocco, 2004; Manna et al., 2004; Silverman et al., 2004). An alignment of the first 200

base pairs of mammalian promoters for mouse, rabbit, sheep, pig, and horse shows the

locations are conserved for 2 SF-1, 1 GATA, and 2 C/EBP response elements. One of

the SF-1 sites, in fact, is only about 12 base pairs upstream from the TATA box. The

promoter alignments indicate how important those binding sites are in mediating the

transcriptional activity of StAR, however, more studies are necessary before the function

or even the identification of many of the response elements is determined.

It is well characterized in the mammalian system that dbcAMP upregulates StAR

transcription via PKA phosphorylation of SF-1 (Aesoy et al., 2002). The magnitude of

activation of StAR by SF-1 is partially contingent on surrounding response elements and

the proteins that they bind. C/EBP interaction with SF-1 can amplify the cAMP

induction of StAR (Reinhart et al., 1999), however, when DAX-1 and SF-1 were co-

transfected in HTB9 cells, a bladder carcinoma cell line devoid of endogenous steroid

production, SF-1 could no longer stimulate StAR in response to cAMP (Sandhoff and

McLean, 1999). The SF-1 data underscores the importance and complexity of protein-

protein and protein-DNA interactions in StAR transcriptional regulation.