Role of IGF-I as a Potential Mediator for the Skeletal Effects of Bone Anabolic Agents

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Title:
Role of IGF-I as a Potential Mediator for the Skeletal Effects of Bone Anabolic Agents
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1 online resource (234 p.)
Language:
english
Creator:
Bassit, Ana C
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
Wronski, Thomas J
Committee Members:
Pozzi, Antonio
Samuelson, Don A
Holliday, Lexie S
Horodyski, Marybeth

Subjects

Subjects / Keywords:
biomechanical -- bone -- expression -- factor -- gene -- gh -- growth -- healing -- histomorphometry -- igf-i -- osteoporosis -- pge2 -- pqct -- pth -- sclerostin
Veterinary Medicine -- Dissertations, Academic -- UF
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Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Growth hormone (GH) has a critical role in the regulation of longitudinal bone growth, skeletal maturation, and maintenance of adult bone mass.  Insulin-like growth factor I (IGF-I) is considered the prime mediator of the skeletal effects of GH, and may also mediate the skeletal effects of bone anabolic agents.  Parathyroid hormone (PTH) is such an agent and its use is approved in the United States by the Food and Drug Administration.  Prostaglandin E2 (PGE2) can also induce cortical and trabecular bone formation in humans and animal models.  The role of IGF-I as a potential mediator for the bone anabolic effects PTH and PGE2 is still controversial; in vivo and in vitro studies yielded conflicting results. The objectives of this study were: 1- Evaluate the dwarf rat (dw-/dw-) as an animal model for studies of the effects of GH and IGF-I deficiency on the skeleton and bone metabolism, as presented in clinical conditions where synthesis is decreased but not abolished; 2- Compare the skeletal effects of PTH and PGE2 treatment in dwarf rats and their background strain, Lewis rats; 3- Determine the expression of genes related to bone formation and bone resorption.  At 9 weeks of age, female Lewis and dwarf rats were injected SC daily for 2 weeks with vehicle,  hPTH 1-34 at a dose of 50 mg/kg body weight , or PGE2 at a dose of 3 mg/kg body weight (N=7-10/group).  Serum IGF-I was measured by ELISA, bone histomorphometry was performed in the lumbar vertebral body, tibial metaphysis and diaphysis.  Peripheral quantitative computerized tomography (pQCT) was performed at femur cancellous and cortical bone; lumbar vertebrae were used for biomechanical testing. Serum levels of IGF-I were markedly lower in dwarf rats compared with Lewis rats regardless of treatment.  Histomorphometry and pQCT revealed overall increased parameters for bone mass and bone formation with both treatments, mainly PTH. Sclerostin is a potent negative regulator of osteogenesis and SOST(sclerostin gene) was downregulated in PTH- and PGE2-treated dwarf rats, suggesting that sclerostin inhibition could be related to the persistent bone anabolic effects of PTH and PGE2, despite low IGF-I serum levels in dwarf rats.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Ana C Bassit.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Wronski, Thomas J.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 ROLE OF IGF I AS A POTENTIAL MEDIATOR FOR THE SKELETAL EFFECTS OF BONE ANABOLIC AGENTS By ANA CRISTINA FERREIRA BASSIT 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 2012

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2 2012 Ana Cristina Ferreira Bassit

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3 To Cary Rodrigo and Ricardo

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4 ACKNOWLEDGMENTS I am thankful for the CAPES/Fulbright sponsorship for graduate studies, w hich made my participation in this program possible, and for the support of the Office of Research and Graduate Studies at the University of Florida College of Veterinary Medicine and the University of Florida International Center. I am also thankful to m y advisor Dr. Thomas J. Wronski and to my Committee members: Dr Don Samuelson, Dr. Shannon Holliday, Dr MaryBeth Horodyski and Dr. Antonio Pozzi. I am grateful to all who collaborated with me in any way during their stay at the Bone Research Lab: Jose Ignacio Aguirre, Molly Altman, John Stabley, Martha Leal, Sarah Franz, Tamashbeen Rahman, Jenni fer Pingel, Sandra Tisdelle, Al y Williams, Alicia Leeper and Kat i e Neuville In addition, I would like to express my gratitude to Dr Linda Hayward, for her ment oring and great support with the molecular biology experiments. In this particular area, I am also thankful to Dr Charles Wood and Dr Maureen Keller Wood, who kindly made available their expertise and laboratories, where I had the opportunity to learn t he necessary techniques from Lisa Fang and Dr. Elaine Sumners ; my sincere thanks to them as well Another part of my research was developed at the Comparativ e Orthopedic and Biomechanical Laboratory, at the College of Veterinary Medicine, with the support of Dr MaryBeth Horodyski, Dr Antonio Pozzi and soonto be engineer, Dan Barousse. The collaboration of Dr Ste phen Borst and Dr. Joshua Yarrow from the VA H os pital, who kindl y allowed us to use their equipment s to process bone samples for molecular bi ology analysis is also appreciated. My gratitude is also due to my former and present colleagues at the graduates office: Pei Ying Sarah Chan, Joslyn Algree n, Karen Porter, Carie Reynolds, Kate Pate,

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5 Vipa Bernhardt, HsiuWen Irene Tsai, Sherry Adams Poon am Ja i swal, and also Noel Takeuchi. Their support and friendship are always going to be remembered. Last but not least, I am deeply thankful to my family, to my mother and my sister, Angelina and Leticia, who were always close despite the distance, to my sons Rodrigo and Ricardo, who faced the challenge with me, and to my husband, Cary, who j oined us and made it all worthwhile And above all, thanks be to God, With God all things are possible, Mat thew19:26.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 22 ABSTRACT ................................................................................................................... 26 CHAPTER 1 INTRODUCTION .................................................................................................... 28 Osteoporosis and Osteopenic Conditions ............................................................... 28 Growth Hormone (GH) and InsulinLike Growth Factor I (IGF I) ............................. 30 Parathyroid Hormone .............................................................................................. 34 Prostaglandin E2 ..................................................................................................... 37 The GH/IGF I Axis and Animal Models ................................................................... 38 Specific Aims .......................................................................................................... 44 2 THE EFFECT S OF A DEPRESSED GH/IGF I AXIS ON BONE STRUCTURE AND BONE TURNOVER IN DWARF RATS ........................................................... 47 Introduction ............................................................................................................. 47 Materials and Methods ............................................................................................ 49 Animal Model .................................................................................................... 49 Experimental Design ........................................................................................ 50 Vehicle Treatment ............................................................................................ 50 Bone Formation Markers .................................................................................. 50 Euthanasia and Tissue Sample Distribution ..................................................... 50 IGF I Se rum Levels .......................................................................................... 51 Evaluation of Body Weight and Femoral Length .............................................. 52 Peripheral Quantitative Computerized Tomography ......................................... 53 Bone Histomorphometry ................................................................................... 54 Biomechanical Testing ..................................................................................... 57 Statistical Analyses .......................................................................................... 59 Results .................................................................................................................... 59 Body Weight and Femoral Length .................................................................... 59 IGF I Serum Levels .......................................................................................... 59 Peripheral Quantitative Computerized Tomography ......................................... 60 Histomorphometric Findings ............................................................................. 60 Cancellous bone measurements in lumbar vertebrae ................................ 60 Cancellous bone measurements in proximal tibiae .................................... 61 Cortical bone measurements in the tibial diaphysis ................................... 62 Biomechanical Testing ..................................................................................... 62

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7 Discussion .............................................................................................................. 63 IGF I Serum Levels .......................................................................................... 63 Body Size and Development ............................................................................ 64 Peripheral Quantitative Computerized Tomography ......................................... 65 Bone Histomorphometry and Biomechanics ..................................................... 67 Conclusions ............................................................................................................ 67 3 THE BONE ANABOLIC EFFECTS OF PTH TREATMENT IN GH/IGF I DEFICIENT DWARF RATS AND THEIR BACKGROUND STRAIN, LEWIS RATS. ..................................................................................................................... 96 Introduction ............................................................................................................. 96 Materials and Methods ............................................................................................ 99 Animal Models .................................................................................................. 99 Experimental Design ........................................................................................ 99 Vehicle and PTH Treatment ............................................................................. 99 Bone Formation Markers ................................................................................ 100 Results .................................................................................................................. 100 Body Weight and Femoral Leng th .................................................................. 100 IGF I Serum Levels ........................................................................................ 101 Peripheral Quantitative Computerized Tomography ....................................... 101 Histomorphometric Findings ........................................................................... 102 Cancellous bone measurements in lumbar vertebrae .............................. 102 Cancellous bone measurements in proximal tibiae .................................. 103 Cortical bone measurements in tibial diaphysis ....................................... 103 Biomechanical Testing in Lumbar Vertebral Body .......................................... 104 Discussion ............................................................................................................ 104 Conclusions .......................................................................................................... 107 4 COMPARISON OF PGE2 TREATMENT IN GH/IGF I DEFICIENT DWARF RATS AND THEIR BACKGROUND STRAIN, LEWIS RATS. ............................... 133 Introduction ........................................................................................................... 133 Materials and Methods .......................................................................................... 134 Animal Models ................................................................................................ 134 Experimental Design ...................................................................................... 134 Vehicle and PGE2 Treatment ......................................................................... 135 Bone Formation Markers ................................................................................ 135 Results .................................................................................................................. 136 Body Weight ................................................................................................... 136 IGF I Serum Levels ........................................................................................ 136 Peripheral Quantitative Computerized Tomography ....................................... 136 Histomorphometric F indings ........................................................................... 137 Cancellous bone measurements in the lumbar vertebrae ........................ 137 Cancellous bone measurements in the proximal tibiae ............................ 138 Cortical bone measurements in the tibial diaphysis ................................. 139 Biomechanical Testing in Lumbar Vertebral Body .......................................... 139

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8 Discussion ............................................................................................................ 140 Conclusions .......................................................................................................... 142 5 CHANGES IN GENE EXPRESSION RELATED TO BONE FORMATION AND BONE R ESORPTION IN PTH AND PGE2TREATED RATS. ............................. 167 Introduction ........................................................................................................... 167 Insulin Like Growth Factor I (IGF I, IGF1, Somatomedin C) ........................... 167 Collagen Type I .............................................................................................. 168 Osteocalcin ..................................................................................................... 169 Osterix ............................................................................................................ 169 RANKL ........................................................................................................... 170 Osteoprotegerin (OPG) .................................................................................. 171 Sclerostin ........................................................................................................ 171 Materials and Methods .......................................................................................... 173 Animal Models and Experimental Design ....................................................... 173 RNA Extraction and cDNA Synthesis ............................................................. 174 Bone tissue .............................................................................................. 174 Hepatic tissue .......................................................................................... 175 Quantitative Real Time PCR .......................................................................... 175 Calculations and Statistical Analysis .............................................................. 176 Results .................................................................................................................. 177 IGF I ............................................................................................................... 177 Bone ........................................................................................................ 177 Liver ......................................................................................................... 177 Collagen Type I .............................................................................................. 177 Osteocalcin ..................................................................................................... 178 Osterix ............................................................................................................ 178 RANKL ........................................................................................................... 178 OPG ............................................................................................................... 178 Sclerostin ........................................................................................................ 179 Discussion ............................................................................................................ 179 Conclusions .......................................................................................................... 181 6 SUMMARY AND CONCLUSIONS ........................................................................ 199 Summary of Experimental Findings ...................................................................... 199 Study 1 Summary ........................................................................................... 199 Study 2 Summary ........................................................................................... 200 Study 3 Summary ........................................................................................... 200 Study 4 Summary ........................................................................................... 2 00 Discussion ............................................................................................................ 201 Conclusions .......................................................................................................... 212 Directions for Future Studies ................................................................................ 212 LIST OF REFERENCES ............................................................................................. 214

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9 BIOGRAPHICAL SKETCH .......................................................................................... 233

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10 LIST OF FIGURES Figure page 2 1 The two strains of Lewis (right) and dwarf (left) rats during their clinical evaluation ........................................................................................................... 69 2 2 rat (right), showing inferior size and ov erall development when compared to Lewis rat (left) ..................................................................................................... 69 2 3 Body weight (g). There was a statistically significant difference between Lewis and dwarf rats (P<0.0001) ........................................................................ 70 2 4 IGF I Serum levels. The calibration curve and slope equation for obtaining the concentration levels for IGF I in ng/mL ......................................................... 71 2 5 Serum IGF I (ng/mL). Dwar f rats showed IGF I serum levels significantly decreased when compared to Lewis rats (P<0.0001) ........................................ 71 2 6 Total Bone Mineral Content (BMC) for Cancellous or Trabecular Bone (mg/mm). Mean value for dwarf rats was significantly lower than for Lewis rats (P<0.0001) ................................................................................................... 72 2 7 Total Bone Mineral Density (BMD) for Cancellous or Trabecular Bone ( mg/cm3). Significant decrease was observed in the mean value for dwarf rats (P<0.0001) ................................................................................................... 72 2 8 Trabecular BMC (mg/mm). A remarkable difference was noted between Lewis and dwarf rats (P<0.0001) ........................................................................ 73 2 9 Trabecular BMD ( mg/cm3). A highly significant difference was observed between Lewis and dwarf rats (P<0.0001) ......................................................... 73 2 10 Total Area for Cancellous Bone (mm2).The values obtain ed were significantly lower for dwarf when compared to Lewis rats (P<0.0001) .................................. 74 2 11 Trabecular Area (mm2). A significant difference between Lewis and dwarf rats was observed with P<0.001 ......................................................................... 74 2 12 Total BMC for Cortical Bone (mg/mm). Mean value for dwarf rats was significantly lower than for Lewis rats (P<0.0001) .............................................. 75 2 13 Total BMD for Cortical Bone ( mg/cm3). A significant difference between Lewis and dwarf rats was observed (P<0.0009) ................................................. 75 2 14 Cortical BMC (mg/mm). A significant decrease was observed in dwarf rats compared to Lewis rats (P<0.0001) .................................................................... 76

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11 2 15 Cortical BMD ( mg/cm3). This was the only pQCT parameter that did not show a significant difference between Lewis and dwarf rats .............................. 76 2 16 Cortical Area (mm2). Significant decrease in dwarf rats compared to Lewis rats (p<0.0001) ................................................................................................... 77 2 17 Cortical Thickness (mm2). Dwarf rats show ed significantly lower values than Lewis rats (P<0.0001) ......................................................................................... 77 2 18 Periosteal Circumference (mm). A significant difference (P<0.0001) was observed between Lewis and dwarf rats ............................................................ 78 2 19 Endosteal Circumference (mm). Dwarf rats showed significantly lower values when compared to Lewis rats (P<0.0001) ............................................... 78 2 20 Vertebral Cancell ous Bone Volume (%). Significantly lower values were observed in dwarf rats (P<0.0001) ..................................................................... 79 2 21 Vertebral Trabecular Number (#/mm). Dwarf rats showed significant lower values, with P<0.005 .......................................................................................... 79 2 22 Vertebral Trabecular Width ( A significant difference between Lewis and dwarf rats was observed with P<0.001 ............................................................... 80 2 23 Vertebral Trabecular Separation ( Dwarf rats exhibited a significant increase compared to Lewi s rats (P<0.0006) ..................................................... 80 2 24 Vertebral Osteoid Surface (%). Dwarf rats did not show a significant difference when compared to Lewis rats ............................................................ 81 2 25 Vertebral Osteoblast Surface (%). There was no significant difference between dwarf and Lewis rats ............................................................................ 81 2 26 Vertebral Osteoclast surface (%). There was no significant difference b etween dwarf and Lewis rats ............................................................................ 82 2 27 values were observed in dwarf rats compared to Lewis rats. (P<0.0001) .......... 82 2 28 Vertebral Mineralizing Surface (%). Dwarf rats showed significantly decreased mean values when compared to Lewis rats (p<0.0001) .................... 83 2 29 Vertebral Bone Formation Rate (10232/d). Mean values were significantly decreased in dwarf rats compared to Lewis rats (p<0.0001) .......... 83 2 30 Tibial Longitudinal Bone Growth ( m/d). Dwarf rats presented significantly lower mean values in comparison to Lewis rats (p<0.0001) ............................... 84

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12 2 31 Proximal tibial metaphyses from Dwarf (A) and Lewis (B) rats. Note the reduced mass of black stained bone indicative of cancellous osteopenia in the Dwarf rat. Von Kossa/ tetrachrome stain, X40 .............................................. 84 2 32 Tibial Cancellous Bone Volume (%). Mean value was markedly lower in the dwarf rats than in the Lewis rats (P<0.0001) ...................................................... 85 2 33 Tibial Trabecular Number (#/mm). Dwarf rats showed lower values than the control group (Lewis rats), with P<0.0001 .......................................................... 85 2 34 Tibial Trabecular Width ( m). Dwarf rats exhibited significantly lower values than Lewis rats, with P<0.0001 ........................................................................... 86 2 35 Tibial Trabecular Separation ( m). The value for dwarf rats was significantly higher than that show ed by Lewis rats, with P<0.016 ......................................... 86 2 36 Total Cortical Bone Tissue Area (mm2). The mean value was markedly lower in dwarf rats than in Lewis rats, with P<0.0001 .................................................. 87 2 37 Cortical Area (mm2). Mean value was significantly lower in the dwarf rats than in the Lewis rats (P<0.0001) ....................................................................... 87 2 38 Marrow Area (mm2). Dwarf rats showed a lower mean value compared to Lewis rats (P<0.02) ............................................................................................. 88 2 39 Periosteal Perimeter (mm). Dwarf rats presented a significantly lower mean value than Lewis rats (P<0.02) ........................................................................... 88 2 40 Endocortical Perimeter (mm). Mean values were significantly lower in the dwarf rats than in the Lewis rats (P<0.04) .......................................................... 89 2 41 Cortical Width (mm). T he value for dwarf rats was significantly lower than that of Lewis rats, with P<0.0001 ........................................................................ 89 2 42 Periosteal Mineralizing Surface (%). Dwarf rats showed significantly lower values than Lewis rats, with P<0.0007 ............................................................... 90 2 43 Periosteal Mineral Apposition Rate ( m/d ). Mean value was significantly lower in the dwarf rats than in the Lewis rats (P<0.0001) ................................... 90 2 44 Periosteal Bone Formation Rate ( 102m3/m2/d ). Dwarf rats showed a lower value compared to Lewis rats (P<0.0001). ......................................................... 91 2 45 Endocortical Mineralizing Surface ( % ) Significantly lower value was observed in dwarf rats compared to Lewis rats (P<0.0015) ................................ 91 2 46 Endocortical Mineral Apposition Rate ( ). Dwarf rats presented a significantly lower value than Lewis rats (P<0.0008) .......................................... 92

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13 2 47 Endocortical Bone Formation Rate ( 102m3/m2/d ). Dwarf rats presented a significantly lower value t han Lewis rats (P<0.0001). ......................................... 92 2 48 Images of lumbar vertebra obtained for area analysis with Image J ................... 93 2 49 Biomechanical Load (N ). Dwarf rats showed significant lower values compared to Lewis rats (P<0.03) ........................................................................ 94 2 50 Biomechanical Stress. There was no significant difference between dwarf and Lewis rats .................................................................................................... 94 2 51 Biomechanical Stiffness (N/mm). No significant difference could be noticed between dwarf and Lewis values ....................................................................... 95 3 1 Body weight (g). Signific ant difference between Lewis and dwarf rats (P<0.0001), but not between VEH and PTH treated rats in either Lewis or dwarf groups ..................................................................................................... 109 3 2 Femur Length (mm). PTH treatment slightly increased mean femur length in Lewis rats, but no difference was observed between VEH and PTH treated rats ................................................................................................................... 109 3 3 Serum IGF I (ng/mL). A significant difference was observed between Lewis and dwarf rats (P <0.0001), but not between VEH and PTH treated rats .......... 110 3 4 Total Bone Mineral Content (BMC) for Cancellous or Trabecular Bone (mg/mm) showed a significant increase in VEH and PTH treated rats f rom both strains, Lewis and dwarf rats (P<0.0001) ................................................. 110 3 5 Total Bone Mineral Density (BMD) for Cancellous or Trabecular Bone ( mg/cm3). Significant increase in dwarf and Lewis rats treated with PTH (P<0.0001) ........................................................................................................ 111 3 6 Trabecular BMC (mg/mm). Significant increase in the trabecular mineral content was noted in both Lewis and dwarf rats treated with PTH (P<0.0001) 111 3 7 Trabecular BMD (mg/cm3). A highly significant increase was observed in trabecular mineral density in Lewis and dwarf rats treated with PTH (P<0.0001) ........................................................................................................ 112 3 8 Total Area for Cancellous Bone (mm2). A significant increase was observed in PTH treated Lewis and dwarf rats (P<0.0001) .............................................. 112 3 9 Trabecular Area (mm2). A significant increase w as observed in the PTH treated rats, dwarf and Lewis (P<0.0001) ......................................................... 113

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14 3 10 Total BMC for Cortical Bone (mg/mm). There was no significant difference in the PTH treated rats, dwarf or Lewis, when compared to their respective vehicle treated controls. .................................................................................. 113 3 11 Total BMD for Cortical Bone (mg/cm3). There was no significant difference between PTH and vehicletreated rats in the two groups of dwarf and Lewis rats ................................................................................................................... 114 3 12 Cortical BMC (mg/mm). No significant difference was found between PTH treated and vehicletreated rats in both dwarf and Lewis rats .......................... 114 3 13 Cortical BMD (mg/cm3). There was a significant difference between PTH treated and vehicletreated rats, but not between Lewis rats (P<0.003) ........... 115 3 14 Cortical Area (mm2). No significant difference observed with PTH treatment in the dwarf and Lewis rats ............................................................................... 115 3 15 Cortical Thickness (mm2). There was no significant difference between PTH and vehicletreated rats in the two groups of dwarf and Lewis rats .................. 116 3 16 Periosteal Circumference (mm). The values were almost identical between PTH treated and vehicletreated rats in dwar f and Lewis rats .......................... 116 3 17 Endosteal Circumference (mm). No significant difference observed with PTH treatment in both groups ................................................................................... 117 3 18 Vertebral Cancellous Bone Volume (%). There was a significant increase in Lewis and dwarf PTH treated rats (P<0.0001), when compared to their VEH control groups. .................................................................................................. 117 3 19 Vertebral trabecular Number (#/mm). There was a significant difference between PTH treated and vehicletreated rats treated with PTH in both groups (P<0.002) .............................................................................................. 118 3 20 Vertebral trabecular Width ( m). There was a significant increase in Lewis and dwarf PTH treated rats, when compared to their VEH control groups. ..... 118 3 21 Vertebral Trabecular Separation ( m). A significant decrease in trabecular separation was observed in Lewis and dwarf rats treated with PTH (P<0.0001). ....................................................................................................... 119 3 22 Vertebral Osteoid Surface (%). A highly significant increase in osteoid surface was noted in Lewis and dwarf rats treated with PTH (P<0.0001). ........ 119 3 23 Vertebral Osteoblast Surface (%). A markedly significant increase in osteoblast surface was noted in Lewis and dwarf rats treated with PTH (P<0.0001). ....................................................................................................... 120

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15 3 24 Vertebral Osteoclast Surface (%). A highly significant increase in osteoid surface was noted in Lewis and dwarf rats treated with PTH (P<0.0001). ........ 120 3 25 Vertebral Mineralizing Surface (%). A highly significant increase in mineralizing surface was noted in Lewis and dwarf rats treated with PTH (P<0.0001). ....................................................................................................... 121 3 26 Vertebral Bone formation Rate (10232/d). A markedly significant increase was noted in Lewis and dwarf rats treated with PTH (P<0.0001). ...... 121 3 27 Vertebral Cancellous Mineral Apposition Rate ( m/d). A significant increase was observed in PTH treated Lewis and dwarf rats (P<0.0001). ...................... 122 3 28 Longitudinal Bone Growth ( m/d). A highly significant increase was observed between VEH and PTH treated Lewis and dwarf rats (P<0.0001) ... 122 3 29 Proximal tibial metaphyses from vehicletreated dwarf (A) and PTH treated dwarf (B) rats. ................................................................................................... 123 3 30 A significant increase in tibial cancellous bone was observed in both Lewis and dwarf rats treated with PTH (P<0.0001). ................................................... 123 3 31 Tibial Trabecular Number (#/mm). A significant increase in PTH treated Lewis and dwarf rats (P<0.0001) was observed. .............................................. 124 3 32 Trabecular Width ( m). A significant increase was observed in the PTH treated rats (P<0.00 01). .................................................................................... 124 3 33 Tibial Trabecular Separation ( m). A significant decrease was observed mainly in the PTH treated rats (P<0.0001). ...................................................... 125 3 34 Total Cortical Bone Tissue Area (mm2). Significant difference between the two strains, but not with the PTH treatment in both groups .............................. 125 3 35 Total Cortical Area (mm2). No significant difference with PTH treatment in both groups, only the difference between the two strains of dwarf and Lewis could be observed ............................................................................................ 126 3 36 Cortical Bone Marrow Area (mm2). There was a significant difference with PTH treatment in both groups with P<0.013 ..................................................... 126 3 37 Periosteal Perimeter (mm). Significant difference between dwarf and Lewis groups, but not with the PTH treatment ............................................................ 127 3 38 Endocortical Perimeter (mm). No significant difference with PTH treatment in both groups, only the difference between the two strains of dwarf and Lewis could be observed ............................................................................................ 127

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16 3 39 Cortical Width (mm). The cortical width decreased in both groups, dwarf and Lewis with PTH treatment, with P<0.001 and P<0.01, respectively .................. 128 3 40 Periosteal Mineralizing Surface (%). A significant increase was observed in the PTH treated rats (P<0.0001) from both groups (P<0.0001) ....................... 128 3 41 Periosteal Mineral Apposition Rate ( m/d). PTH treatment led to a significant increase in both groups (P<0.0001) ................................................................. 129 3 42 Periosteal Bone Formation Rate (102 m3/ m2/d). Significant increase in both group was observed with PTH treatment (P<0.0001) ............................... 129 3 43 Endocortical Mineralizing Surface(%). A significant increase was observed in the PTH treated rats (P<0.0001) from both groups (P<0.0001) ....................... 130 3 44 Endocortical Mineral Apposition Rate (um/d). Significant increase was observed in both groups with PTH treatment (P<0.0001) ................................. 130 3 45 Endocortical Bone Formation Rate (102 m3/ m2/d). A significant increase was observed in the PTH treated rats from both groups (P<0.0001) ............... 131 3 46 Biomechanical Load (N). Significant increase was observ ed only in the PTH treated Lewis rats, (P<0.0001) ......................................................................... 131 3 47 Biomechanical Stress (N*mm2 or MPa). Significant increase was observed in both groups, dwarf (P<0.0011) and Lewis (P<0.010) with PTH treatment .... 132 3 48 Biomechanical Stiffness (N/mm). Only PTH treated Lewis rats showed a significant increase with the treatment (P<0.0001), a trend was observed in dwarf rats .......................................................................................................... 132 4 1 Body Weight (g). There was a significant decrease in the body weight of PGE2treated Lewis rats, but not in PGE2 treated dwarf rats ............................ 143 4 2 IGF I Serum Levels (ng/mL). There was a significant difference between Lewis and dwarf rats (P<0.0001). PGE2treated rats showed significantly lower values compared to vehicle treated rats (P<0.0003) ............................... 143 4 3 Total Bone Mineral Content (BMC) for Cancellous or Trabecular Bone (mg/mm) in the distal femoral metaphysis. There was no significant difference with PGE2 treatment in dwarf and Lewis rats .................................. 144 4 4 Total Bone Mineral Density (BMD) for Cancellous or Trabecular Bone ( mg/cm3). No significant change was observed with PGE2 treatment .............. 144 4 5 Trabecular BMC (mg/mm). There was a significant increase in PGE2treated dwarf and Lewis rats ......................................................................................... 145

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17 4 6 Trabecular BMD (mg/cm3). A significant increase was observed in trabecular mineral density in Lewis and dwarf rats tr eated with PGE2 (P<0.0001) ........................................................................................................ 145 4 7 Total Area for Cancellous Bone (mm2). No significant difference was noted with PGE2 treatment ......................................................................................... 146 4 8 Trabecular Area (mm2). There was no significant difference with PGE2 treatment in dwarf and Lewis rats ..................................................................... 146 4 9 Total BMC for Cortical Bone (mg/mm). The mean values were significantly lower in the PGE2treated rats, dwarf and Lewis, when compared to their respective vehicletreated controls .................................................................. 147 4 10 Total BMD for Cortical Bone (mg/cm3). Dwarf rats treated with PGE2 showed significant lower values than vehicle controls. No difference in Lewis rats with PGE2 treatment ......................................................................................... 147 4 11 Cortical BMC (mg/mm). Significant lower mean values in the PGE2 treated rats, dwarf an d Lewis ........................................................................................ 148 4 12 Cortical BMD (mm/cm3). PGE2 treated rats showed significant lower mean values in both groups ....................................................................................... 148 4 13 Cortical A rea (mm2). Dwarf treated with PGE2 showed significant lower mean values, while there was no difference between Lewis groups ................ 149 4 14 Cortical Thickness (mm2). PGE2 treated dwarf rats showed significant lower mean values, while there was no difference between Lewis groups ................ 149 4 15 Periosteal Circumference (mm). Significant lower values in dwarf and Lewis rats treated with PGE2 ...................................................................................... 150 4 16 Endosteal Circumference (mm). Significant lower mean values in PGE2 treated Lewis rats, but no significant difference between PGE2 treated dwarf and their controls .............................................................................................. 150 4 17 Vertebral Cancellous Bone Volume (%). Significant higher mean values in PGE2 treated Lewis rats, but no significant difference between PGE2 treated dwarf and their controls (only a trend) .............................................................. 151 4 18 Vertebral Trabecular Number (#/mm). A significant increase was observed in Lewis and dwarf rats treated with PGE2 (P<0.0001) ......................................... 151 4 19 Ver tebral Trabecular Width ( m). There was no significant difference in PGE2 treated rats, dwarf and Lewis .................................................................. 152

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18 4 20 Vertebral Trabecular Separation (um). Significant lower mean values in PGE2 treated rats, dwarf and Lew is, compared to their controls ...................... 152 4 21 Vertebral Osteoid Surface (%). No significant difference was observed with PGE2 treatment in dwarf and Lewis rats ........................................................... 153 4 22 Vertebral Osteoblast Surface (%). There was no significant difference in PGE2 treated rats, dwarf and Lewis .................................................................. 153 4 23 Vertebral Osteoclast Surface (%). There was no significant difference with PGE2 treatment in dwarf and Lewis rats ........................................................... 154 4 24 Vertebral Mineralizing Surface (%). PGE2 treatment caused a significant increase in dwarf and Lewis rats comp ared to their controls. ........................... 154 4 25 Vertebral Bone Formation Rate (10-2 m3/ m2/d). Only PGE2 treated Lewis rats showed mean values significantly increased (P<0.05) compared to vehicle treated controls ..................................................................................... 155 4 26 Vertebral Cancellous Mineral Apposition Rate ( m/). Dwarf and Lewis rats treated with PGE2 showed decreased values compared to their controls ........ 155 4 27 Longitudinal Bone Growth ( m/d). PGE2 treated dwarf and Lewis rats showed significant increas e in the mean values ............................................... 156 4 28 Proximal tibial metaphyses from VEH treated dwarf (A) and PGE2treated dwarf (B) rats. ................................................................................................. 156 4 29 Ti bial Cancellous Bone Volume (%). PGE2 caused a significant increase only in dwarf rats compared to their controls(P<0.001) .................................... 157 4 30 Tibial Trabecular Number (#/mm). PGE2 treated dwarf rats presented a significant increase compared to their controls (P<0.0005), but not PGE2 treated Lewis rats ............................................................................................. 157 4 31 Tibial Trabecular Width ( m). PGE2 treated Lewis rats showed significant lower v alues compared to their controls, while there was no difference between dwarf rats ........................................................................................... 158 4 32 Tibial Trabecular Separation (um). PGE2 treated dwarf rats showed markedly significant lower values compared to their controls, while Lewis rats had no difference .............................................................................................. 158 4 33 Total Cortical Bone Tissue Area (mm2). There was no significant difference between the two groups of dwarf rats with PGE2 treatment, while Lewis rats showed a significant t decrease between treated and controls ........................ 159

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19 4 34 Cortical Area (mm2). Lewis rats showed a significant t decrease between PGE2 treated and controls. No difference between the two groups of dwarf rats ................................................................................................................... 159 4 35 Marrow Area (mm2). PGE2 treated dwarf rats showed significantly increased values compared to their controls, while Lewis rats showed no difference ....... 160 4 36 Periosteal Perimeter (mm). PGE2 treated Lewis rats showed significantly decreased values compared to their controls, while dwarf rats showed no difference .......................................................................................................... 160 4 37 Endocortical Perimeter (mm). PGE2 treated dwarf rats showed significantly increased values compared to their controls, while Lewis rats showed no difference .......................................................................................................... 161 4 38 Cortical Width (mm). PGE2 treated Lewis rats showed significantly decreased values compared to their controls, while dwarf rats showed no difference .......................................................................................................... 161 4 39 Periosteal Mineralizing Surface (%). There was no significant difference with PGE2 treatment in dwarf and Lewis rats ........................................................... 162 4 40 Periosteal Mineral Apposition Rate (um/d). PGE2 treatment s ignificantly increased the mean values in dwarf and Lewis rats ......................................... 162 4 41 No significant differences in dwarf and Lewis rats with PGE2 treatment ........... 163 4 42 Endocortical Mineralizing Surface (%). PGE2 treatment did not show any significant effect in dwarf and Lewis rats .......................................................... 163 4 43 Endocortical Mineral Apposition Rate (um/d). T here was a significant increase in PGE2 treated rats compared to their controls (P<0.0001), but not in Lewis rats ...................................................................................................... 164 4 44 Endocortical Bone Formation Rate (102um3/um2/d). PGE2 treated dwarf rats presented a significant increase, no significant effect in Lewis rats .................. 164 4 45 Biomechanical Load (N). Dwarf and Lewis PGE2 treated rats did not show significant difference ......................................................................................... 165 4 46 Biomechanical stress (N*mm2). There was a significant increase in PGE2 treated rats. No change presented by dwarf treated rats .................................. 165 4 4 7 Biomechanical Stiffness (N/mm). No significant difference observed with PGE2 treatment in both groups ......................................................................... 166 5 1 Extractor (A) and freezer mill (B), used to pulverize the bone tissue ................ 182

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20 5 2 RNA pellets obtained from bone samples ........................................................ 182 5 3 Bioanalyzer results indicating the RNA quality and integrity ............................. 183 5 4 IGF I primers and probe sequences obtained with Primer Express 3.0 ............ 184 5 5 Collagen Type I primers and probe sequences obtained with Primer Expres s 3.0 .................................................................................................................... 185 5 6 Osteocalcin primers and probe sequences obtained with Primer Express 3.0 186 5 7 Osterix primers and probe sequences obtained with Primer Express 3.0 ........ 187 5 8 RANKL primers and probe sequences obtained with Primer Express 3.0 ........ 188 5 9 Osteoproteger in primers and probe sequences obtained with Primer Express 3.0 .................................................................................................................... 189 5 10 Sclerostin primers and probe sequences obtained with Primer Express 3.0 .... 190 5 11 Fold change in bone IGF I expression in dwarf rats ......................................... 191 5 12 Fold change in bone IGF I expression in Lewis rats ......................................... 191 5 13 Fold change in liver IGF I expression in dwarf rats ........................................... 192 5 14 Fold change in liver IGF I expression in Lewis rats .......................................... 192 5 15 Fold change in collagen type I gene expression in dwarf rats .......................... 193 5 16 Fold change in collagen type I gene expression in Lewis rats .......................... 193 5 17 Fold change in osteocalcin gene expression in dwarf rats ............................... 194 5 18 Fold change in osteocalcin gene expression in Lewis rats ............................... 194 5 19 Fold change in osterix gene expression in dwarf rats ....................................... 195 5 20 Fold change in osterix gene expression in Lewis rats ...................................... 195 5 21 Fold change in RANKL gene expression in dwarf rats ..................................... 196 5 22 Fold change in RANKL gene expression in Lewis rats ..................................... 196 5 2 3 Fold change in OPG gene expression in dwarf rats ......................................... 197 5 24 Fold change in OPG gene expression in Lewis rats ......................................... 197 5 25 Fold change in sclerostin gene expression in dwarf rats .................................. 198

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21 5 26 Fold change in s clerostin gene expression in Lewis rats .................................. 198

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22 LIST OF ABBREVIATION S ALS acid labile subuni t AN anorexia nervosa ANOVA analysis of variance ASBMR American Society for Bone and Mineral Research ACTH adrenocorticotro pic hormone AVMA American Veterinary Medical Association BFR bone formation rate BGFLAP bone gamma carboxyglutamic acidcontaining pr otein BMC bone mineral content BMD bone mineral density BMP bone morphogenetic protein BMUs basic multicellular units BS bone surface BV bone volume cDNA complementary DNA CKD chronic kidney disease COX 2 cyclooxygenase 2 Ct cycle threshold DAN differentia l screening selected gene aberrant neuroblastoma DKK1 Dickkopf 1 DXA dual x energy x ray absorptiometry ELISA enzyme linked immune sorbent assay EP receptors for E series of prostaglandins FDA Food and Drug Administration

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23 FGF fibroblast growth factor GH gr owth hormone GHRH growth hormonereleasing hormone GPCRs G proteincoupled receptors HBM high bone mass syndrome HET heterozygous hPTH human parathyroid hormone IACUC Institutional Animal Care and Use Committee IEMA immunoenzymometric assay IGFBP insulin l ike growth factor binding proteins IGF I insulin like growth factor I IRS 1 insulin receptor substrate1 LH luteinizing hormone LID liver deficient LRP lipoprotein receptor related protein MAR mineral apposition rate M CSF macrophage colony stimulating fac tor mRNA messenger RNA MS mineralizing surface NAMS North American Menopause Society NHANES National Health and Nutrition Examination Survey N CBI National Center for Biotechnology Information NOF National Osteoporosis Foundation Ob.S osteoblast surface Oc. S osteoclast surface

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24 ODF osteoclast differentiation factor OPG osteoprotegerin OPGL osteoprotegerin ligand OPPS osteoporosis pseudoglioma syndrome OS osteoid surface Osx osterix PCR polymerase chain reaction PGE2 prostaglandin E2 PGs prostaglandins pQCT peripheral quantitative computerized tomography PRL prolactin PTH parathyroid hormone PTHrP PTH related protein qPCR quantitative polymerase chain reaction RANK receptor activator of nuclear kappa RANKL receptor activator of nuclear kappa ligand rhGH recom binant human GH rhIGF I recombinant human IGF I RIN RNA integrity number RT PCR reverse transcriptase polymerase chain reaction Runx 2 runt related transcriptional factor 2 SEM scanning electron microscopy SOST sclerostin gene Tb.N trabecular number TB.Sp trabecular separation

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25 Tb. Wi trabecular width TGF transforming growth factor beta TH thyroid hormone TIDM type I diabetes mellitus TNF tumor necrosis factor TRANCE tumor necrosis factor related activationinduced cytokine TSH thyroid stimulating hormone VEH vehicle solution Wnt wingless/mammalian homologue of drosophila gene wingless

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26 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 ROLE OF IGF I AS A POTENTIAL MEDIATO R FOR THE SKELETAL EFFECTS OF BONE ANABOLIC AGENTS By Ana Cristina Ferreira Bassit August 2012 Chair: Thomas Joseph Wronski Major: Veterinary Medical Sciences Growth hormone (GH) has a critical role in the regulation of longitudinal bone growth skeleta l maturation, and maintenance of adult bone mass. Insulin like growth factor I (IGF I) is considered the prime mediator for the skeletal effects of GH Parathyroid hormone (PTH) has potent anabolic effects and its use as an osteoporosis therapy is approved by the Food and Drug Administration. Prostaglandin E2 ( PGE2) can also induce cortical and trabecular bone formation in animal models The role of IGF I as a potential mediator for the bone anabolic effects of PTH and PGE2 is controversial as in vivo an d in vitro studies have yielded conflicting results. The objective s of this study w ere: 1Evaluate the dwarf rat (dw-/dw-) as an animal model for the effects of GH and IGF I deficiency on the skeleton, as related to clinical conditions in which serum levels of IGF I are decreased but not abolished; 2C ompare the skeletal effects of PTH and PGE2 treatment in dwarf rats and their background strain, Lewis rats ; 3 Determine the expression of genes related to bone formation and resorption At 9 weeks of age, female Lewis and dwarf rats were treated daily for 2 weeks with vehicle, hPTH 134 at a dose of 50 g/kg body weight, or PGE2 at a dose of 3 mg/kg body weight (N=710/group). Serum IGF I was measured by ELISA, and bone

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27 changes were analyzed by histomorph ometry p eripheral quantitative computerized tomography (pQCT) and biomechanical testing. RNA was isolated from bone tissue for evaluation of gene expression by RT PCR. Dwarf rats exhibited markedly lower IGF I serum levels, and decreased bone mass, stre ngth, and formation compared to Lewis rats. PTH and PGE2 treatment increased bone mass and formation in both dwarf and Lewis rats. Sclerostin, a potent negative regulator of bone formation, was downregulated in PTH and PGE2treated dwarf rats, suggesting that sclerostin inhibition could be related to the persistent bone anabolic effects of PTH and PGE2, despite low IGF I serum levels in dwarf rats. Therefore, under the conditions of this study, normal serum levels of IGF I are not essential for the bone anabolic effects of PTH and PGE2.

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28 CHAPTER 1 INTRODUCTION Osteoporosis and Osteopenic C onditions Osteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD), deterioration of bone tissue with disruption of bone architectur e, compromised bone strength and an increase in the risk of fracture. It affects primarily the elderly, and, in particular women, representing a major public health problem It is responsible for an estimated 90% of all hip and spine fractures in white A merican women ages 65 to 84, according to the 2010 evidencebased position statement published by The North American Menopause Society (NAMS, 2010) Based on data from the National Health and Nutrition Examination NOF Survey III (NHANES III), the NOF (National Osteoporosis Foundation) has estimated that more than 10 million Americans have osteoporosis and an additional 33.6 million have low bone density of the hip with related fractures having an estimated cost of $17 billion in 2005 (NOF, 2008) Furthermore, osteopenia, or low bone mineral density, is not only found in postmenopausal women, but also in other estr ogen deficient bone loss conditions, such as women with anorexia nervosa and hypothalamic amenorrhea, as well as in the juvenile onset of osteoporosis (Grinspoon et al., 1999, Giustina et al., 2008, Hofman et al., 2 009) Oncological patients under treatment must also be considered, as chemotherapy adversely affects bone metabolism and osteoblast activity (Davies et al., 2002, Hurson et al., 2007) Glucocorticoidinduced os teoporosis (GIOP) is another significant problem, as these potent anti inflammatory drugs can cause rapid bone loss and increase the risk of fractures (Hofbauer et al., 1999, Kream et al., 2008)

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29 Osteopenia and/or osteoporosis can also be seen in renal osteodystrophy, as a consequence of an extra skeletal systemic disorder of mineral and bone metabolism, which occurs in patients with chronic kidney disease (CKD) (Martin and Gonzalez, 2007, Kalantar Zadeh et al., 2010) Alcohol abuse, immobilization, reduced physical activity, and low dietary calcium levels mainly during periods of rapid bone growth, all can lead to a decrease in bone mass and osteopenia, with a predispositi on to fractures. These conditions can sometimes be assessed, in clinical situations, through bone biopsy and histomorphometric analysis, that are powerful and informative diagnostic tools for the determination of bone abnormalities More commonly nonin vasive methods, such as bone imaging and serum biomarkers for bone formation and resorption are used to diagnose osteoporosis and monitor the effectiveness of treatments. Bone despite its static appearance, is a dynamic tissue going through constant mode ling and remodeling throughout life. Bone modeling, an uncoupled process of bone formation and resorption, occurs mostly during skeletal growth, and it serves mainly to maintain bone shape and mass. Remodeling is a coordinated process in which osteoclast s resorb bone, followed by bone formation through osteoblast activity. Growth hormone (GH) and insulinlike growth factor I (IGF I) are essential for longitudinal bone growth and can potentially influence the regulation of bone modeling and remodeling as well (Isaksson et al., 1982, Isgaard et al., 1986, Ohlsson et al., 1998, Ohlsson et al., 2009) Depression of the GH/IGF I axis has been commonly associated with disturbances in bone metabolism and mass, and is co nsequently related to many osteopenic conditions in humans and in animal models.

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30 Growth Hormone (GH) and InsulinL ike Growth Factor I (IGF I) Growth hormone (GH) plays an important role in the regulation of longitudinal bone growth, and also in the metabol ism and maintenance of adult bone mass G H or somatotropin is an anabolic peptide hormone released from the somatotrophs, cells in the anterior pituitary gland. GH secretion occurs under the main regulation of GH releasing hormone (GHRH) and somatostatin, the two main hypothalamic regulators of GH, which exert stimulatory and inhibitory influences, respectively. GH is released in a pulsatile mode, under circadian cycle influence, being secreted mainly at night, during sleep. There is an influence of sex steroids as well, and puberty has a great effect on the amplitude of GH releasing pulses, with both estrogens and androgens stimulating higher pulses (Jansson et al., 1985) GH secretion can also be stimulated by ghrelin, a peptide expressed mainly in the stomach, but also found in other organs, such as t he kidney, pancreas, hypothalamus and pituitary (Molina, 2006a) In addition, the secretion of GH can be altered by glucocorticoids, when administered over prolonged periods of time, leading to osteoporosis, as they inhibit the secretion of GH in response to GH RH in healthy subjects (Giustina et al., 1995) There are other hormones, such as thyroid hormone (TH), and hypothalamic peptides a nd neurotransmitters that can affect the regulation of GHRH and somatostatin release, and consequently, GH synthesis (Molina, 2006a) Most important ly GH release is also inhibited by insulin like growth factor I (IGF I), produced mainly in the liver in a classic negative feedback mechanism of hormone control (Ohlsson et al., 1998, Rosen, 2008) According to the classical concept of the Somatomedin Hypothesis, proposed by Salmon and Daughaday about fifty years ago, GH indirectly stimulates IGF I (or somatomedinC, as initially denominat ed) synthesis

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31 and secretion by the liver, which acts as a mediator of the growth and metabolic effects of GH (Salmon and Burkhalter, 1997, Daughaday, 2000) G H plays an important role in the regulation of longitudinal bone growth, and also in the metabolism and maintenance of adult bone mass, acting directly on GH receptors in bone cells and indirectly stimulating IGF I synthesis by the liver. IGF I is an important anabolic growth factor for bone, and is considered the prime mediator of the skeletal effects of GH (Ohlsson et al., 1998, Giustina et al., 2008, Ohlsson et al., 2009) Discovered 50 years ago, it was first described as a soluble factor induced by GH that had ins ulin like properties and acted on body growth (Rosen and Niu, 2008) IGF I is a small 7 kDa peptide that still presents new questions about its metabolic effects and action pathways in several tissues, with special emphasis on bone growth and bone formation (Rajaram et al., 1997, Rosen, 2008) The highest expression of IGF I occurs in the liver, wher e it is primarily secreted and transported to other tissues to act as an endocrine hormone (Ohlsson et al., 2009) Outside the liver, bone is the richest source of IGF I and IGF II in mamm alian organisms (Rosen and Niu, 2008) It is also synthesized by a variety of other tissues, exerting then tissuespecific paracrine and autocrine effects. In bone tissue, IGF I is synthesized by many cells, including cells of the osteoblastic lineage, as well as chondroblasts and osteoclasts (Molina, 2006a, BouGharios and Crombrugghe, 2008) IGF I can stimulate bone formation, protein synthesis, increases replication of cells of the osteoblastic lineage, enhances fibr oblast proliferation, increases type I collagen production by osteoblasts, enhances matrix apposition rates, and decreases collagen

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32 degradation in calvariae (Thorngren and Hansson, 1975, Rajaram et al., 1997, BouGh arios and Crombrugghe, 2008, Rosen, 2008) A group of six insulinlike binding proteins (IGFBP 1 to 6) regulate the biological actions of IGF I in circulation and tissue, constituting a complex system for regulating IGF I activity. IGF BPs can both have stimulatory and/or inhibitory effects on IGF I function. Binding of IGFB P s to IGF I usually impedes the interaction of IGF I to its receptor (IGFR), and therefore inhibits IGF I actions. On the other hand, binding to IGFB P s also avoids the proteolytic degradation of IGFs by specific serine proteases, increasing their bioavailability in local tissues. In this manner IGFBPS have a central position in the bioavailability and distribution of IGFs in the extracellular environment (Kelley et al., 1996) The predominant or most abundant form is IGFBP 3, which is the main binding protein in serum, carrying IGF I in a ternary complex consisting of one molecule of IGF I, one molecule of IGBP 3 and one molecule of a protein named acid labile subunit (ALS). The association of these three proteins forms a complex that prolongs the half life of the IGFs in the circulation, acting like a reservoir in the organism. Other IGFBPs can bind to IGF I, forming binary complexes able to cross the capillary barrier and achieve selective transport to different tissues. IGFBPs are produced in several tissues involving intricate regulatory mechanisms (Molina, 2006a) IGF B P 3 is also the predominant binding protein in bone, and hepat ic IGFBP 3 and synthesis of ALS are under GH stimulation (Rosen and Niu, 2008) Low circulating levels of IGF I are linked to low bone mineral density (BMD), predisposing to the development of osteoporosis and a greater risk of fractures (Niu and Rosen, 2005) In fact, both GH secretion amplitudes and IGF I and IGFBP 3 serum

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33 levels decrease with age, as the GH/IGF I axis undergoes natural changes over the life span. Several of these changes in the GH/IGF I axis have been observed in patients with osteoporosis, as a correlation occurs between low serum IGF I and IGFBP 3 levels in females with postmenopausal osteoporosis and the likelihood of hip and spinal fractures (Sugimoto et al., 1997) Patients with anorexia nervosa, a condition characterized by low body weight and amenorrhea, also show a markedly decreased BMD, with a high propensity for fractures, and resistance to GH and est rogen treatment. In fact, there is a twoto three fold increase in fractures in patients with anorexia, associated with low levels of IGF I, osteopenia and abnormal bone microarchitecture (Lawson et al., 2010, Jac obsonDickman and Misra, 2010) IGF I circulating levels are also dramatically reduced in patients with diabetic bone disease. In children with new onset of type I diabetes (TIDM) a 73 % fall in IGF I serum levels was observed accompanying the downregul ation of hepatic IGF I release (Bereket et al., 1995) Chronic immobilization, as well as space flight, leads to bone loss due to inhibited bone formation. It was proposed that skelet al unloading interferes with the proliferation of osteoblasts and their precursors by inhibiting IGF I signaling pathways (Sakata et al., 2004) GH has been successfully used to treat children with growth impairment, resulting in increas ed sk eletal growth. Regarding therapeutic applications, there is one indication of treatment with IGF I approved in the United States, for Laron syndrome, in which patients lack the GH receptor (GHR) and present very low levels of IGF I, but high levels of GH. Despite this very specific condition, recombinant human IGF I (rhIGF I) and recombinant human GH (rhGH) are not usually indicated for the treatment of

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34 osteoporosis or other osteopenic conditions Nonetheless, GH/IGF I axis depression is associated with many of the dis orders involving bone loss, and the influence of low GH and IGF I levels on the action of the drugs routinely used must be better investigated. Studying these growth factors, deeply involved in bone metabolism, is of great importance to improve the available armamentarium of drugs used in the treatment of osteoporosis. The most commonly used drugs, such as estrogen, raloxifene, biphosphonates, and calcitonin, inhibit osteoclast mediated bone loss, but are unable to stimulate bone formation and restore bone mass to normal levels. Parathyroid Hormone Parathyroid hormone (PTH) has this targeted bone anabolic capability, and is currently approved by the FDA for the treatment of osteoporotic patients, but it is used only for high fracture risk patients, as it requires daily injections. Interestingly, PTH was considered, at first, only as a primarily catabolic hormone, stimulating bone resorption and the release of calcium into the circulation in hypocalcemic conditions, in order to reestablish calcium homeostasis. Yet, in the kidney, PTH promotes calcium re abs orption and inorganic phosphorus excretion in the urine. Through indirect actions on the gastrointestinal tract, PTH also leads to greater calcium absorption, stimulating the hydroxylati on of 25 hydroxyvitamin D3, at the 1 position, leading to the active form 1,25dihydroxyvitamin D, or calcitriol. Even very small changes in calcium serum levels can induce the parathyroid glands to increase remarkably PTH secretion. The anabolic effects of PTH were initially noticed in animal studies using parathyroid gland extract, about 80 years ago, and when hPTH started to be pharmacologically synthesized, studies about the potential bone anabolic actions of PTH were undertaken. After initial skepti cism about the paradoxical actions of PTH, being both catabolic and anabolic, it

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35 was accepted that continuous high serum levels or doses lead to catabolic effects, while intermittent low doses are associated with anabolic effects on bone (Kousteni and Bilezikian, 2008) Several studies extensively demonstrated the anabolic effects of PTH on the skeleton, increasing bone formation and remodeling, increasing bone density and impr oving the quality of skeletal microarchitecture (Dempster et al., 1993, Finkelstein et al., 1994, Gunness, 1995, Jilka, 2007, Canalis et al., 2007) M echanisms for PTH to induce bone formation include stimulation o f growth factors, especially IGF I. Transient treatment of rat calvarial cell cultures with PTH stimulated collagen synthesis by osteoblasts, but IGF I neutralizing antibodies prevented this effect (Canalis et al., 1989) Gene expression for IGF I was also found to be upregulated in bone cell cultures and in bone tissue from PTH treated rats and IGF I was then considered as a potential mediator for the actions of PTH in bone tissue, possibly regulating the coupling of bone formation and bone resorption (Linkhart et al., 1989) However, PTH treatment of hypophysectomized (HYPOX) rats deficient in GH/IGF I yielded conflicting results as to whether IGF I is essential for the bone anabolic effects of PTH possibly due to the poor physical condition of the rats and deficiencies in other boneactive hormones Hock and Fonseca reported that the GH/IGF I axis is essential for the bone anabolic actions of PTH (Hock, 1990) In contrast, Schmidt et al. performed a similar study in HYPOX rats and concluded th at GH/IGF I is not required for a PTH induced stimulation of bone formation (Schmidt et al., 1995) Gunness and Hock reported that the PTH induced increase in bone mass did not respond to supplementati on with GH or IGF I when the rats were fed an energ y restricted or ad libitum diet (Gunness, 1995)

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36 Dempster (1993) compared the effects of intermittent doses of PTH to the effects of mi ld primary hyperparathyroidism, in which there is an increase in the rate of bone remodeling, with new ly formed BMUs (basic multicellular units) presenting more osteoblasts and osteoclasts, but with a balance in favor of bone formation. That could explain the dual mechanism of PTH, but there were still many questions to be answered concerning PTH mechanisms and the interaction of PTH with other drugs and/or other growth factors such as IGF I, IGF II, GH, and TGF (Dempster et al., 1993) PTH activates osteoblasts, resulting in increased expression of genes with important roles in the production of growth factors, including IGF I (S warthout et al., 2002) Based on previous in vitro results, Miyakoshi et al. (2001) then worked with IGF I knockout mice, finding that PTH treatment had no significant effects on serum bone formation markers, and did not increase bone mineral density com par ed to their wild type controls H is findings strongly suggested that IGF I was required for the bone anabolic effects of PTH (Miyakoshi et al., 2001) In another study, two strains of mice, null for insulin receptor substrate1 (IRS 1) and null for insulin receptor 2 (IRS 2), were treated with PTH and compared to their wildtype controls. The PTH anabolic effects were blunted in the IRS 1 / mice, indicating that the activation of IRS 1 was essential for PTH actions on bone (Yamaguchi et al., 2005) In a comprehensive review, Jilka (2007) considered the increase in the number of matrix synthesizing osteoblasts, increased osteoblastogenesis, attenuation of osteoblasts apoptosis, and activation of quiescent lining cells as explanations for the anabolic effects of PTH on bone. N evertheless, the molecular and cellular mechanisms

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37 underlying these skeletal effects are not completely clarified and the role IGF I as a potential mediator for the bone anabolic eff ects of PTH is controversial (Jilka, 2007) Prostaglandin E2 Prostaglandins (PGs) are synthesized by bone cells and can affect both bone formation and bone resorption, acting as local and m ultifunctional regulators of skeletal metabolism. PGs are produced by cyclooxygenase, and their production is regulated by various local and systemic factors that ultimately induce cyclooxygenase 2 (COX 2) expression (Harada et al., 1995, Machwate et al., 2001) Through their anti inflammatory actions, glucocorticoids can substantially inhibit PGs production, as they also inhibit COX 2 expression (Pilbeam et al., 2008) Prostaglandins, especially prostaglandin E2 (PGE2), act on multiple receptors, and th ere are at least nine G proteincoupled receptors (GPCRs ) for PGs mediating their actions. The multiple receptors for PGE2 are associated with four classes of GPCRs, named EP1, EP2, EP3 and EP4. Their ability to activate different signaling pathways make s it difficult to elucidate the effects on skeletal tissues (Pilbeam et al., 2008) Prostaglandin E2 ( PGE2) is the most abundant product in bone cells and in vivo experiments showed that it increased bone mass in the metaphysis of the proximal and distal tibia and on endocortical and periosteal surfaces of the tibial shaft; it also stimulated bone formation and resorption evaluated by histomorphometric anal yses (Lin et al., 1995) The anabolic effects of PGE2 in cancellous bone could be observed in adult and even in aged rats (Ito et al., 1993, Cui et al., 2001) PGE2 is a potent inducer of cortical and trabecular bone formation in humans and animal models with strong evidence that the bone anabolic effects are mediated by the EP4 receptor, as the admini stration of an antagonist to EP4 suppressed the action of

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38 PGE2 in increasing trabecular bone volume and bone formation indices In addition, PGE2 can improve fracture healing, stimulating both bone remodeling and angiogenesis, and induces the production o f IGF I, which modifies the rate of type I collagen synthesis (Pilbeam et al., 2008) Fracture healing and bone formation stimulated by PGE2 are also correlated with mechanical loading. As observed by Jee and Ma (1997), the anabolic actions of PGE2 are influenced by the impact of mechanical usage and the skeletal adaptation to this usage. Another inf luence was exerted by the source and abundance of osteoblast lineage cells (Jee and Ma, 1997) PGE2 stimulates the synthesis of IGF I in osteoblast cultures from fetal r at bone, increasing IGF I transcripts by 2.2 fold (McCarthy et al., 1991) In another study (Harada et al., 1995) in vivo and in vitro effects were observed; cancellous bone formation in the proximal tibial metaphysis was enhanced by PGE2 treatment in ovariectomized rats and in vitro PGE2 stimulated IGF I expression. The IGF I mRNA expressi on was correlated with osteogenesis (Harada et al., 1995) Despite the above evidence that PGE2 upregulates gene expression for IGF I in bone cells, the skeletal effects of PGE2 in IGF I deficient animal models have not been studied to date. The GH/IGF I Axis and Animal Models Different animal models have been used to address the questions related to the GH/IGF I axis and its influence on bone metabolism by inducing IGF I deficiency. However, many of these surgically or genetically modified rodents have low survival rates, deficiencies in other boneactive hormones, and general health complications that might affect the interpretation of collected data. Hypophysectomized (HYPOX or HX) rats. Hypophysectom y of immature rats results in decreased bone growth and osteopenia, due to decreased bone formation in

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39 the presence of continued bone resorption. Growth hormone is considered the most effective growth stimulating pituitary hormone and has a doseresponse effect on longitudinal bone growth in hypophysectomized rats (Thorngren and Hansson, 1975, Thorngren and Hansson, 1977, Thorngren et al., 1977, Schoenle et al., 1982) However, hypophysectomized rats are markedly underweight and show diminished nonspecific immune responses with increased susceptibility to infections; as an example of this effect on the immune system, the macrophages from hypophysectomized rats exhibited 50% reduced capacity to kill Salmonella when compared to those derived from intact rats (Kooijman et al., 1996) These animal s also present decreased erythropoiesis, resulting in anemia, leucopenia and thrombocytopenia (Yeh et al., 1999) GH and IGF I administration can lead to resumption of growth and formation of red blood cells with increased incorporation of Fe (Kurtz et al., 1988) The intake of food is decreased in hypophysectomized rats, causing nutri tional deficiencies as well. The muscle growth is also affected by the lack of GH; rats are less active in their cages, and this mechanical disuse leads to an overall effect similar to animals under partial immobilization or underloading (Yeh et al., 1997) Pituitary hormones are important not only for longitudinal bone growth, but also for the radial growth of bone and maintaining cancellous bone balance (Isgaard et al., 1986, Kidder et al., 1997) The absence of hypophyseal hormones causes a marked decrease and delay in osteogenesis, affecting the quality of the newly formed bone, with decreased periosteal bone formation and depressed bone turnover, with more suppression in bone formation than in bone resorption, leading to an overall loss of cancellous bone mass (Yeh et al., 1999)

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40 Nevertheless, hypophysectomized rats have been used mostly for studies of the effects of GH and IGF I on longitudinal bone growth (Guler et al., 1988, Zapf et al., 1989, Turner et al., 2010) In addition, the effects of hypophysectomy are related to the age at the time of surgery and whether or not it is complete (Thorngren et al., 1980) Heterozygous (HET) mice. Heterozygous mice have one functional IGF I allele (IGF I + / -), and were developed by Powell Braxton and collaborators in 1993 to study the IGF I involvement in embryonic and postnatal growth. At that time as IGF I showed low levels in the embryo, it was thought to have an effective role o nly in postnatal growth and development. Compared to their wildtype littermates, the phenotype for the heterozygous animals included a 10 to 20% smaller size at birth and throughout their development, associated with a reduction in the size and weight of their organs, although there were no abnormalities in the analyzed tissues. There was no significant difference in the function of the growth plate, labeled with tetracycline, when compared to the wild type mice. The IGF I serum levels were approximatel y 37 % lower without affecting other serum components, and male and female heterozygotes were fertile and healthy, and could be intercrossed to generate homozygous IGF I / mice (Pow ellBraxton et al., 1993) In another study with heterozy gous mice, further analysis of bone morphometry revealed reduced femur length (46 %), reduced femur cross sectional area of cortical bone, marrow space and subperiosteal area. Through microcomput ed tomography, the femoral bone mineral density showed a decrease of 712% in both genders, when evaluated from 118 months, in comparison with wildtype mice. These findings were correlated to a 20 30% decrease in the IGF I serum levels and body weight ( 1020%).

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41 The IGF I protein levels were reduced by approximately 40% in the heterozygous cultures of neonatal calvarial osteoblasts, compared to the wildtype cells (He et al., 2006) Mohan and Baylink also observed that during puberty, the rate of gain in femoral bone mineral density was decreased by 25 % in heterozygous IGF I mice comparing with the control group, suggesting that IGF I has an important role in bone acquisition during puberty (Mohan and Baylink, 2005) The IFG I haploinsufficiency was then considered as a suitable condition for studying the changes in bone that occur when IGF I levels are only slightly decreased. IGF I knockout mice Homozygous IGF I knockout mice were also generated by the Powell Braxton group, through the disruption of the mouse IGF I gene and the intercross breeding of heterozygous IGF I mice (IGF I + / -). These animals were significantly smaller than their wild type littermates, demonstrat ing a clear impairment of skeletal growth, with a 60% reduction in their body weight. They also showed a generalized muscular dystrophy, with decreases in muscle mass and maturation, mainly in the diaphragm, heart and tongue; atelectatic and less histologically organized lungs; hypoplastic epidermis, and no significant difference in bone development, between homozygous and wildtype mice. These changes could be correlated with the high degree of perinatal lethality observed (> 95%) that, once again, confirmed the important role of IGF I in embryonic and postnatal growth. Initially, all IGF I / mice were found dead at birth. H owever, approximately 5% of these mutant animals survived after birth up to four months, but were not fertile (Powell Braxton et al., 1993) When using histomorphometry, peripheral computerized tomography (pQCT) and microcomputerized tomography ( QCT) to evaluate more deeply the effects of this

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42 mutation on bone tissue, it was found that 4 month old IGF I knockout mice were 24 % of the body weight of the wildtype mice, and their tibiae were 28 % and the lumbar vertebrae (L1) 26 % of the size of the control animals bones. In contrast, trabecular bone volume in the proximal tibia was increased in the IGF I mice, compared to the wild type, while the tibial bone periosteal formation rate and cortical thickness were decreased 27 % and 17 %, respectively. The growth plate and longitudinal bone growth were not evaluated in this study (Bikle et al., 2001) In summary, the IGF I knockout mouse has the advantage of complete absence of IGF I, but is a difficult animal model to work with due to limited viability and the poor general health. In addition, IGF I knockout mice have high serum levels of GH, which may complicate data interpretation. Liver IGF I D eficient (LID) mice. In 1999, Yakar and colleagues created another model to study the GH/IGF I axis, by targeting the IGF I gene specifically in the liver, obtaining liv er IGF I deficient (LID) mice (Yakar et al., 1999) These mutant animals have a complete ablation of liver IGF I gene expression and were developed through the Cre/loxP System, a conditional knockout system of genetic recombination (Liu et al., 2000) This new mutation provided ways to test one of the statements of the Somatomedin Hypothesis, that implicated liver derived IGF I as the main factor responsible for postnatal g rowth and skeletal development, acting as a mediator for GH. This new animal model suggested that local IGF I could support postnatal growth and development. The reduction in IGF I serum levels w as about 75%, confirming the liver as the major source of circulating IGF I. The remaining 25% of serum IGF I represent s the contribution of IGF I synthesis by nonhepatic tissues. The GH serum levels,

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43 howev er, showed markedly increased values in the LID mice, probably due to a negative feedback mechanism, in which the lack of circulating IGF I leads to an increase in GH synthesis by the hypophysis. Surprisingly, the body length, as well as the femur length, was not significantly different between the LID mice and their controls. The LID mice did not show growth retardation, nor changes in skeletal IGF I production, and were viable for at least 34 months. The average wet weight s of targeted organs such as liver heart, spleen and kidney, were similar between the LID and control mice. They were also fertile, and could give birth to normal sized pups (Yakar et al., 1999) Liu also correlated the 75% reduction in serum IGF I levels to a 50100 fold decrease in IGF I mRNA in the liver when compared to wildtype mice, while the body growth curves showed no difference with the wildtype mice, despite an increase in GH levels and a slight enlargement in the liver (Liu et al., 2000) On the other hand, when analyzing adult LID mice, there was a reduction in the periosteal circumf erence, femoral crosssectional area, cortical thickness and total volumetric BMD, resulting in slender and more fragile bones with age (Yakar et al., 2009a) Once established, the LID strain was used in cross breeding with other mutant strains, e.g. knockout mice for the ALS (acid labile subunit), or mice with inactivation of IGF IBP3 (IGF I binding protein 3), producing double or triple mutant strains, according to the endocrine and /or skeletal phenotype to be investigated (Yakar et al., 2009b) In a recent review, more than 50 different mutant strains of mice were li sted, according to their target action, genetic background, skeletal phenotype, and human counterpart (if there is a similar condition or disease in humans) (Yakar et al., 2010)

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44 Dwarf rats The dwarf rat (dw 4/dw 4 ) has potential as an animal model for studies of the effects of GH/IGF I deficiency on bone structure and function. This mutation, inherited as an autosomal recessive, arose spontaneously in a breeding colony of Lewis rats at the Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford, U.K., in 1985. Based on reports about their basic physiological characteristics by Charlton et al. (1988), the body growth in the mutant is retarded such that at 3 months of age, both males and females weigh approximately 40% less than their normal littermates, and continue to grow at a slower rate GH synthesis in the pituitary is reduced to 10% of normal levels in males and 6% in females, and the GH mRNA levels are 2025% of that normal, although the structural GH gene apparently is unaltered (Skottner et al., 1989, Charlton et al., 1988) Despite these hormonal changes, dwarf rats are outwardly healthy without any skeletal malformations, and therefore, appear to be a promising animal model for studies of GH/IGF I deficiency. Specific Aims The proposed research focused on the role of circulating IGF I in skeletal acquisition and whether or not low levels of serum IGF I, sometimes found in osteopenic conditions, would affect the anabolic responses to PTH and PGE2. The research project was, therefore, designed to evaluate, initially, the levels of serum IGF I and t he consequent changes in bone mass and indices of bone formation and resorption in GH/IGF I deficient dwarf rats ( dw 4/dw 4, autosomal recessive). Following this first analysis to establish the dwarf rat as an animal model for our studies, we tested the hypotheses that IGF I acts as a potential mediator for the bone anabolic effects of PTH and PGE2. In order to characterize the influence of this growth factor, the

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45 responses to PTH and PGE2 treatments were evaluated in two different strains of rats: dwarf rats and their background strain, Lewis rats. As mentioned, the dwarf mutation arose in an inbred colony of Lewis rats, thus allowing an appropriate direct comparison between the two strains (Charlton et al., 1988) Specific a im 1 Evaluate the skeletal consequences of GH/IGF I deficiency in dwarf rats by determining the IGFI serum levels and its effects on bone structure and turnover. We proposed the dwarf rat as an animal model to study the GH/IGF I axis deficiency and its importance for normal bone metabolism. We m easured the skeletal changes in both Lewis and dwarf rats and established baseline characteristics for further studies to compare the relative contribution of the GH/IGF I axis to the bone anabolic effects of hormones and drugs. These changes were measured at the organ level by pQCT analyses, quantitative bone histomorphometry, and biomechanical testing. Specific Aim 2 Ev aluate the influence of GH/IGF I deficiency on the effects of PTH tre a tment on bone mass and formation. This experiment was designed t o evaluate the influence of GH/IGF I axis deficiency upon the bone anabolic effects of intermittent administration of PTH. We hypothesized that the ability of PTH to increase bone mass and stimulate bone formation would be diminished in dwarf rats with low IGF I serum levels. This hypothesis was tested at the organ level by pQCT analyses, quantitative bone histomorphometry, and biomechanical testing. Specific Aim 3 Evaluate the influence of GH/IGF I deficiency on the effects of PGE2 on bone mass and for mation. In this experiment the actions of PGE2 on bone tissue were tested in dwarf rats with low IGF I serum levels, with the objective of

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46 clarifying whether or not this deficiency can impair the potential therapeutic use of PGE2 for osteoporosis. We hypothesized that the ability of PGE2 to increase bone mass and stimulate bone formation would be diminished in dwarf rats with low IGF I serum levels. This hypothesis was tested at the organ level by pQCT analyses, quantitative bone histomorphometry, and bi omechanical testing. Specific Aim 4 Evaluate the influence of GH/IGFI deficiency on the bone anabolic effects of PTH and PGE2 at the molecular level by analysis of gene expression. In order to study the molecular mechanisms involved in the responses to PTH and PGE2 treatments in IGF I deficient dwarf rats, we evaluated, by real time RT PCR, the expression of genes involved in bone formation and bone resorption, as listed below: Bone formation: IGF I, Collagen type I, Osteocalcin, Osterix, and Sclerostin. Bone resorption: RANKL and Osteoprotegerin (OPG). In addition, the gene expression of IGF I was also evaluated in hepatic tissue samples, to differentiate the expression of circulating IGF I produced by the liver, from the local IGF I synthesized by bone cells.

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47 CHAPTER 2 THE EFFECTS OF A DEPRESSED GH/IGF I AXIS ON BONE STRUCTURE AND BONE TURNOVER IN DWARF RATS Introduction The laboratory rat Ratus norvegicus has been a primary model in biomedical research, and a constantly increasing number of mutant st rains have been providing diseasetarget ed models for scientific investigations (Steen et al., 1999, Pearce et al., 2007, Lelovas et al., 2008) Particularly in bone research, the rat has been used to successfully reproduce osteoporosis/ osteopenic conditions and growth related diseases, and even though it does not ex hibit the same pattern of Haversian systems in cortical bone as seen in humans and higher mammals, it still provides data that can be translated to other species (Lelovas et al., 2008) Moreover, the amount of available information about the rat skeleton and the rat genome, protocols for inducing osteopeni a, orthopedic testing techniques, and reliable information that can be applied to different pathological conditions make the rat an adequate animal model in this field of science, having contributed to many therapeutic advances in bone disease management. GH stimulates the synthesis of IGF I in the liver, as well as in almost every tissue, including bone, and most of its anabolic action is mediated by IGF I, an important growth factor in the skeleton. IGF I has multiple physiological functions, acting through endocrine, paracrine and autocrine pathways, and is essential for both embryonic and postnatal growth (Powell Braxton et al., 1993, Liu et al., 1993, Bikle et al., 2001, Iida et al., 2005) The use of animal models has been crucial for understanding the numerous mechanisms and molecular pathways involved in the GH/IGF I axis.

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48 The dwarf rat has been used as an animal model in other studies to evaluate growth responses to GH and IGF I (Skottner et al., 1989) and as pr eviously mentioned, this model could allow a direct comparison of bone parameters with its background strain, the Lewis rat. Although GH synthesis is substantially reduced in dwarf rats, differently from h eterozygous and/or knockout animals, these rats ar e healthy without skeletal malformations and the concentration s of other trophic hormones such as luteinizing hormone (L H ), thyroid stimulating hormone (TSH), prolactin (PRL) and adrenocorticotropic hormone (ACTH), do not differ from unaffected heterozygotes (Charlton et al., 1988) In addition, b one mass, bone growth, and osteoblast activity are decreased in dwarf rats These characteristics make dwarf rats suitable for the study of osteoporosis, as well as other osteopenic syndromes in which the GH/IGF I serum levels are decreased but not abolished, as seen in most clinical conditions. The use of this mutant would mimic the GH/IGF I deficiency as it appears in clinical cases of postmenopausal osteopenia and other bone loss conditions, such as anorexia nervosa, cortico id therapy induced osteopenia, and chemotherapy Lange et. al. (2004) also found that there was a qualitative and quantitative difference, between dwarf and Lewis rats, in the morphology and arrangement of collagen microfibrils of femurs evaluated by SEM (sc anning electron microscopy) (Lange et al., 2004) IGF I and IGFBP3 were significantly decreased in dwarf rats, compared to the control group, in addition to decreased bone biomechanical properties: ultimate stress stiffness and energy at load. These findings were correlated with the decreased strength and increased occurrence of fractures observed in GH deficient patients.

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49 For all the characteristics presented in C hapter 1, we consider that the dwarf rat is suit able to address the importance of IGF I for the effectiveness of certain pharmacological agents in stimulating bone formation. The present study was, therefore, designed to evaluate, initially, the levels of serum IGF I and the consequent changes in bone mass and levels of bone formation and resorption in GH/IGF I deficient dwarf rats ( dw 4/dw 4, autosomal recessive). For th is study two hypotheses were tested: 1. The GH/IGF I deficiency in dwarf rats results in a significant decrease in IGF I serum levels, compared to those observed in HYPOX rats (85%), LID mice (75%) and HET mice (20 30%). 2. The GH/IGF I deficiency would similarly reduce body weight, decrease bone growth and cause osteopenia as observed in the animal models mentioned above. Materials and Meth ods Animal Model In order to compare the two different strains of rats: dwarf rats and their background strain, we used gender and agematched Lewis controls. The animals five week old female dwarf and Lewis rats, were obtained from the same supplier, Ha rlan Laboratories (UK ). They were housed two per cage, with relative humidity, air quality, illumination (12h light/12h dark cycles) and temperature controlled according to the criteria established by the Institute for Laboratory Animal Research and the National Research Council (2011) The protocol for us e of rats in this research pr oject w as approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida.

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50 Experimental Design Both groups of Lewis and dwarf rats consisted of 13 animals. Since dwarfism is not reliably noticed until 45 weeks of age, the rats were maintained in the same nutritional and environmental conditions for four weeks before starting treatment. D uring this period, all rats had their clinical status evaluated daily and were weighed weekly. Vehicle Treatment Starting at nine weeks o f age, the rats were injected daily subcutaneously, with vehicle solution, for two weeks. The preparation of the vehicle solution involved: Dissolving 0.1 ml of concentrated hydrochloric acid in 1000 ml pyrogen free distilled water, to get a 0.001N HCl so lution. Addition of 97.9 ml of sterile saline (0.9g NaCl) to 0.1 ml of the previous solution, resulting in a 0.015M NaCl solution, followed by filtration with #22 Milipore filter. Addition of 2 mL of heat inactivated rat serum. Bone Formation Markers All r ats received intraperitoneal injections of fluorochrome compounds, declomycin and calcein (Sigma Chemical Co., St. Louis, MO) at a dose of 15 mg / kg body weight, ten and three days prior to euthanasia respectively, in order to label actively forming bone surfaces. Euthanasia and Tissue Sample Distribution The animals were euthanized in accordance with the AVMA (American Veterinary Medical Association) Guidelines on Euthanasia2007, and following the protocol already approved by the IACUC. They were anest hetized with ketamine (Ketaset Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (AnaSed Lloyd Laboratories, Shenandoah, IA), at doses of 50 and 10 mg/kg, respectively. Once a deep level of

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51 anesthesia was achieved, exanguination from the abdomi nal aorta was performed, followed by cervical dislocation to confirm death. S erum samples were stored at 80 C for fut ure analyses. The animal carcasses were examined for macroscopic pathological changes. Both tibiae, right femur and left distal femur, t he first, second, third and fourth lumbar ver t ebrae and the fifth and sixth caudal vertebrae as well as the liver, were collected at the time of necropsy. All bone specimens were stripped of musculature. The tibiae, first and second lumbar vertebrae a nd the caudal vertebrae were placed in 10% phosphatebuffered formalin (pH 7.4) for 24 hours. These bone samples were then transfer r ed to 70% ethanol, and further processed undecalcified for quantitative bone histomorphometry. The right femora were placed in 70% ethanol until evaluated for total length and bone mineral by peripheral quantitative computerized tomography The left distal femora and liver samples were snap frozen in liquid ni trogen, and transfer r ed to a 80C freezer for future gene expressi on analys e s. The third and fourth lumbar vertebrae were also stored in a 80 C freezer for biomechanical analysis. IGF I Serum Levels The ELISA (EnzymeLinked Immuno Sorbent Assay) method was performed to obtain the IGF I serum levels for all animals. IG F I, although highly conserved between species, shows some differences when comparing human to rat or mouse IGF I. There are three amino acid substitutions that distinguish rat from human IGF I, and although there are only a small number of substitutions, most human IGF I assays are not able to measure rat or mouse IGF I. The antibody must recognize the specific peptide of the specie s being anlayz ed, and for these reasons, we used the Rat/ M ouse IGF I ELISA kit

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52 (IDS Immuno Diagnostic Systems), a specific immunoenzymometric assay (IEMA) for quantitative determination of Insulinlike Growth Factor I in rat and mouse serum or plasma. The serum samples were incubated briefly with a reagent to inactivate binding proteins, and then diluted for the assay. A pur ified monoclonal anti Rat IGF I is coated onto the i nner surface of microtitre wells, where the samples were then incubated together with biotinylated polyclonal rabbit anti Rat IGF I, and shaken for two hours at room temperature. The well s were then washed and enzyme (horse radish peroxidase) labelled avidin was added, binding to the biotin complex. After a further wash, a simple component chromogenic substrate ( a formulation of tetramethyl benzidine) was add ed to develop color. The absorbance (at 450 nm, with reference 620 nm) of the reaction, for each well, was read in a microtitre plate reader, with color intensity being proportional to the amount of rat IGF I present in the samples. The analysis was achieved with the Ascent Multiscan ELISA Software. The mean absorbance for each calibrator was used to generate a calibration curve, and obtain the slope of the equation to calculate the concentration of serum IGF I in ng/mL. Evaluation of Body Weight and Femoral Length The evaluation of body weight and femoral leng th were performed as an initial approach to ver ify the effects of GH/IGF I def iciency on the overall development and growth of dwarf rats, in comparison to Lewis rats. For this purpose, the animals were weighed, since their arrival in the ani mal care fac i litiy with the same precision scale. After necropsy, t he length of the right femur was measured with an electronic digital caliper (Fisher Scientific), considering the femoral head and the femoral condy les as proximal and distal reference points for measurement, respectively.

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53 Peripheral Quantitative Computerized Tomography Peripheral Quantitative Computerized Tomography (pQCT) is a noninvasive, sensitive and reproducible method to monitor changes in cancellous and cortical bone mass, bone density, and geometric properties. Moreover, this technology determines some indicators of bone properties that are relevant to bone strength: mass, mechanical quality and spatial distribution of bone material (Ferretti, 1999) Its precision and accuracy can be comp ared to histology and microcomputed tomography (CT) (Schmidt et al., 2003) and yet, as it measures volumetric density, it should not be affected by bone size, as with DXA (Dual X energy x ray absorptiometry), a method that measures areal bone mineral density. The pQCT captures a 3dimensional image, and allows the analysis of cortical and cancellous bone separately. Th is method has an accuracy of 92 to 98% and should be viewed as a complimentary technique to static and dynamic histo morphometry (Lelovas et al., 2008) The right femurs were scanned and analyzed by pQCT with a Stratec XCT Research M instrument (Norland Medical Systems, For t Atkinson, WI). The difference i n femur length between the two strains was considered when determining the region of interest at both sites the distal femoral metaphysis which is rich in trabecular bone, and the femoral shaft which is composed entirel y of cortical bone. Trabecular bone measurements were taken at a distance of 5mm and 4mm from the distal end of the femur, for Lewis and dwarf rats, respectively Similarly, when evaluating cortical bone in the femoral shaft the images were obtained at distances of 16 mm and 14 mm from the distal end of the femur in Lewis and dwarf rats, respectively.

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54 B one structural parameters measured by pQCT at the distal femoral metaphysis were : 1. Total bone mineral content ( total BMC mg/mm ) ; 2. T otal bone mineral density ( total BMD mg/cm3); 3. T rabecular bone mineral content (trabecular BMC, mg/mm); 4. Trabecular bone mineral density (trabecular BMD, mg/cm3); 5. Total area (mm2); 6. T rabecular area (mm2). B one structural parameters measured at the femoral diaphysis (shaft) were: 1. Tota l bone mineral content ( total BMC, mg/mm); 2. Total bone mineral density ( total BMD, mg/ cm3); 3. Cortical bone mineral content (cortical BMC mg/mm); 4. Cortical bone mineral density (cortical BMD mg/cm3); 5. Cortical area ( mm2); 6. C ortical thickness (mm); 7. Perioste al circumference (mm); 8. Endosteal (endocortical) circumference (mm). Bone Histomorphometry Bone histomorphometry is a histological method that provides quantitative data about bone structure/ organization and bone remodeling, by using a computer assisted analysis of microscopic images. It accurately quantifies the level of cellular activity and bone mass from undecalcified bone biopsy specimens or bone samples, representing an important tool in both clinical assessment of bone health status and research. Fluorochrome markers, such as tetracycline, declomycin, and calcein, are also used to obtain dynamic or kinetic parameters that yield important information on bone formation. The right proximal tibia and first lumbar vertebra were collected from each rat dissected free of surrounding soft tissue, dehydrated in increasing concentrations of ethanol (70 %, 95%, and 100%), and processed undecalcified in modified methyl metacrylate (Baron et al., 1983) The samples were sectioned longitudinally with Jung

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55 2065 and 2165 microtomes (Leica Corp., Rockleigh, NJ) at thicknesses of 4 and 8 m. The thinner secti ons were stained according to the von Kossa method with a tetrachrome counterstain (Polysciences, Warrington, PA), whereas the 8 m thick sections remained unstained for collecti on of fluorochrome based data. Bone measurements were performed in cancellous bone tissue of the proximal tibial metaphysis beginning at a distance of 1 mm from the growth platemetaphyseal junction to exclude the primary spongiosa. The region of interest within the first lumbar vertebral body excluded the primary spongiosa within 0.5 mm of the cranial and caudal growth plates. The bone variables were measured with the Osteomeasure System (Osteometrics, Atlanta, GA) and the Bioquant Bone Morphometry System (R&M Biometrics Corp., Nashville, TN), bonespecific co mputer image analysis systems, and using a Nikon microscope equipped with u ltraviolet lighting. The bone histomorphometric indices were described according to the standardiz ed nomenclature, symbols and units proposed by Parfit t et al. (1987) for the ASBMR (American Society of Bone and Mineral Research) Histomorphometry Nomenclature Committee (Parfi t et al., 1987) and included: 1. Cancellous Bone Volume (BV/TV, %): percent of cancellous bone in the tissue area analyzed. 2. Trabecular Width (Tb.Wi, m): mean width of trabeculae. 3. Trabecular Number (Tb. N, #/mm): number of trabeculae per unit distance. 4. Trabe cular separation (Tb. Sp., m): mean distance between trabeculae. 5. Osteoid Surface (OS/BS, %): percent of bone surface covered by osteoid layer. 6. Osteoblast Surface (Ob.S/BS, %): percent of bone surface with adjacent osteoblasts.

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56 7. Osteoclast Surface (Oc.S/BS, %): percent of bone surface with adjacent osteoclasts. In addition, the following fluorochromebased data were collected from unstained, 8 m thick bone sections with the same quantitative systems described above, to derive the kinetic or dynamic variables of bone formation: 1. Rate of Longitudinal Bone Growth (m/day): rate at which the proximal tibia grows in length each day. 2. Mineralizing Surface (MS/BS, %): percent of bone surface with fluorochrome labels (doublelabeled plus half the singlelabeled), representing active mineralization and bone formation. 3. Mineral Apposition Rate (MAR, m/day): distance between the labels divided by the time interval between their administration This is an index of osteoblastic activity. 4. Bone Formation Rate (BFR /BS 102m3/m2/day): amount of bone formed in unit time per unit of bone surface. It is calculated by multiplication of mineral apposition rate and m ineralizing surface. For the cortical analyses the right tibia from each animal w as dissected free of muscle cu t in half cross sectionally with a handheld saw and placed in 10% phosphatebuffered formalin for 24 hours for tissue fixation, followed by storage in 70% ethanol. The distal halves of the tibiae were dehydrated and defatted in 10 changes of 100% ethanol and 10 changes of acetone (at least 2 hours per change), followed by embedding undecalcified in a styrene monomer that polymerizes into a polyester resin (Tap Plastics, San Jose, CA). The tibial diaphyses were sawed 12 mm proximal to the tibio fibular junction into approximately 100 m thick cross sections with an Isomet low speed saw (Buehler, Lake Bluff, IL). These cross sections of cortical bone were then histomorphometric measurements also performed with t he Osteomeasure System

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57 (Osteometrics, Atlanta, GA). The following structural measurements were performed in one cross section per animal at a magnification of 2X. 1. Cortical Bone Tissue Area (mm2): cortical bone area plus the bone marrow area. 2. Marrow Area (mm2): area of the bone marrow cavity. 3. Cortical Bone Area (mm2): calculated by subtracting the marrow area from cortical bone tissue area. 4. Periosteal Perimeter (mm) : distance around the outer surface of cortical bone. 5. Endocortical Perimeter (mm) : distance around the inner surface of cortical bone. 6. Cortical Width ( m): measured by averaging the distance from the periosteal surface to the endocortical surface at 8 different locations around the bone. Kinetic values for periosteal and endocortical bone formation were also obtained through collection of double and sing le label fluorochromebased data, measured at 200X under UV illumination, as follows: 1. Perios teal Mineralizing Surface (%): calculated as the percent of doublelabeled surface plus one half the percent of single labeled surface. 2. Periosteal Min eral Appositio n Rate (m/day): calculated by dividing the interlabel distance by the time interval between administration of labels (7 days). 3. Periosteal Bone Formation Rate (102 m3/m2/day): calculated by multiplying the mineralizing surface by the mineral apposition rate. 4. Endocortical Mineralizing Surface (%); 5. Endocortical Mineral Apposition Rate (m/day); 6. Endocortical Bone Formation Rate (102 m3/m2/day). Biomechanical Testing An increase in bone mass does not always translate into a decrease in the occurrence of fractures, making it important to also evaluate the mechanical strength of bones. However, the most common tests, such as threepoint bending, 4point bending and torsion, assess the strength of the diaphysis of long bones, at sites where

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58 osteoporotic fractures occur less frequently. For this reason, we performed compression testing of the lumbar vertebral body a more common site for osteoporotic fractures, with an 858 MTS Mini Bionix II machine. At necropsy lumbar vertebrae 3 and 4 were snap frozen, an d stored at 80oC until ready to be tested when they were thawed at room temperature. In order to ensure uniformity between specimens, and to prevent lateral deformation, each vertebral body was cut with parallel edges making sure there was no remaining tissue from intervertebral disks This was accomplished using a custom machined wood block to hold the specimens and an 80/20 (Columbia City, IN) adjustable swing arm with an attached Dremel Multitool (Mount Prospect, IL) for cutting the parallel surfaces. Diamond cutting blades were used for the most precise cut. The vertebrae were irrigated with cooling saline during the cutting process to prevent a change in the molecular structure of the tissue due to heat. After cutting, the height of e ach vertebr a l body was measured with an electronic digital caliper (Fisher Scientific) and photographed with a highresolution digital camera (Nikon Inc., Melville, NY). Afterwards, the images were analyzed with image software (Image J) to obtain the area of the vertebra l body Compression t esting was done with the 858 MTS Mini Bionix II machine (Eden Prairie, MN). Each specimen was placed onto a flat steel plate and then loaded in compression until failure at a rate of 3 mm/sec All failure data were recorded internally by the MTS load cell and then exported to a spreadsheet. Failure was defined by a decrease in detected force on the load cell, or a yield point. Maximum stress was calculated using the area measurements f rom the images captured earlier and the m aximum force recorded by the MTS Stiffness was

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59 calculated using the height measurements and the maximum force recorded by the MTS In summary, the parameters used for biomechanical analysis of the vertebrae were: 1. Load (N) 2. Stress (N*mm2 or MPa) 3. Stiffnes s (N/mm) Statistical Analyses Data are presented as the mean SD for each group, unless stated otherwise. Statistical differences among groups were determined by analysis of variance (ANOVA), followed by a multiple comparison test (Scheffe post hoc anal ysis, StatView SAS Software Institute Inc.). Differences were considered significant at P<0.05. Alternatively, if the data were not normally distributed, statistical differences were evaluated with the non parametric Kruskal Wallis test, which is valid for both normal and nonnormal data distributions. Results Body Weight and Femoral Length Visible phenotypic differences between Lewis and dwarf rats could be observed b efore the beginning of treatment, with Lewis rats showing more increase in body size a nd weight (Figures 21 and 22), as expected. At the time of euthanasia, the mean body weight of the Lewis rats was 52.9 % greater than the dwarf rats (187.6 10.9 g vs. 122.7 5.6 g, P<0.0001) as seen in Figure 23 and their right femurs were 14.8 % l onger (3 2 .32 0.42 mm vs. 27.28 0.32 mm) IGF I Serum Levels Serum IGF I concentration values, calculated in ng/mL, based on the equation of the calibration curve: y= 0.140 + (0.000230 X), as seen in Figure 2 4, revealed that the

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60 serum levels of IGF I were nearly 2.5fold lower in dwarf rats compared with Lewis rats (353.36 153.15 ng/mL vs. 845.22 169.45 ng/mL), as seen in Figure 25. Peripheral Quantitative Computerized Tomography The pQCT analyses of the distal femoral metaphysis revealed that bone structural values for the parameters total BMC, total BMD, trabecular BMC, and trabecular BMD were significantly lower in dwarf rats compared to Lewis rats (P<0.0001), as shown in Figures 26 to 211. All the values obtained at the femoral shafts for total BMC, total BMD, cortical BMC, cortical area, cortical thickness, periosteal a nd endocortical circumferences were also significantly lower in dwarf rats, with the exception of cortical BMD (Figures 212 to 2 19). Histomorphometric Findings Cancellous bone measurements in lumbar vertebrae Cancellous bone volume was significantly lower in the dwarf rats than in the Lewis rats (15.99 4.40 % vs. 26.40 6.44 %, P<0.0001), and this cancellous osteopenia was associated with decreased trabecular number (5.44 0.86 #/mm vs. 6.57 1.01 #/mm, P<0.0053) and width (34.86 5.18 m vs. 48.50 12.30 m, P<0.0012), and increased trabecul ar separation (158.70 29.6 m vs. 115.18 26.28 m, P< 0.0006), as observed in Figures 220 to 223. When considering cancellous bone cellular surface parameters (Figures 224 to 2 26), there w ere no statistically significant differences between the two groups; in fact, the mean values for osteoclast surface, an index of bone resorption, were nearly identical in Lewis and dwarf rats.

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61 Although mean values for osteoblast and osteoid surfaces were not significantly different between the two groups, when analyzing bone kinetic variables (Figures 227 to 2 29), it was found that the cancellous bone mineral apposition rate, an index of o steoblast activity, was significantly lower in dwarf rats compared to Lewis rats (0.88 0.09 m/d vs. 1.07 0.12 m/d, P<0.0001). In addition, the mineralizing surface in the dwarf rats ( 14.03 6.78 %) was also significantly lower than that observed in the Lewis rats (29.96 6.05 %, P<0.0001) F or bone formation rate, the mean value for the dwarf rats (12.15 5.87 102 m3/ 2/d) w as lower (P<0.0001) than tha t of Lewis rats ( 32.37 8.34 102 m3/ 2/d) Cancellous bone measurements in proximal tibiae As observed before, dwarf rats showed lower values for body weight and femoral length, and not surprisingly, tibial longitudinal bone growth was threefold lower in the dwarf rats compared to the Lewis rats (27.29 8.67 m/d vs. 70.88 5.55 m/d, P<0.0001) as seen in Figure 230. The effect of a depressed GH/IGF I axis on cancellous bone structure is even more noticeable in the histologic images of proximal tibial sections from dwarf and Lewis r ats i n Figure 231. Among the cancellous bone structural values for the tibiae (Figures 232 to 235), cancellous bone volume (2.77 1.83 % vs. 19.4 2.75 %, P<0.0001), trabecular number (1.52 0.93 #/mm vs. 6.19 0.90 #/mm, P<0.0001), and trabecular width (21.35 3.0 m vs. 37.99 6.3 m, P<0.0001), were significantly lower in the dwarf rats compared to the Lewis rats, respectively. As a consequence, the trabecular separation values were higher in the dwarf rats than in the control strain (1738.88 2241.08 m vs. 133.26 23.88 m, P<0.016).

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62 Cortical b one measurements in the tibia l diaphysis Dwarf mutations effects could also be observed in the left tibial diaphyses, as sho wn in Figures 2 3 6 to 2 4 1 Dwarf rats cortical bone structural measurements were significantly lower than those of Lewis rats, including the total bone tissue area (2.49 0.16 mm2 vs. 3.67 0.27 mm2, P<0.0001), cortical area (1.95 0.10 mm2 vs. 3.04 0.20 mm2, P<0.0001), marrow area (0.54 0.08 mm2 vs. 0.63 0.10 mm2, P<0.02), periosteal perimeter (5.86 0.17 mm vs. 7.06 0.30 mm, P<0.0001), endocortical perimeter (2.69 0.20 mm vs. 2.89 0.24 mm, P<0.044 ) and cortical width (504.55 27.61 m vs. 669.75 30.92 m, P<0.0001). Regarding cortical bone kinetic indices, the periosteal mineralizing surface (49.44 17.79 % vs. 84.02 25.25 %, P<0.0007), periosteal mineral apposition rate (1.15 0.30 m /d vs. 2.40 0.52 m /d, P<0.0001) and per iosteal bone formation rate (60.38 31.68 102 m3/ 2/d vs. 211.41 73.48, P<0.0001) as well as the endocortical mineralizing surface (50.52 19.77 % vs. 82.29 21.95 %, P<0.0015), endocortical mineral apposition rate (0.83 0.18 m /d vs. 1.49 0.5 5 m /d, P<0.0008), and endocortical bone formation rate (42.82 19.56 102 m3/ 2/d vs. 128.94 59.64 102 m3/ 2/d P<0.0001), were all significantly lower in d warf rats than in Lewis rats, respectively as shown in Figures 24 2 to 2 4 7 Biomechanical Testing The area s of the lumbar vertebrae w ere measured in the obtained photographs with Image J software as shown in Figures 2 48 and 249. These values (not shown) were used only for calculation of the stress parameter, and the heights of the vertebrae (data not shown) were used only to calculate stiffness Biomechanical load values were significant ly lower in dwarf rats compared to Lewis rats ( P = 0.038), but no differences

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63 w ere observed for biomechanical stress and stiffness between the two groups, as seen in Figures 24 9 to 2 51. Discussion IGF I Serum L evels The hypophysectomized rat was considered, at first, the classical model to study GH actions and deficiencies, exhibiting IGF I serum levels 85% lower than intact animals. The dwarf rats presented a 60% reduction in the IGF I serum levels when compared to Lewis rats O bviously the effect was not as dramatic as that observed in the hypophysectomized rats. However, in addition to GH, h ypophysectomized rats lack all other hormones synthesized by t he hypophysis, that also act on growth and bone metabolism. Due to this abrogation of other bone acting hormones and related growth factors, it is difficult to interpret data obtained from hypophysectomized rats Another drawback to be considered is the possibility of remaining hypophyseal tissue in the sella turcica Thorngre n et al. screened the completeness of hypophysectomy through serial microscopic sections of the sella turcica and measurements of longitudinal bone growth by tetracycline label ing 15 days after surgery (Thor ngren et al., 1980) The longitudinal bone growth parameters were correlated with hypophyseal tissue re mnants in the sella turcica (Thorngren et al., 1980) In contrast the recessive mutation of the dwarf rats results in partial deficiency in GH production, but with normal levels of the other trophic hormones such as luteinizing hormone (LH ), thyroid stimulating hormone (TSH), prolactin (PRL) and adrenocorticotropic hormone (ACTH) (Skottner et al., 1989) Therefore, the dwarf rat has fewer variables in systemic hormone circulation to potentially complicate data interpretation.

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64 The circulating IGF I values obtained for the dwarf rats were also higher than those expressed by LID mice (75% reduction ) and by IGF I knockout mice, whose levels are at or even below detection levels (22 ng/ mL). However, as mentioned before, only 5 % of the IGF I knockout mice survive up to four months (Powell Braxton et al., 1993) LID mice, and other transgenic strains developed to eluc idate the contributions of local and systemic IGF I, are obviously a great tool in terms of elucidating the molecular pathways involved in the GH/IGF I axis, but th e markedly higher serum GH levels in the LID mice may complicate data interpretation. The he terozygous (HET) mice have their IGF I serum levels reduced by only 2030% when compared to their wildtype littermates (He et al., 2006) a reduction that may be considered too small to compare to the 54 % decreased IGF I serum levels found in patients with anorexia nervosa or the 73% drop in IGF I serum levels in children with type I diabetes mellitus (Gianotti et al., 1998, Grinspoon et al., 1999) Body Size and D evelopment When analyzing the development and body size of Lewis and dwarf rats through their weight and femoral length, we verified that the body weight of Lewis rats was 52.9% great er than that of the dwarf rats, and their left femurs were 14.8% longer compared to the dwarf rats. These results were comparable to other rodent models of IGF I deficiency but were no t evident at birth; in fact, the observed changes were noticed after 45 weeks of age demonstrating that postnatal skeletal development and bone growth were clearly affected by the dwarf mutation. In contrast, IGF I knockout mice were born at 60% of normal weight, and postnatal growth curves indicated that the surviving IGF I ( / ) mutants continued to grow with a retarded rate after birth in comparison to their controls and were 30% of normal weight as adults (Baker et al.,

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65 1993) When knockout mice were first described in 1993 by Baker, Liu, and Powell Braxton, 80 to 95% lethality was reported. Liu (1993) reported that 10 to 68% of IGF I ( / ) mutants survived, and attributed the difference in survival rates to the genetic background, whereas Powell Braxton (1993) considered the position where the IGF I gene was interrupted as the possible source of difference (Liu et al., 1993, Powell Braxton et al., 1993) Bikle (2001) compared the skeletal structure of IGF I deficient, heterozygous and homozygous, mice to their wildtype controls, and by 12 weeks o f age, female and male IGF I knockout mice were approximately 30 and 20%, respectively, of the weight of their wildtype controls (Bikle et al., 2001) Recently, i n a comprehensive study about IGF I mutant mouse models Yakar (2010) considered total inactivation of the IGF I gene to result in 80% perinatal lethality, wit h surviving pups 50% smaller than the wildtype controls (Yakar et al., 2010) Peripheral Quantitative Computerized Tomography Osteopenia in human patients routinely is evaluated by measurem ents of bone mineral density by the DXA method, and is confirmed when values are under 2.5 standard deviations from normal. In this research project the pQCT parameters for Lewis rats were considered normal for evaluation purposes. There was a clear ost eopenic condition in dwarf rats revealed by the pQCT data The total and trabecular cancellous bone mineral density were significantly decreased in the distal fem oral metaphysis, as well as reduced values for bone mineral content (p<0.0001). The low BMD values in femoral cancellous bone in dwarf rats (12%) could be compared to those observed in young heterozygous mice (712 % ) (Mohan and Baylink, 2005, He et al., 2006)

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66 The only parameter that was not significantl y lower in the dwarf rats was cortical bone mineral density. A possible explanation for this observation comes from the fact that the animals in our study were young, and bone modeling related to growth and changes i n bone shape due to mechanical loading were still occurring. Since Wolffs law first introduced the concept that every change in the form and function of a bone, or its function alone, is followed by certain definite changes in its external conformation (Stedman, 1990) several studies examined the various aspects of bone responses to loading in anatomical studies of paleontological skeletal remains (Pearson and Lieberman, 2004, Ruff, 2005) in vitro (Klein Nulend et al., 1995, Mullender et al., 2004) and in vivo m ainly in clinical studies Most of the se clinical studies support the general conclusion that cortical bone responses to loading occur primarily in the juveni le state leading to macroscopic effects on long bone cross sections and increased bone density, and that there are agerelated differences in the ability of bone to r espon d to exercise and load (Prior et al., 1996, Ruff, 2003, Pearson and Lieberman, 2004, Greene and Naughton, 2006, Judex et al., 2008) The majority of cells in cortical bone are osteocytes. Although the molecular mechanisms and pathways involved in mechanosensation and mechanotra nsduction are complex and not yet identified, osteocytes are considered mechanosensors of bone (Klein Nulend et al., 1995, Judex et al., 2008, KleinNulend and Bonewald, 2008) In addition, cortical remodeling would not yet be taki ng place during this experiment, as the transition time from modeling to remodeling in the rat skeleton would be around 3 to 6 months for endocortical bone, in the lumbar vertebra, and 9 to 12 months in the proximal tibial metaphysis (Lelovas et al., 2008)

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67 Bone H istomorphometry and Biomechanics The histomorphometric findings were consistent with the pQCT data The lower values in dwarf rats for total BMD an d trabecular BMD in the distal femoral metaphysis were fully consistent with the low cancellous bone volume, decreased trabecular number and width (p<0.0001), and increased trabecular separation (p<0.0001) observed in the structural analysis of histologic sections of the proximal tibial metaphysis and lumbar vertebral body. The lower vertebral bone mass resulted in decreased bone strength in dwarf rats, as indicated by significantly lower load to failure in the lumbar vertebral body of these IGF I deficien t animals Although bone cellular surface parameters (osteoblast and osteoclast surfaces) did not show significant differences between Lewis and dwarf rats, dynamic indices of bone formation at the cancellous, periosteal, and endocortical bone surfaces were all significantly decreased in dwarf rats compared to Lewis rats. These findings suggest that the osteopenia detected in dwarf rats is due primarily to an inhibition of bone formation. Since osteoclast surface was nearly identical in dwarf and Lewis r ats, bone resorption did not appear to be affected by IGF I deficiency. Yet, the dwarf rat expressed an evident osteopenic phenotype, characterizing it as a good model to reproduce the observed osteopenia in IGF I deficiency related conditions. Conclusions The results addressed S pecific Aim 1, and the major findings were: 1. The dwarf rat expressed significantly decreased IGF I serum levels compared to its back ground strain, representing a major reduction of 60 % in circulating IGF I. Therefore, the dwarf r at is a reliable model to study the physiological changes seen in IGF I deficienc y 2. GH/IGF I deficiency in the dwarf rat has profound negative effects on bone growth, accumulation of bone mass, bone strength, and bone formation in both

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68 cortical and cancell ous bone. For this reason, it is a valid animal model to study the osteopenic effects caused by this deficiency on bone structure and metabolism 3. Since IGF I may mediate the skeletal effects of bone anabolic agents, the dwarf rat is a lso a promising animal model for studies of these interactions.

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69 Figure 2 1 The two strains of Lewis (right) and dwarf (left) r ats during their clinical evaluation. Photo courtesy of Dr. Ana Cristina F. Bassit Figure 2 2 Dwarf rat (right ), showing inferior size and overall development when compared to Lewis rat (left) Photo courtesy of Dr. Ana Cristina F. Bassit

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70 Figure 23. Body weight (g). There was a statistically significant difference between Lewis and dwarf rats (P<0.0001) 0 25 50 75 100 125 150 175 200 225 Body Weight (g) Dwarf Lewis VEH

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71 Figure 24 IGF I Serum Levels. The calibration curve and slope equation for obtaining the concentration levels for IGF I in ng/mL Figure 25. Serum IGF I (ng/mL). Dwarf rats showed IGF I serum levels significantly decreased when compared to Lewis rats (P<0.0001) y = 0.0002x + 0.1401 R = 0.9858 0 0.2 0.4 0.6 0.8 1 1.2 0 1000 2000 3000 4000 5000Absorbance (nm) Concentration (ng/ml) 0 200 400 600 800 1000 1200Serum IGF-I (ng/mL) Dwarf Lewis VEH

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72 Figur e 26. Total Bone Mineral Content (BMC) at distal femoral metaphysis (mg/mm). Mean value for dwarf rats was significantly lower than for Lewis rats (P<0.0001) Figure 27. Total Bone Mineral Density (BMD) at distal femoral metaphysis ( mg/cm3). A s i g nificant decrease was observed in dwarf rats (P<0.0001) 0 2 4 6 8 10 12Total BMC (mg/mm) Dwarf Lewis VEH 0 100 200 300 400 500 600 700 800Total BMD (mg/cm3) Dwarf Lewis VEH

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73 Figure 28. Trabecular BMC (mg/mm). A remarkable difference was noted between Lewis and dwarf rats (P<0.0001) Figure 29. Trabecular BMD ( mg/cm3). A highly significant difference was observed between Lewis and dwarf rats (P<0.0001) 0 .25 .5 .75 1 1.25 1.5 1.75 2 2.25Trabecular BMC (mg/mm) Dwarf Lewis VEH 0 50 100 150 200 250 300 350 400 450 500Trabecular BMD (mg/cm3) Dwarf Lewis VEH

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74 Figure 210. Total Area for Cancellous Bone (mm2). The mean value was significantly lower for dwarf rats compared to Lewis rats (P<0.0001) Figure 211. Trabecular Area (mm2). A significant diffe rence between Lewis and dwarf rats was observed with P< 0.001 0 2 4 6 8 10 12 14 16 18Total Area Cancellous Bone (mm2) Dwarf Lewis VEH 0 1 2 3 4 5 6Trabecular Area (mm2) Dwarf Lewis VEH

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75 Figure 212. Total BMC at femoral diaphysis (mg/mm). Mean value for dwarf rats was significantly lower than for Lewis rats (P<0.0001) Figure 213. Total BMD at femoral diaphysis ( mg/cm3). A significant difference between Lewis and dwarf rats was observed (P<0.0009) 0 1 2 3 4 5 6 7 8Total BMCCortical Bone (mg/mm) Dwarf Lewis VEH 0 100 200 300 400 500 600 700 800 900 1000Total BMD Cortical Bone (mg/cm3) Dwarf Lewis VEH

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76 Figure 214. Cortical BMC (mg/mm). A significant decrease was observed in dwarf rats compared to Lewis rats (P<0.0001) Figure 215. Cortical BMD ( mg/cm3). This was the only pQCT parameter that did not show a significant difference between Lewis and dwarf rats 0 1 2 3 4 5 6 7 8Cortical BMC (ng/mm) Dwarf Lewis VEH 0 200 400 600 800 1000 1200 1400Cortical BMD (mg/cm3) Dwarf Lewis VEH

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77 Figure 216. Cortical Area (mm2). Significant decrease in dwarf rats compared to Lewis rats (p<0.0001) Figure 217. Cortical Thickne ss (mm). Dwarf rats showed a significant ly lower mean va lue than Lewis rats (P<0.0001) 0 1 2 3 4 5 6Cortical Area (mm2) Dwarf Lewis 0 .1 .2 .3 .4 .5 .6 .7 .8Cortical Thickness (mm) Dwarf Lewis VEH

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78 Figure 218. Periosteal Circumference (mm) A significant difference (P<0.0001) was observed between Lewis and dwarf rats Figure 219. Endosteal Circumfe rence (mm). Dwarf rats showed a significantly lower mean value compared to Lewis rats (P<0.0001) 0 2 4 6 8 10 12Periosteal Circumference (mm) Dwarf Lewis VEH 0 1 2 3 4 5 6 7Endocortical Circumference (mm) Dwarf Lewis VEH

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79 Figure 220. Vertebral Cancellous Bone Volume (%). A s ignificant ly lower mean value w as observed in dwarf rats (P<0.0001) Figure 221. Vertebra l Trabecular Number (#/mm). Dwarf rats showed a significant ly lower mean value, with P<0.005 0 5 10 15 20 25 30 35Vert. Cancellous Bone Volume (%) Dwarf Lewis VEH 0 1 2 3 4 5 6 7 8 Trabecular number (#/mm) Dwarf Lewis VEH

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80 Figure 222. Vertebral Trabecular Width ( A significant difference between Lewis and dwarf rats was observed, with P<0.001 Figure 223. Vertebral Trabecular S eparation ( Dwarf rats exhibited a significant increase compared to Lewis rats (P<0.0006) 0 10 20 30 40 50 60 70Trabecullar Width (um) Dwarf Lewis VEH 0 20 40 60 80 100 120 140 160 180 200 Trabecular Separation (um) Dwarf Lewis VEH

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81 Figure 224. Vertebral Oste oid Surface (%). Dwarf rats did not show a significant difference compared to Lewis rats Figure 225. Vertebral Osteoblast Surface (%). There was no significant difference between dwarf and Lewis rats 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5Osteoid Surface (%) Dwarf Lewis VEH 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25Osteoblast Surface (%) Dwarf Lewis VEH

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82 Fig ure 226. Vertebral Osteoclast S urface (% ). There was no significant difference between dwarf and Lewis rats Figure 227. A s ignificantly lower mean value w as observed in dwarf rats compared to Lewis rats. (P<0.0001) 0 .2 .4 .6 .8 1 1.2 1.4Osteoclast Surface (%) Dwarf Lewis VEH 0 .2 .4 .6 .8 1 1.2 1.4Vert. Mineral Apposition Rate (um/d) Dwarf Lewis VEH

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83 Figure 228. Vertebral Mineralizing Surface (%). Dwarf rats showed a significantly decreased mean value compared to Lewis rats (p<0.0001) Figure 229. Vertebral Bone Formation Rate (1023 m2/d). Mean value w as significantly decreased in dwarf rats compared to Lewis rats (p<0.0001) 0 5 10 15 20 25 30 35 40Vert. Mineralizing Surface (%) Dwarf Lewis VEH 0 5 10 15 20 25 30 35 40 45Vert. BFR/BS (10-2um3/um2/d) Dwarf Lewis VEH

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84 Figure 230. Tibial Longitudinal Bone Growth ( m/d). Dw arf rats presented a significantly lower mean value in comparison to Lewis rats (p<0.0001) A B Figure 231. Proximal tibial metaphyses from Dwarf (A) and Lewis (B) rats. Note the reduced mass of black stained bone indicative of cancellous osteopenia in the Dwarf rat. Von Kossa/ tetrachrome stain, X40 Photos courtesy of Dr. Thomas J.Wronski 0 10 20 30 40 50 60 70 80Tibial Longitudinal Bone Growth (um/d) Dwarf Lewis VEH

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85 Figure 232. Tibial Cancellous Bone Volume (%). Mean value was markedly lower in dwarf rats than in Lewis rats (P<0.0001) Figure 233. Tibial Trab ecular Number (#/mm). Dwarf rats showed a lower mean value than Lewis rats with P<0.0001 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25Tibial Cancellous Bone Vollume (%) Dwarf Lewis VEH 0 1 2 3 4 5 6 7 8Tibial Trabecular Number (#/mm) Dwarf Lewis VEH

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86 Figure 234. Tibial Trabecular Width ( m). Dwarf rats exhibited a significantly lower mean value than Lewis rats, with P<0.0001 Figure 235. Tibial Trab ecular Separation ( m). The mean value for dwarf rats was significantly higher than that for Lewis rats, with P<0.016 0 5 10 15 20 25 30 35 40 45 50TIbial Trabecular Width(um) Dwarf Lewis VEH 0 500 1000 1500 2000 2500 3000 3500 4000 4500Tibial Trabecular Separation (um) Dwarf Lewis VEH

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87 Figure 236. Total Cortical Bone Tissue Area (mm2). The mean value was markedly lower in dwarf rats than in Lewis rats with P<0.0001 Figure 237. Cortical Bone Area (mm2). Mean value was significantly lower in dwarf rats than in Lewis rats (P<0.0001) 0 .5 1 1.5 2 2.5 3 3.5 4 4.5Total Cort. Bone Tissue Area (mm2) Dwarf Lewis VEH 0 .5 1 1.5 2 2.5 3 3.5Cortical Bone Area (mm2) Dwarf Lewis VEH

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88 Figure 238. Marrow Area (mm2). Dwarf rats showed a lower mean value compared to Lewis rats (P<0.02) Figure 239. Periosteal Perimeter (mm). Dwarf rats presented a significantly lower mean value than Lewis rats (P<0.02) 0 .1 .2 .3 .4 .5 .6 .7 .8 Marrow Area (mm2) Dwarf Lewis VEH 0 1 2 3 4 5 6 7 8 Periosteal Perimeter (mm) Dwarf Lewis VEH

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89 Figure 240. Endocortical P erimeter (mm). Mean value w as significantly lower in dwarf rats than in Lewis rats (P<0.0 4 ) Figure 24 1. Cortical Width ( m). The mean value for dwarf rats was significantly lower than that of Lewis rats, with P<0.0 001 0 .5 1 1.5 2 2.5 3 3.5Endocortical Perimeter (mm) Dwarf Lewis VEH 0 100 200 300 400 500 600 700 800Cortical Width (um) Dwarf Lewis VEH

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90 Figure 242. Periosteal Mineralizing Surface (%). Dwarf rats showed a significantly lower mean value than Lewis rats, with P<0.0 0 07 Figure 243. Periosteal Mineral Apposition Rate ( m/d ). Mean value was significantly lower in dwarf rats than in Lewis rats (P< 0.0 001) 0 5 10 15 20 25 30 35 40Periosteal Mineralizing Surface (%) Dwarf Lewis VEH 0 .5 1 1.5 2 2.5 3Periosteal Mineral Apposition Rate (um/d) Dwarf Lewis VEH

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91 Figure 244. Periosteal Bone Formation Rate ( 102m3/m2/d ). Dwarf rats showed a lower mean value compared to Lewis rats (P< 0.0 001 ) Figure 245. Endocortical Mineralizing Surface ( % ). A s ignificant ly lower mean value w as observed in dwarf rats compared to Lewis rats (P< 0.0 015) 0 50 100 150 200 250 300Periosteal BFR/BS (10-2um3/um2/d) Dwarf Lewis VEH 0 20 40 60 80 100 120Endocortical Mineralizing Surface(%) Dwarf Lewis VEH

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92 Figure 246. Endocortical Mineral Apposition Rate ( ). D warf rats presented a significantly lower mean value than Lewis rats (P< 0.0 008 ) Figure 247. Endocortical Bone Formation Rate ( 102m3/m2/d ). D warf rats presented a significantly lower mean value than Lewis rats (P< 0.0 001 ) 0 .25 .5 .75 1 1.25 1.5 1.75 2 2.25Endocortical MAR (um/d) Dwarf Lewis VEH 0 20 40 60 80 100 120 140 160 180 200Endocortical BFR/BS (10-2um3/um2/d) Dwarf Lewis VEH

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93 Figure 248. Images of lumbar vertebral body obtained for area analysis with Image J software Photo courtesy of Dan Barousse

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94 Figure 249. Biomechanical Load (N). Dwarf rats showed a significant ly lower mean value compared to Lewis rats (P<0.03) Figu re 2 50. Biomechanical Stress (N*mm2. or MPa) There was no significant difference between dwarf and Lewis rats 0 50 100 150 200 250 300 Load (N) Dwarf Lewis VEH 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25Stress (N*mm2 or MPa) Dwarf Lewis VEH

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95 Figure 251. Biomechanical Stiffness (N/mm). No significant difference was observed between dwarf and Lewis rats 0 200 400 600 800 1000 1200Stiffness (N/mm) Dwarf Lewis VEH

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96 CHAPTER 3 THE BONE ANAB OLIC EFFECTS OF PTH TREAT MENT IN GH/IGF I DEFICIENT DWARF RATS AND THEIR BACKGROUND STRAIN, L EWIS RATS. Introduction Parathyroid hormone ( PTH ) is an 84 amino acid polypeptide synthesized by parathyroid gland cells, and highly conserved among mammalian spec ies It induces b one anabolic effects when administered intermittently at low doses (Dempster et al., 1993, Canalis et al., 2007, Jilka, 2007) Teriparatide [(PTH (134)] is the synthetic or recombinant segment o f human parathyroid hormone, amino acid sequence 1 through 34, of the complete molecule containing 84 amino acids, and has the same anabolic properties as PTH in humans (Bilezikian and Rubin, 2006) and rats (Mosekilde et al., 1991) The full length PTH (184) is available only in Europe, while the foreshortened aminoterminal form is available and approved in the United States by the Food and Drug Administration (FDA) since November 2002, for the treatment of osteoporosis in postmenopausal women and men at high risk for fractures. In this condition are included patients with a T score determined by dual energy x ray ab sorptiometry (DXA) lower than 3.0, patients with glucocorticoidinduced osteoporosis (GIOP), osteoporotic patients resistant to other available treatments and patients with previous fragility fracture s. The indications for PTH therapy are restricted to these specific conditions mainly because of the high cost of treatment and the need for daily subcutaneous administration (Canalis et al., 2007) PTH is the most important endocrine regulator in calcium homeostasis in mammals, involving different receptors and multiple signal ing mechanisms (Kousteni and Bilezikian, 2008) Similar to other peptide hormones, PTH effects are mediated through a G proteincoupled receptor system in the cells of targeted tissues that

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97 promotes the activation of intracellular events and various biochemical pathways. Three PTH receptors, PTHR1, PTHR2, and PTHR3, have been identified, but the majority of the physiological actions of PTH are mediated by PTHR1 (Molina, 2006b) Although PTHR1 is primarily and abundantly present in osteoblasts, it is also expressed by osteocytes and bone lining cells (Dempster et al., 1993, Swarthout et al., 2002, Poole and Reeve, 200 5, Keller and Kneissel, 2005) Intermittent PTH promote s osteoblastogenesis and increases the number of osteoblasts by regulating the ir proliferation, differentiation and survival (Dempster et al., 1993, Canalis et al., 2007, Jilka, 2007, Jilka et al., 2009, Jilka et al., 2010) PTH stimulates the response of osteocytes to mechanical strain and shear forces, and preserves osteocyte viability through anti apoptotic actions (Bilezikian and Rubin, 2006, Kousteni and Bilezikian, 2008) Additionally, PTH regulates the production of sclerostin (SOST), an osteocy te produced antagonist of bone formation, by inhibiting SOST expression in vitro and in vivo (Keller and Kneissel, 2005, Poole and Reeve, 2005) Apparently the effects o n bone resorption are more likely to be indirect, upregulating RANKL (receptor activator of nuclear factor ligand) and the expression of osteoprotegerin (OPG), which activates and inhibits osteoclastogenesis (Huang et al., 2004, Bilezikian and Rubin, 2006, Kousteni and Bilezikian, 2008) PTH related protein (PTHrP) is related in function and structure to PTH, sharing 13 amino acids with the aminoterminal of the hormone, and binds to one of the PTH receptors, PTHR1. Therefore, PTHrP can induce the same e ffects as PTH in bone and kidney. PTH or PTHrP binding to the receptor stimulates the synthesis of cAMP and activation of protein kinase A and the phosphor y lation of targeted proteins, that once

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98 activated, lead to the induction of gene transcription. A ct ivation of the receptor can also involve additional signaling pathways, activating phospholipase C. These two pathways regulate some osteoblast functions, although the exact signaling pathway and the in vivo response to PTH are still not completely determ ined, and may explain whether the hormone has catabolic or anabolic effects in the skeleton (Bilezikian and Rubin, 2006, Canalis et al., 2007) PTH treatment has been evaluated in many different animal models and pathological conditions, such as the ovariectomized rat model for postmenopausal bone loss (Wronski et al., 1993, Wronski and Yen, 1994) fracture healing (Nozaka et al., 2008, Barnes et al., 2008) and in many other genet ically modified rodents models, such as IGF I knockout mice (Bikle et al., 2002) mice with deletion of the genes for insulin receptor substrate 1 and 2 (IRS.1 / and IRS.2/ ) (Yamaguchi et al., 2005) and mice with IGF I receptor null mutation (Wang et al., 2007) In order to evaluate the influence of the components of the ternary IGF I complex on the response to PTH treatment Yakar et. al. (2006) generated three types of mutant mice: with deletion of the liver specific IGF I gene (LID mice), with global deletion of the acidlabile subunit (ALS) gene ( ALSKO mice), and with both li ver and ALS inactivated genes ( LA mice ) (Yakar et al., 2006) In general these studies showed that IGF I is essential for PTH to induce bone anabolic effects. Nevertheless, the mechanisms for the skeletal response to PTH are not completely understood, and the role of I GF I as a potential mediator for the bone anabolic effects of PTH is still controversial (Jilka, 2007, Bikle, 2008)

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99 Materials and Methods Animal Models In the first experiment, dwarf rats expressed significantly decreased IGF I serum levels compared to their back ground strain, Lewis rats, which supported their use as a reliable model to reproduce the physiological changes seen in IGF I deficienc y Therefore, the dwarf and Lewis strains of rats were used to evaluate the skeletal response to PTH treatment. Based on the same criteria observed for the animals from the first experiment, fiveweeks old female dwarf and Lewis rats, obtained from Harlan Laboratories (UK ), were kept in a temperature and humidity controlled environment (25 C), and on a 12h light/12h dark cycle. The experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida. Experimental Design Fo llowing the first analysis to establish the dwarf rat as an animal model for our studies we test ed the hypothesis that IGF I acts as a potential mediator for the bone anabolic effects of PTH The four groups (N=13, for vehicletreated dwarf and Lewis rats, and N=710 for PTH treated dwarf and Lewis rats) were ma intained in the same nutritional and environmental conditions until 9 weeks of age, when treatment started. They were routinely evaluated and weighed once a week, for monitoring their health status. Vehicle and PTH Treatment The rats from the first experi ment, treated with vehicle solution (VEH), formed the control groups, for comparison with the PTH treated dwarf and Lewis groups. Preparation of t he vehicle solution was described above for experiment one The PTH solution was prepared as follows:

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100 1. H eat i nactivated serum was used to prepare the PTH stock solution with human synthetic PTH 1 34 hPTH (Bachem Laboratory, Torrance, CA), according to the respective strain. S erum from Lewis rats was used to prepare the PTH stock solution for Lewis rats, and in the same way, we used serum from dwarf rats for the preparation of the PTH stock solution to treat dwarf rats. 2. The PTH concentration was adjusted to 1 mg/mL, by adding vehicle. 3. The rats were injected, subcutaneously, daily for 2 weeks with hPTH 134 at a d ose of 50 g/kg body weight Bone F ormation M arkers We used the same markers as for experiment one, to be consisten t with the methodology All rats received subcutaneous injections of fluorochrome compounds, declomycin and calcein (Sigma Chemical Co., St. Louis, MO ) at a dose of 15 mg/ kg of body weight, ten and three days prior to euthanasia respectively, in order to label actively forming bone surfaces. Following these initial steps, the methods are the same as described in C hapter 2 ( pages 3441), including necropsy procedures, tissue processing, data collection and statistical analysis Results Body Weight and Femoral Length The evident phenotypic differences between Lewis and dwarf rats remained proportionally the same after treatment with PTH. At the time of e uthanasia, the mean body weight s between the Lewis and the dwarf rats w ere significantly different, as detected in the previous experiment but not between the VEH and PTH treated rats, regardless if the rats were in the Lewis (194.5 12.91g vs. 203 13.5g) or dwarf groups (126.5 6.8g vs. 121.9 8.2g), as seen in Figure 3 1.

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101 Femur length values were decreased in dwarf rats, when compared to Lewis rats but no significant difference was observed between VEH and PTH treated dwarf rats (27.28 0.32mm v s. 27.64 0.67mm). PTH treated Lewis rats showed a slight, but significant increase in mean femur length compared to VEH treated Lewis rats (32.13 0.63mm vs. 31.19 0.51mm, respectively, P<0.00 26) as seen in Figure 32. IGF I Serum Levels Serum levels of IGF I were markedly lower in VEH treated dwarf rats compared with VEH treated Lewis rats (353.36 153.15 ng/mL vs. 845.22 169.45 ng/mL, P<0.0001), as seen in experiment 1. R egardless of treatment there were no significant differences in IGF I seru m levels in either PTH treated Lewis rats compared to their VEH treated controls (755.00 202.00 ng/mL vs. 845.22 169.45 ng/mL ), or in PTH treated dwarf rats compared to their VEH treated controls ( 265.53 95.28 ng/mL vs. 353.36 153.15 ng/mL), as sho wn in Figure 33. Peripheral Quantitative Computerized Tomography The pQCT analyses of the distal femoral metaphysis revealed that cancellous bone structural parameters (total BMC, total BMD, trabecular BMC, and trabecular BMD) were significantly higher in PTH treated dwarf and Lewis rats, when compared to vehicle treated rats, as shown in Figures 34 to 39 The trabecular density measurements demonstrated a significant interaction between group and treatment. Post hoc analysis found that PTH treated dwarf rats had a greater increase in trabecular BMD when compared to PTH treated Lewis rats, similar to the results obtained for longitudinal bone growth (see below) When considering cortical bone parameters ( Figures 310 to 3 17) almost all values obtained at the femoral shafts for total BMC, total BMD, cortical BMC, cortical

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102 area, cortical thickness, periosteal and endocortical circumferences did not show any PTH tr eatment effect s in either group, with the exception of cortical BMD which showed a slight, but significant decrease in PTH treated dwarf rats (P=0.0013). Histomorphometric Findings Cancellous bone measurements in lumbar vertebrae PTH significantly increased vertebral cancellous bone volume in both dwarf (25.16.7% vs. 15.94.4%, P = 0.0016) and L ewis rats (37.06.4% vs. 26.46.4%, P<0.0001) when compared to vehicletreated rats, as shown in Figure 318 and this effect was associated with increased trabecular width in PTH treated dwarf (P=0.0229) and Lewis (P=0.0320) rats. There was a slight, but non significant increase in trabecular number between PTH and vehicletreated rats in both groups, and a strong trend for decreased trabecular separation in PTH treated dwarf (P=0.0567) and Lewis rats (P=0.063), as seen in Figures 319 to 321. PTH treatm ent increased cancellous bone surface parameters osteoid and osteoblast surfaces to the same extent in both dwarf and Lewis rats. There was no significant difference in the percentage increase between the two strains but a highly significant increase when PTH treated dwarf and Lewis rats were compared to their vehicle treated controls, as seen in Figures 3 22 and 3 2 3 The osteoclast surface was significantly increased only in PTH treated dwarf rats (Figure 324). When analyzing bone kinetic variables we found that PTH treated dwarf rats exhibited 7and 13fold increases in mineralizing surface and bone formation rate, respectively, compared to vehicletreated dwarf rats. PTH treated Lewis rats showed 3and 4fold increases in mineralizing surface and bone formation rate compared to vehicle treated Lewis rats, as seen in Figures 325 and 32 6 Cancellous bone mineral

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103 apposition rate, an index of osteoblast activity, was increased i n PTH treated dwarf rats PTH treated Lewis rats (1.350.08 vehicle treated groups (Figure 3 27). Cancellous bone measurements in proximal tibiae Wh en compared to vehicle treatment t ibial longitudinal bone growth was significantly higher in both PTH treated groups of dwarf rats (27.29 8.67 m/d vs. 72.79 6.11 m/d, P<0.0001) and Lewis rats (70.88 5.55 m/d vs. 82.27 12.66 m/d, P = 0.0 321 ) as seen in Figure 328 The effect of PTH treatment on tibial cancellous bone structure is even more noticeable in the histologic images from vehicle and PTH treated dwarf rats i n Figure 3 29 Among the cancellous bone structural values for the proximal ti bia, we observed the same positive effect of PTH on tibial cancell ous bone volume, in both dwarf (2.78 1.83 % vs. 8.78 2.78 % P = 0.00 16 ) and Lewis rats ( 19.36 2.75 % vs. 24.58 4.33% P<0.00 23) (Figure 3 30). The effect of PTH on bone volume was asso ciated with increased trabecular number in dwarf rats (1.52 0.93 #/mm vs. 3.48 0.88 #/mm, P = 0.0001) but not in Lewis rats (6.19 0.90 #/mm vs. 6.82 0.30 #/mm P = 0. 3484) (Figure 3 31), and with increased trabecular width only in dwarf rats ( 21.35 3.0 m vs. 29.96 2.21 m, P = 0.0 183) (Figure 3 32) In addition no significant differences were observed in trabecular separation in dwarf and Lewis rats treated with PTH (Figure 3 33) although a strong trend for an increase in this parameter was observ ed in the former animals Cortical b one measurements in tibia l diaphysis The effects of PTH treatment on cortical bone were observed in the analysis of the left tibial diaphyses, as shown in Figures 3 3 4 to 3 39. The total cortical bone tissue

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104 area, corti cal area, marrow area, periosteal perimeter, and endocortical perimeter showed no significant effects of PTH treatment either in dwarf or Lewis rats However, cortical width was slightly but significant ly decreased in PTH treated dwarf rats (P = 0.0 116). A ll the dynamic measurements at the tibial diaphysis, periosteal and endocortical mineralizing surface, mineral apposition rate and bone formation rate were markedly increased in PTH treated dwarf rats PTH treated Lewis rats showed significantly increased periosteal mineral apposition and bone formation rates (P=0.0072 and P=0.0026, respectively) as seen in Figures 340 to 345. Biomechanical Testing in Lumbar Vertebral Body The biomechanical parameter of stress in the lumbar vertebrae of PTH treated dwa rf rats w as increased when compared to vehicletreated dwarf rats (P = 0.0 115 ). The same increase was not observed in PTH treated Lewis rats (P = 0.0 818 ). PTH treatment did not cause a significant increase in dwarf rats in the other parameters, load and sti ffness but PTH treated Lewis rats showed a significant increase in load, with P = 0.0004 as seen in Figures 346 to 348. Discussion Our findings indicate that PTH stimulates bone formation and increases cancellous bone mass in dwarf rats with low serum levels of IGF I. These results may be considered to contrast with the majority of studies that postulate IGF I as an essential mediator for the bone anabolic effects of PTH, since initial in vitro experiments indicated that PTH stimulate s IGF I release from calvarial osteoblasts (Linkhart et al., 1989) and that IGF I antibody inhibits PTH induced stimulation of collagen synthesis in bone cultures (Canalis et al., 1989) Furthermore, PTH has been report ed to participate in

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105 the regulation of local and serum IGF I levels and to stimulate IGF I synthesis by osteoblasts (Dempster et al., 1993, He et al., 2006) Bone mass and bone matrix IGF I increased in rats treat ed with PTH, but there w as no effect on serum IGF I levels, suggesting that IGF I act s as a local mediator of PTH anabolic effects (Pfeilschifter et al., 1995) In a study to detect the early effects of short term PTH administration, decreased IGF I serum concentrations were observed throughout the study, which contrast ed with previous ly published data (Toromanoff et al., 1998) K nockout mice with global deletion of the ac id labile subunit (ALS), named ALSKO, provided evidence that the IGF I ternary complex is important for bone remodeling and for the anabolic response to PTH treatment (Yakar et al., 2006) Dwarf rats are also deficient in GH, and the anabolic effects of PTH (134) may depend on GH, as suggested by studies with hypophysectomized rats (Hock and Fonseca, 1990) However, PTH increased bone mass by stimulating bone formation in aged female rats, regardless of GH treatment (Gunness, 1995) Despite low serum IGF I and GH levels, we found that PTH treatment increased cancellous bone mass and stimulated bot h cancellous and cortical bone formation in dwarf rats. The pQCT data from the distal femoral metaphysis revealed increased mineral density and mineral content of cancellous bone with PTH treatment in both Lewis and dwarf rats; in fact, dwarf rats showed a somewhat greater increase in trabecular bone density compared to Lewis rats, with a significant grouptreatment interaction. At the femoral shaft, only the cortical BMD was significantly higher in the PTH treated dwarf rats In most studies the anaboli c effects of PTH are more pronounced in cancellous

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106 bone than in cortical bone (Dempster et al., 1993) Our results from t he distal femoral metaphysis and femoral shaft are in agreement with this finding PTH increases the number of osteoblasts and their activity, which results in thicker trabeculae and improves trabecular connectivity, restoring bone microarchitecture (Hodsman et al., 2005, Bilezikian and Rubin, 2006) The bone measurements in the lumbar vertebrae and proximal tibiae of PTH treated dwarf and Lewis rats, in accordance with previous studies showed significant increases in bone volume, associated with increased trabecular width. In addition to the PTH induced increase in bone density and mineral content observed with pQCT, biomechanical testing revealed that the lumbar vertebrae in PTH treated dwarf rats did not show a significant increase in load and stiffness, but the observed increase in the parameter stress (N*mm2), which includes the area in its calculation, seems more relevant in this analysis, as the vertebrae in dwarf rats were very small. Nevertheless, vertebr al load, which was significantly increased in PTH treated Lewis rats, would undoubtedly have also been increased in PTH treated dwarf rats with a longer treatment period. In contrast to antiresorptive agents used in the treatment of osteoporosis, PTH has t he capability of stimulat ing bone turnover (Bilezikian and Rubin, 2006, Canalis et al., 2007, Kousteni and Bilezikian, 2008) Consist ent with this concept, osteoclast surface, an index of bone resorption, was significantly increased in PTH treated dwarf rats. Furthermore, mineralizing surface and bone formation rate were notably increased in PTH treated dwarf and Lewis rats compared to their vehicle treated controls Th e anabolic effect of PTH in dwarf rats may have been superior to th at observed in PTH treated Lewis rats Moreover, c ancellous bone mineral apposition rate, an index of

PAGE 107

107 osteoblast activity, was increased in PTH treated dwarf and Lewis rats compared to their respective control groups. Therefore, P TH stimulated both cancellous bone resorption and formation in dwarf and Lewis rats. Regardless of IGF I status, the increment in bone formation must have exceeded the increment in bone resorption for the observed PTH induced gain in bone mass to occur. I GF I is decreased, but not abolished in the dwarf rat, which is different from mice with global deletion of the IGF I gene. This may explain why PTH fails to induce a bone anabolic response in IGF I knockout mice, but has a strong anabolic effect in dwarf rats. Different responses to PTH can occur, as mixed genetic background can affect bone acquisition and response to PTH treatment (Yakar et al., 2006) and not all patients and not all bones respond to intermittent treatment with PTH in the same manner (Bikle, 2008) Human patients with IGF I gene deletion have been identified, but cases are rare (Yakar et al., 2006) w hile IGF I deficiency is present in several osteopenic conditions. Different responses to PTH treatment have also been observed in healthy inbred strains of mice with differences in skeletal IGF I synthesis (Bilezikian and Rubin, 2006) Yet, in addition to the GH and IGF I skeletal effects, the participation of the six IGF I binding proteins must also be considered. The different cellular signaling pathways that are concomitantly influenced by the GH/IGF I axis and PTH, suggest that several levels of regulation might be involved in the role of IGF I as a mediator for the bone anabolic effects of PTH in vivo (Pereira and Canalis, 1999) Nevertheless, our findings indicate that PTH has a strong stimulatory effect on bone formation and augments bone mass even in the presence of low circulating levels of IGF I. Conclusions The results addressed S pecific Aim 2, and the major findings were:

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108 1. PTH did not affect body weight or serum IGF I levels in either Lewis or dwarf rats. 2. PTH induced highly significant anabolic effects in vertebral and tibial cancellous bone in dwarf rats, and s timulated both cancellous and cortical bone formation, despite low circulating levels of IGF I.

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109 Figure 3 1 Body weight (g). Significant difference between Lewis and dwarf rats (P<0.0001), but not between VEH and PTH treated rats in either the Lewis or dwarf groups Figure 32. Femur Length (mm). PTH treatment slightly increased mean femur length in Lewis rats (P=0.0026) but no difference was observed between VEH and PTH treated dwarf rats 0 25 50 75 100 125 150 175 200 225Body Weight (g) Dwarf Lewis VEH PTH 0 5 10 15 20 25 30 35Femur Length (mm) Dwarf Lewis VEH PTH

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110 Figure 33. Serum IGF I (ng/mL) A significa nt difference was observed between Lewis and dwarf rats (P<0. 0001), but not between VEH and PTH treated rats in either group Figure 34. Total Bone Mineral Content (BMC mg/mm) in distal femoral metaphysis S ignificant increases w ere observed in PTH treated rats from both groups (P<0.0001) 0 200 400 600 800 1000 1200Serum IGF-I (ng/mL) Dwarf Lewis VEH PTH 0 2 4 6 8 10 12 14Total BMC (mg/mm) Dwarf Lewis VEH PTH

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111 Figure 35. Total Bone Mineral Density (BMD) in the distal femoral metaphysis ( mg/cm3) S ignificant increases were observed in dwarf and Lewis rats treated with PTH (P<0.0001) Figure 36. Tr abecular BMC (mg/mm). Significant increases were observed in both Lewis and dwarf rats treated with PTH (P<0.0001) 0 100 200 300 400 500 600 700 800Total BMD (mg/cm3) Dwarf Lewis VEH PTH 0 .5 1 1.5 2 2.5 3Trabecular BMC (mg/mm) Dwarf Lewis VEH PTH

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112 Figure 37 Trabecular BMD (mg/cm3). H ighly significant increases w ere observed in dwarf (P<0.0001) and Lewis (P=0.0022) rats treat ed with PTH Figure 38. Total Area for distal femoral metaphysis (mm2). A significant increase was observed in PTH treated dwarf rats (P =0.0202) 0 100 200 300 400 500 600Trabecular BMD (mg/cm3) Dwarf Lewis VEH PTH 0 2 4 6 8 10 12 14 16 18 20Total Area (mm2) Dwarf Lewis VEH PTH

PAGE 113

113 Figure 39. Trabecular Area (mm2). A significant increase was observed in PTH treated Lewis rats (P=0.0169) Figure 310. Total BMC for femoral diaphysis (mg/mm). There w ere no significant differences in the PTH treated rats, dwarf or Lewis, when compared to their respective vehicletreated controls 0 1 2 3 4 5 6Trabecular Area (mm2) Dwarf Lewis VEH PTH 0 1 2 3 4 5 6 7 8Total BMC (mg/mm) Dwarf Lewis VEH PTH

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114 Figure 311. Total BMD for femoral diaphysi s (mg/cm3). There w ere no significant differences between PTH and vehicletreated groups in dwarf and Lewis rats Figure 312. Cortical BMC (mg/mm). No significant differences w ere found between the PTH and vehicletreated groups for both dwarf and L ewis rats 0 100 200 300 400 500 600 700 800 900 1000Total BMD (mg/cm3) Dwarf Lewis VEH PTH 0 1 2 3 4 5 6 7 8Cortical BMC (mg/mm) Dwarf Lewis VEH PTH

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115 Figure 313. Cortical BMD (mg/cm3). There was a significant difference between PTH and vehicletreated dwarf rats (P=0.0013) but not between these groups of Lewis rats Figure 314. Cortical Area (mm2). No significant differences wer e observed with PTH treatment in dwarf and Lewis rats 0 200 400 600 800 1000 1200 1400Cortical BMD (mg/cm3) Dwarf Lewis VEH PTH 0 1 2 3 4 5 6Cortical Area (mm2) Dwarf Lewis VEH PTH

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116 Figure 315. Cortical Thickness (mm). There w ere no significant differences between PTH and vehicletreated groups for dwarf and Lewis rats Figure 316. Periosteal Circumference (mm). The values were almost identical between PTH and vehicletreated groups for dwarf and Lewis rats 0 .1 .2 .3 .4 .5 .6 .7 .8Cortical Thickness (mm) Dwarf Lewis VEH PTH 0 2 4 6 8 10 12Periosteal Circumference (mm) Dwarf Lewis VEH PTH

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117 Figure 317. Endosteal Circumference (mm). No significant differences were observed with PTH treatment in both groups Figure 318. Vertebral Cancellous B one Volume (%). There w ere significant increases in PTH treated dwarf (P=0.0016) and Lewis (P<0.0001) rats when compared to their vehicle control groups 0 1 2 3 4 5 6 7Endosteal Circumference (mm) Dwarf Lewis VEH PTH 0 5 10 15 20 25 30Vertebral Cancellous Bone Volume (%) Dwarf Lewis VEH PTH

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118 Figure 319. Vertebral T rabecular Number (#/mm). There was a slight, but non significant increase between PTH and vehicletreated rats in both groups Figure 320. Vertebral T rabecular Width ( m). There was a significant increase in PTH treated dwarf (P=0.0229) and Lewis (P=0.0320) rats, when compared to their vehicle control groups 0 1 2 3 4 5 6 7 8 9Vertebral Trabecular Number (#/mm) Dwarf Lewis VEH PTH 0 10 20 30 40 50 60 70Vertebral Trabecular Width (um) Dwarf Lewis VEH PTH

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119 Figure 321. Vertebral Trabecular Separation ( m). S trong trends for decreases w ere observed in dwarf (P=0 .00567) and Lewis (P=0.0603) rats treated with PTH Figure 322. Vertebral Osteoid Surface (%). H ighly significant increases w ere noted in dwarf (P<0.0001) and Lewis (P=0.0027) rats treated with PTH 0 20 40 60 80 100 120 140 160 180 200Vertebral Trabecular Separation (um) Dwarf Lewis VEH PTH 0 5 10 15 20 25 30 35 40 45Vertebral Osteoid Surface (%) Dwarf Lewis VEH PTH

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120 Figure 3 23. Vertebral Osteoblast Surface (%) M arkedly significant increases w ere noted in dwarf (P<0.0001) and Lewis (P=0.0011) rats treated with PTH Figure 324. Vertebral Osteoclast Surface (%). A highly significant increase was noted only in dwarf rats treated with PTH (P = 0.0 125 ) 0 5 10 15 20 25 30 35 40 45 50Vertebral Osteoblast Surface (%) Dwarf Lewis VEH PTH 0 .25 .5 .75 1 1.25 1.5 1.75 2 2.25Vertebral Osteoclast Surface (%) Dwarf Lewis VEH PTH

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121 Fig ure 325. Vertebral Mineralizing Surface (%). H ighly significant increases w ere noted in dwarf and Lewis rats treated with PTH (P<0.0001) Figure 326. Vertebral Bone F ormation Rate (10232/d) M arkedly significant increas es w ere noted in dwarf and Lewis rats treated with PTH (P<0.0001) 0 10 20 30 40 50 60 70Vertebral Mineralizing Surface (%) Dwarf Lewis VEH PTH 0 10 20 30 40 50 60 70 80 90Vertebral Bone Formation Rate (10-2um3/um2/d) Dwarf Lewis VEH PTH

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122 Figure 327. Vertebral Cancellous Mineral Apposition Rate ( m/d). S ignificant increases w ere observed in PTH treated dwarf and Lewis rats (P<0.0001) Figure 328. Longitudinal Bone Growth ( m/d). S i gnificant increases w ere observed in PTH treated dwarf (P<0.0001) and Lewis (P=0.0321) rats 0 .2 .4 .6 .8 1 1.2 1.4 1.6Vertebral Cancellous MAR (um/d) Dwarf Lewis VEH PTH 0 10 20 30 40 50 60 70 80 90 100Longitudinal Bone Growth (um/d) Dwarf Lewis VEH PTH

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123 A B Figure 329. Proximal tibial metaphyses from vehicle treated dwarf (A) and PTH treated dwarf (B) rats. Note the reduced mass of black stained bone indicative of cancellous osteopenia in the vehicle treated d warf rat compared to the increased number of thicker trabeculae in the PTH treated dwarf rat Von Kossa/ tetrachrome stain, X40 Photos courtesy of Dr. Thomas J.Wronski Figure 330. Tibial Cancellous B one Volume (%). S ignificant increase s w ere observed in both dwarf (P=0.0016) and Lewis (P=0.0023) rats treated with PTH 0 5 10 15 20 25 30Tibial Cancellous Bone Volume (%) Dwarf Lewis VEH PTH

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124 Figure 331. Tibial Trabecular Number (#/mm). A significant incr ease was observed only in PTHtreated dwarf rats (P = 0.0001) Figure 332. Trabecular Width ( m). A significant increase was observed in PTH treated dwarf rats (P = 0.0 183 ) but not in PTH treated Lewis rats 0 1 2 3 4 5 6 7 8Tibial Trabecular Number (#/mm) Dwarf Lewis VEH PTH 0 10 20 30 40 50 60Tibial Trabecular Width (um) Dwarf Lewis VEH PTH

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125 Figure 333. Tibial Trabecular Separation ( m). No significant d ifference s w ere observed in PTH treated dwarf and Lewis rats Figure 334. Total Cortical Bone Tissue Area in Tibial Diaphysis (mm2). No s ignificant differences were observed with PTH treatment in dwarf and Lewis rats 0 500 1000 1500 2000 2500 3000 3500 4000 4500Tibial Trabecular Separation (um) Dwarf Lewis VEH PTH 0 .5 1 1.5 2 2.5 3 3.5 4 4.5Total Cort. Bone Tissue Area (mm2) Dwarf Lewis VEH PTH

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126 Figure 335. Cortical Bone Area in Tibial Diaphysis (mm2). No significant differences were observed w ith PTH treatment in dwarf and Lewis rats Figure 336. Bone Marrow Area (mm2). There were no significant differences with PTH treatment in both groups 0 .5 1 1.5 2 2.5 3 3.5 Cortical Area (mm2) Dwarf Lewis VEH PTH 0 .1 .2 .3 .4 .5 .6 .7 .8 Marrow Area (mm2) Dwarf Lewis VEH PTH

PAGE 127

127 Figure 337. Periosteal Perimeter (mm). No s ignificant differences were observed with PTH treatm ent in both dw arf a n d Lewis rats Figure 338. Endocortical Perimeter (mm). No significant difference s were observed with PTH treatment in both dwarf and Lewis rats 0 1 2 3 4 5 6 7 8Periosteal Perimeter (mm) Dwarf Lewis VEH PTH 0 .5 1 1.5 2 2.5 3 3.5Endocortical Perimeter (mm) Dwarf Lewis VEH PTH

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128 Figure 339. Cortical Width ( m). C ortical width decreased in PTH treated dwarf rats (P=0.0116), but not in PTH treated Lewis rats Figure 340. Periosteal Mineralizing Surface (%). A significant increase was observed in PTH treated dwarf rats (P<0.0001) 0 100 200 300 400 500 600 700 800Cortical Width (um) Dwarf Lewis VEH PTH 0 20 40 60 80 100 120Periosteal Mineralizing Surface (%) Dwarf Lewis VEH PTH

PAGE 129

129 Figure 341. Periosteal Mineral Apposition Rate ( m/d). PTH treatment induced significant increases in both dwarf (P<0.0001) and Lewis (P=0.0072) rats Figure 342. Periosteal Bone Formation Rate (102 m3/ m2/d). S ignificant increase s in PTH treated dwarf (P<0.0001) and Lewis (P=0.0026) rats w ere observed 0 .5 1 1.5 2 2.5 3 3.5 4Periosteal MAR (um/d) Dwarf Lewis VEH PTH 0 50 100 150 200 250 300 350 400Periosteal Bone Formation Rate (10-2um3/um2/d) Dwarf Lewis VEH PTH

PAGE 130

130 Figure 343. Endocortical Mineralizing Surface(%). A significant increase was observed in PTH treated dwarf rats (P = 0.0022 ) Figure 344. Endocortical Mineral Apposition Rate (um/d). A s ignificant increase was observed in dwarf rats with PTH treatment (P = 0.0 00 6 ) 0 20 40 60 80 100 120Endocortical Mineralizing Surface (%) Dwarf Lewis VEH PTH 0 .5 1 1.5 2 2.5 3Endocortical MAR (um/d) Dwarf Lewis VEH PTH

PAGE 131

131 Figure 345. Endocortical Bone Formation Rate (102 m3/ m2/d). A significant increase was observed in PTH treated dwarf rats (P = 0.000 2 ) Figure 346. Biomechanical Load (N). A s ignificant increase was observed only in PTH treated Lewis rats (P = 0.0004 ) 0 25 50 75 100 125 150 175 200 225 250Endocortical Bone Formation Rate (10-2um3/um2/d) Dwarf Lewis VEH PTH 0 50 100 150 200 250 300 350 400Load (N) Dwarf Lewis VEH PTH

PAGE 132

132 Figure 347. Biomechanical Stress (N*mm2 or MPa). A s ignificant increase was observed only in PTH treated dwarf rats (P=0.0115) Figure 348. Biomechanical Stiffness (N/mm). No significant differences were observed with PTH treatment in both dwarf and Lewis rats 0 5 10 15 20 25 30Stress (N*mm2 or MPa) Dwarf Lewis VEH PTH 0 250 500 750 1000 1250 1500 1750 2000 2250 2500Stiffness (N/mm) Dwarf Lewis VEH PTH

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133 CHAPTER 4 COMPARISON OF PGE2 TREATMENT IN GH/IGF I DEFICIENT DWARF RA TS AND THEIR BACKGROUND STRAIN LEWIS RATS. Introduction Prostaglandins (PGs) are critical regulators of both skeletal physiologic and pathologic responses, acting in local tissues as paracrineautocrine factors (Raiz and Lorenzo, 2006) The synthesis of PGs by bone cells is regulated by local factors, as well as systemic hormones that participate in bone metabolism including PTH and vitamin D3 (Pilbeam et al., 2008) Prostaglandins are considered dual regulators of bone metabolism, stimulating both bone formation and bone resorption in vivo (Lin et al., 19 95) but with a positive balance in favor of bone formation (Harada et al., 1995, Jee and Ma, 1997) Prostaglandins are synthesized by many cells in the skeleton and prostaglandin E2 (PGE2) is abundant in bone c ells acting mainly through receptor EP4 to stimulate bone formation, as demonstrated in vitro and in vivo (Ke et al., 2006, Aguirre et al., 2007, Downey et al., 2009) In vitro studies showed both stimulatory and inhibitory effects of PGE2 on bone formation in cell and organ culture, and IGF I was also considered as a mediator for the effects of PGE2 on osteoblasts (Pilbeam et al., 2008) Machwate et al. (2001) examined the effect of an EP 4 specific antagonist, EP4A, on bone formation induced by PGE2 in young rats. T hey found that EP4A suppressed the increase in bone mass induced by PGE2. This effect is accompanied by a reduction in the extent of calcein labeled surface and trabecular number, suggesting that EP4 is the main receptor through which PGE2 induces bone formation in rats (Machwate et al., 2001) The bone anabolic effect of PGE2 has been compared to th e effects of PTH (Harada et al., 1995) In vivo systemic injection of PGE2 in rats increases both cortical

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134 and cancellous bone formation, and produces substantial increases in bone mass, similar to the effects of PTH (Li et al., 1992) Like PGE2, PTH also increases the production of IGF I by osteoblasts in vitro (McCarthy et al., 1991) and the anabolic effects of PTH may be mediated by IGF I (Canalis et al., 1989) Taking these findings in to consideration, we decided to evaluate the bone anabolic effects of PGE2 in IGF I deficient dwarf rats, to determine whether low serum levels of IGF I affect the ability of PGE2 to stimulate bone formation and augment bone mass. Materials and M ethods Animal Models The s ame groups of vehicletreated dwarf and Lewis rats, analyzed in the first experiment were used as controls, for evaluat ion of the effects of PGE2 on bone. IGF I serum levels in dwarf rats were significant ly lower compared to their back ground strain, Lewi s rats, reproduci ng the physiological changes seen in IGF I deficiency which validates their use as a reliable model Therefore, the dwarf and Lewis strains of rats were used to evaluate the effects of P GE2 treatment on bone m ass and turnover Based on the same criteria for animals from the first and second experiment s, five week old female dwarf and Lewis rats, obtained from Harlan Laboratories (UK ), were kept in a temperature and humidity controlled environment (25 C), and on a 12h light/12h dark cycl e. The experiment was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. Experimental Design Following the first analysis to establish the dwarf rat as an animal model for our studies we test ed the hypothesi s that IGF I acts as a potential mediator for the bone anabolic effects of PGE2. The four groups (N=13, for vehicletreated dwarf and Lewis

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135 rats, and N= 7 for PGE2treated dwarf and Lewis rats) were maintained in the same nutritional and environmental conditions until 9 weeks of age, when treatment started. All rats were routinely examined and weighed once a week, for monitoring their health status. Vehicle and P GE2 Treatment The rats from the first experiment, treated with vehicle solution (VEH), formed th e control groups, for comparison with the PGE2treated dwarf and Lewis groups. The vehicle solution was already described for experiment one. The PGE2 solution was prepared as follows: 1. PGE2 (Cayman Chemical Co., Ann Arbor MI) was supplied as a crystalli ne solid and the stock solution (15mg/mL) was prepared by dissolving 45 mg of PGE2 in 3 mL of 100% ethanol 2. For the injections, 0.5 mL of the stock solution was then diluted with sterilized distilled water, obtaining a final concentration of 3 mg/ mL The r ats were injected, subcutaneously, daily for 2 weeks with PGE2 at a dose of 3 mg /kg body weight Bone Formation M arkers We used the same fluorochrome markers as described for experiment one. All rats received subcutaneous injections of the fluorochrome c ompounds, declomycin and calcein (Sigma Chemical Co., St. Louis, MO) at a dose of 15 mg/kg body weight, ten and three days prior to euthanasia respectively, in order to label actively forming bone surfaces. Following these initial steps, the methods are the s ame as described in C hapter 2 (pages 3441), including necropsy procedures, tissue processing, data collection and statistical analysis

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136 Results Body Weight The evident phenotypic differences between Lewis and dwarf rats r emained proportionally the s ame At the time of euthanasia, the mean body weight s for the Lewis and dwarf rats w ere significantly different, as already observed in the previous experiment s. PGE2treat ed Lewis rats however, weighed significantly less than vehicle treated rats Lewis rats (194.54 12.90g vs. 167.28 9.75g, respectively P<0.0001), but this was not weight loss due to PGE2 treatment. Rather, the PGE2treated Lewis rats weighed less than the vehicletreated Lewis rats at the beginning of treatment, and this difference in body weight was maintained during the 2 week treatment period. No significant difference in body weight was observed in dwarf rats treated with PGE2 or vehicle, as seen in Figure 4 1. IGF I Serum Levels Serum levels of IGF I were markedly lower in dwar f rats compared with Lewis rats as seen in experiment 1. PGE2 did not increase serum IGF I levels in either Lewis or dwarf rats. In fact, PGE2treated Lewis rats even showed significant ly lower IGF I serum levels than their vehicle treated controls (Fig ure 4 2). Peripheral Quantitative Computerized Tomography The pQCT analyses of the distal femoral metaphysis (Figures 4 3 to 4 8 ) revealed that cancellous bone structural parameters such as trabecular BMC and trabecular BMD were significantly higher in P GE2treated dwarf rats, when compared to vehicletreated dwarf rats (P = 0.0 251 and P=0.0011). Total BMD exhibited a significant decrease only in PGE2treated dwarf rats (P<0.0001). Total BMC, total area, and

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137 trabecular area showed no significant differenc e with PGE2 treatment in dwarf and Lewis rats when compared to their vehicle treated controls. When considering cortical bone (Figures 4 9 to 4 16) however, almost all the parameters measured at the femoral shafts showed significant ly lower values in the PGE2treated groups T otal BMC and cortical BM D had significant ly lower values in the PGE2treated groups dwarf and Lewis, compared to their controls Total BMD, cortical BMC, and cortical area exhibited significant lower values in PGE2treated dwarf ra ts. Endocortical circumference and periosteal circumference presented significant lower values only in PGE2 treated Lewis rats. C ortical thickness did not show significant differences in PGE2 treated dwarf and Lewis rats, when compared to their respect ive vehicle treated controls. Histomorphometric Findings Cancellous bone measurements in the lumbar vertebrae W hen comparing the effects of PGE2 and vehicle treatment on vertebral cancellous bone volume, there were no significant increases in Lewis and dwarf rats treated with PGE2, although a trend was evident as shown in Figure 4 17 However, PGE2 treatment induced a significant increase in trabecular number associated with decreased trabecular separation in dwarf and Lewis rats compared to their controls (Figures 4 1 8 and 4 2 0 ), but no significant changes occurred in trabecular width (Figure 4 19) When analyzing cancellous bone surface parameters, PGE2 treatment did not induce any significant difference in the osteoid, osteoblast, and osteoclast surfaces in dwarf and Lewis rats as seen in Figures 4 2 1 to 4 23.

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138 Considering bone kinetic variables ( Figures 424 to 4 26 ) PGE2treated Lewis rats exhibited significantly increase d mean values for vertebral cancellous bone mineralizing surface compared to vehicle treated Lewis rats PGE2treated dwarf rats showed only a trend for increased vertebral cancellous bone mineralizing surface and a decreased cancellous bone mineral apposit ion rate The mean values for vertebral bone formation rate did not show sig nificant differences in PGE2treated dwarf and Lewis rats compared to their vehicletreated controls Cancellous bone measurements in the proximal tibiae Tibial longitudinal bone growth was significantly higher in PGE2treated dwarf rats compared to vehicl e treated dwarf rats (27.29 8.67 m/d vs. 72.79 6.11 m/d, P<0.0001) whereas PGE2treated Lewis rats (70.88 5.55 m/d vs. 82.27 12.66 m/d, P =0.0881 ) only exhibited a trend for increased longitudinal bone growth, as seen in Figure 4 2 7 The effect of PGE2 treatment on tibial cancellous bone structure is even more noticeable in histo logic images from vehicle and PGE2treated dwarf rats i n Figure 4 28. Regarding cancellous bone structural parameters in the proximal tibia, we observed an increase in the tibial cancell ous bone volume in dwarf rats, as a response to PGE2 treatment, but not in Lewis rats as seen in Figure 4 29. The PGE2 effect on bone volume was associated with increased trabecular number only in dwarf rats, but not in Lewis rats when compared to vehicletreated controls (Figures 430). T rabecular width, however, was decreased in PGE2treated Lewis rats, but showed no significant difference in dwarf rats in response to PGE2 treatment (Figure 431) Tibial trabecular separation (Figur e 432) was not significant ly different in PGE2treated dwarf and Lewis

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139 rats compared to their vehicletreated controls, although a strong trend for an increase in this variable was observed in PGE2treated dwarf rats. Cortical b one measurements in the tib ia l diaphysis When analyzing the effects of PGE2 treatment on bone structural parameters i n the left tibial diaphyses (Figures 4 33 to 438) there was no significant change in the total cortical bone tissue area in both PGE2treated dwarf and Lewis rats compared to vehicle treated rats, but t he cortical bone area and width showed significant ly lower mean values in Lewis rats treated with PG E2 No differences in these parameters were observed in PGE2 treated dwarf rats compared to their vehicle treated co ntrols. However, PGE2treated dwarf rats exhibited an increase in marrow area and periosteal perimeter Regarding periosteal dynamic measurements in the tibial diaphysis (Figures 439 to 4 4 1) only the periosteal mineral apposition rate was increased in response to PGE2 treatment in dwarf rats Periosteal mineral izing surface and bone formation rate did not show any difference due to PGE2 treatment in the two strains. At the endocortical surface (Figures 442 to 444), no effect of PGE2 treatment was ob served in mineralizing surface in dwarf and Lewis rats compared to their vehicle treated controls. PGE2 treatment increased endocortical mineral apposition rate and bone formation rate only in dwarf rats, but not in PGE2treated Lewis rats. Biomechanical Testing in Lumbar Vertebral Body The biomechanical analysis revealed an increase in the stress parameter only in PGE2treated Lewis rats compared to vehicletreated Lewis rats No significant differences w ere detected for load and stiffness parameters in dwarf and Lewis rats

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140 treated with PGE2, compared to their respective vehicletreated controls, as seen in Figures 4 4 5 to 4 4 7 Discussion Our results indicate th at PGE2 treatment augments bone mass in dwarf rats, mainly in cancellous bone, despite lower s erum IGF I levels. This finding is somewhat surprising in that other studies have suggested that IGF I or the IGF I receptor may mediate the stimulatory effects of PGE2 on bone formation (Harada et al., 1995, McCarthy et al., 1991, Raisz et al., 1993a) However, our findings show that low serum levels of IGF I are adequate to support a PGE2induced increase in cancellous bone mass. PGE2treated rats Lewis rats did not gain as much weight as PGE2treated rats. Thi s finding may be related to the gastrointestinal side effects that occur during PGE2 therapy (Jee and Ma, 1997) In a study with aged rats, Cui (2001) also observed body weight loss due to diarrhea and reduced activity that persisted 2 to 4 hours after injection (Cui et al., 2001) The weight loss, however did not affect bone growth in our young rats, as our results showed a significant increase in tibial longitudinal bone growth in the two strains treated with PGE2. The pQCT data obtained in our study revealed a positive anabolic effect of PGE2 treatment on metaphyseal cancellous bone, with increased trabecular BMC and BMD, but the same response to PGE2 treatment was not observed in cortical bone, where almost all of the parameters presented lower values Our results diverge from those observed in another in vivo study that reported increased cortical bone mass in the tibial shaft, using the same PGE2 dose of 3mg/kg, but for a longer treatment period (30 days) in aged rats (Cui et al., 2001) The weak response to PGE2 treatment in cortical bone

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141 was also noticed in the histomorphometric analysis we performed in the tibial diaphysis where the cortical area and the cortical width were dec reased (PGE2 treated Lewis rats) or did not show any significant difference (PGE2treated dwarf rats). The lack of a strong anabolic effect of PGE2 on cortical bone in the current study may be due to the relatively short treatment period (14 days) or the relat ively young age of the dwarf and Lewis rats at the beginning of treatment (9 weeks). Ito (1993) observed differences in the response to PGE2 treatment depending upon cancellous bone sites (proximal and distal tibial metaphysis) and in a dosedependent mann er (Ito et al., 1993) We noticed that the values for cancellous bone volume in the lumbar vertebrae were not increased in PGE2treated dwarf rats, whereas in the proximal tibial metaphysis, cancellous bone volume was significantly increased in these animals This finding may be a consequence of a more rapid response to PGE2 treatment in the long bone metaphysis, which has a higher rate of cancellous bone turnover compared to the lumbar vertebral body Jee (1997) proposed that the amount of bone formation was higher in the tibial shaft than in cancellous bone of the distal tibial metaphysis, followed by the distal femoral metaphysis, proximal tibial metaphysis and lumbar vertebral body, in decreasing sequence (Jee and Ma, 1997) Although PGE2 has been reported to stimulate bone formation at the perioste al and endocortical surfaces (Raiz and Lorenzo, 2006) we found no significant differences for periosteal mineralizing surface and bone formation rate in Lewis rats treated with PGE2 O nly the mineral apposition rate indicative of osteoblastic activity, was significantly increased by PGE2 treatment in dwarf rats In addition, t he biomecha nical analysis of the lumbar vertebral body showed a

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142 significant increase only in the parameter stress and exclusively in Lewis rats that received PGE2 treatment As mentioned above, the treatment period may not have been of sufficient duration for PGE2 to induce biomechanical changes and fully stimulate cortical bone formation. Nevertheless our findings demonstrate that lower l e vels of systemic IGF I do not affect the bone anabolic response to PGE2 treatment. Conclusions 1. PGE2 treatment did not increase body weight or serum IGF I levels in either Lewis or dwarf rats. 2. PGE2treated rats exhibited significant ly higher bone mass, mostly cancellous bone, wh ereas cortical bone mass was significantly lower. 3. Most importantly, PGE2 induced significant anabolic effects, mainly in cancellous bone of dwarf rats, despite low circulating levels of IGF I.

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143 Figure 41. Body Weight (g). There was a significant decrease in the body weight of PGE2treated Lewis rats (P<0.0001) but not in PGE2 treated dwarf rats Figure 42. IGF I Serum Levels (ng/mL). There was a significant difference between vehicle treated Lewis and dwarf rats (P =0.0115). PGE2treated Lewis rats showed significantly lower values compared to vehicletreated Lewis rats (P =0.0228) 0 25 50 75 100 125 150 175 200 225Body Weight (g) Dwarf Lewis VEH PGE2 0 250 500 750 1000 1250 1500 1750 2000 2250 2500IGF-I Serum Levels (ng/mL) Dwarf Lewis VEH PGE2

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144 Figure 43. Total Bone Mineral Content (BMC) (mg/mm) in the distal femoral metaphysis There w ere no significant differences with PGE2 treatment in dwarf and Lew is rats Figure 44. Total Bone Mineral Density (BMD) ( mg/cm3) A significant decrease was observed in PGE2treated dwarf rats (P<0.0001), but not in Lewis rats treated with PGE2 0 1 2 3 4 5 6 7 8Total BMC (mg/mm) Dwarf Lewis VEH PGE2 0 100 200 300 400 500 600 700 800 900Total BMD (mg/cm3) Dwarf Lewis VEH PGE2

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145 Figure 45. Trabecular BMC (mg/mm). There was a significant incr ease in PGE2treated dwarf rats (P=0.0251) but not in PGE2treated Lewis rats Figure 46. Trabecular BMD (mg/cm3). A significant increase was observed in PGE2treated dwarf rats (P=0.0011), but not in Lewis rats treated with PGE2 0 .25 .5 .75 1 1.25 1.5 1.75 2 2.25 2.5Trabecular BMC (mg/mm) Dwarf Lewis VEH PGE2 0 100 200 300 400 500 600Trabecular BMD (mg/cm3) Dwarf Lewis VEH PGE2

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146 Figure 47 Tot al Area for Cancellous Bone (mm2). No significant difference was noted with PGE2 treatment Figure 48 Trabecular Area (mm2). There was no significant difference with PGE2 treatment in dwarf and Lewis rats 0 2 4 6 8 10 12 14 16 18Total Area Cancellous Bone (mm2) Dwarf Lewis VEH PGE2 0 1 2 3 4 5 6Trabecular Area (mm2) Dwarf Lewis VEH PGE2

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147 Figure 49 Total BMC (mg/mm) in the femoral diaphysis The mean values were significantly lower in the PGE2treated dwarf (P=0.0002) and Lewis rats (P=0.0047) when compared to their respective vehicletreated controls Figure 410. Total BMD for (mg/cm3). Dwarf rats treated with PGE2 showed a significant ly lower value than vehicletreated controls (P<0.0001) No difference was observed in Lewis rats with PGE2 treatment 0 1 2 3 4 5 6 7 8Total BMC (mg/mm) Dwarf Lewis VEH PGE2 0 200 400 600 800 1000Total BMD (mg/cm3) Dwarf Lewis VEH PGE2

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148 Figure 411. Cortical BMC (mg/mm). A s ignificant ly lower mean value w as observed in the PGE2 treated dwarf rat s (P=0.0003), but not in PGE2 treated Lewis rats (P=0.0910) Figure 41 2 Cortical BMD (m g /cm3). PGE2treated rats showed significant ly lower mean values in both groups (P<0.0001) 0 1 2 3 4 5 6 7 8Cortical BMC (mg/mm) Dwarf Lewis VEH PGE2 0 200 400 600 800 1000 1200 1400Cortical BMD (mg/cm3) Dwarf Lewis VEH PGE2

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149 Figure 41 3 Cortical Area (mm2). Dwarf rats treated with PGE2 h ad a significant ly lower mean value (P=0.0496) but there was no difference with PGE2 treatment in Lewis rats Figure 41 4 Cortical Thickness (mm). There was no significant difference with PGE2 treatment in dwarf and Lewis rats 0 1 2 3 4 5 6Cortical Area (mm2) Dwarf Lewis VEH PGE2 0 .1 .2 .3 .4 .5 .6 .7 .8Cortical Thickness (mm) Dwarf Lewis VEH PGE2

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150 Figure 41 5 Perios teal Circumference (mm). PGE2treated Lewis rats had a significantly lower mean value (P=0.0038), but there was no difference with PGE2 treatment in dwarf rats (P=0.0826) Figure 41 6 Endosteal Circumference (mm). A s ignificant lower mean value was observed in PGE2treated Lewis rats (P=0.0002) but no t in PGE2treated dwarf rats 0 2 4 6 8 10 12Periosteal Circumference (mm) Dwarf Lewis VEH PGE2 0 1 2 3 4 5 6 7Endosteal Circumference (mm) Dwarf Lewis VEH PGE2

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151 Figure 41 7 Vertebral Cancellous Bone Volume (%). There was no significant difference in PGE2 treated dwarf and Lewis rats Figure 418. Vertebral Trabecular Number (#/mm). A significant increase was observed in Lewis (P=0.0004) and dwarf rats (P=0. 0021) treated with PGE2 0 5 10 15 20 25 30 35 40Vertebral Cancellous Bone Volume (%) Dwarf Lewis VEH PGE2 0 1 2 3 4 5 6 7 8 9 10Vertebral Trabecular Number (#/mm) Dwarf Lewis VEH PGE2

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152 Figure 419. Vertebral Trabecular Width ( m). There was no significant difference in PGE2treated dwarf and Lewis rats Figure 420. Vertebral Trabecular Separation ( m). Significant ly lower mean values were observed in PGE2treated dwarf (P=0.0041) and Lewis rats (P=0.0417) 0 10 20 30 40 50 60 70Vertebral Trabecular Width (um) Dwarf Lewis VEH PGE2 0 20 40 60 80 100 120 140 160 180 200Vertebral Trabecular Separation (um) Dwarf Lewis VEH PGE2

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153 Figure 421. Vertebral Osteoid Surface (%). No significant differences w ere observed with PGE2 treatment i n dwarf and Lewis rats Figure 422. Vertebral Osteoblast Surface (%). There w ere no significant differences in PGE2treated dwarf and Lewis rats 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5Vertebral Osteoid Surface (%) Dwarf Lewis VEH PGE2 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25Vertebral Osteoblast Surface (%) Dwarf Lewis VEH PGE2

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154 Figure 423. Vertebral Osteoclast Surface (%). There w ere no significant differences with PGE2 treatm ent in dwarf and Lewis rats Figure 424. Vertebral Mineralizing Surface (%). Only PGE2treated Lewis rats showed a significantly increase (P=0.0068) compared to their vehicletreated controls 0 .2 .4 .6 .8 1 1.2 1.4 1.6 1.8Vertebral Osteoclast Surface (%) Dwarf Lewis VEH PGE2 0 10 20 30 40 50 60Vertebral Mineralizing Surface (%) Dwarf Lewis VEH PGE2

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155 Figure 425. Vertebral Bone Formation Rate (10-2 m3/ m2/d). There w ere no significant differences with PGE2 treatment in dwarf and Lewis rats Figure 426. Vertebral Cancellous Mineral Apposition Rate ( m/ d ). Only PGE2treated dwarf rats showed a significant decrease (P=0.0030) compared to their v ehicle treated controls 0 5 10 15 20 25 30 35 40 45 50Vertebral BFR/BS (10-2um3/um2/d) Dwarf Lewis VEH PGE2 0 .2 .4 .6 .8 1 1.2 1.4Vertebral Cancellous MAR (um/d) Dwarf Lewis VEH PGE2

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156 Figure 427. Longitudinal Bone Growth ( m/d). Only PGE2treated dwarf rats showed a significant increase (P<0.0001) compared to their vehicletreated controls A B Figure 428. Proximal tibial metaphyses from vehicle t reated (A) and PGE2treated dwarf (B) rats. Note the increased number of black stained trabecular bone spicules and the wider growth plate in the PGE2treated dwarf rat (Von Kossa/ tetrachrome stain, X20) Photos courtesy of Dr. Thomas J.Wronski 0 10 20 30 40 50 60 70 80 90 100Longitudinal Bone Growth (u/d) Dwarf Lewis VEH PGE2

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157 Figu re 4 29. Tibial Cancellous Bone Volume (%). PGE2 induced a significant increase only in dwarf rats compared to their controls (P = 0.0168 ) Figure 430. Tibial Trabecular Number (#/mm). PGE2treated dwarf rats exhibited a significant increase compared to their controls (P = 0.00 56), but not PGE2treated Lewis rats 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25Tibial Cancellous Bone Volume (%). Dwarf Lewis VEH PGE2 0 1 2 3 4 5 6 7 8Tibial Trabecular Number (#/mm) Dwarf Lewis VEH PGE2

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158 Figure 431. Tibial Trabecular Width ( m). PGE2treated Lewis rats showed a significant ly lower value compared to their controls (P=0.0227) whereas there was no difference in PGE2treate d dwarf rats Figure 432. Tibial Trabecular Separation ( m). There w ere no significant differences with PGE2 treatment in dwarf and Lewis rats 0 5 10 15 20 25 30 35 40 45 50Tibial Trabecular Width (um) Dwarf Lewis VEH PGE2 0 500 1000 1500 2000 2500 3000 3500 4000 4500Tibial Trabecular Separation (um) Dwarf LewisCell VEH PGE2

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159 Figure 433. Total Cortical Bone Tissue Area (mm2). There w ere no significant differences with PGE2 tr eatment in dwarf and Lewis rats Figure 434. Cortical Area (mm2). There was no significant difference in dwarf rats treated with PGE2, but PGE2treated Lewis rats showed a significant decrease (P=0.0256) 0 .5 1 1.5 2 2.5 3 3.5 4 4.5Total Cortical Bone Tissue Area (mm2) Dwarf Lewis VEH PGE2 0 .5 1 1.5 2 2.5 3 3.5Cortical Area (mm2) Dwarf Lewis VEH PGE2

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160 Figure 435. Marrow Area (mm2). PGE2tr eated dwarf rats showed a significantly increased mean value compared to their vehicletreated controls (P=0.0111) wh ereas PGE2treated Lewis rats showed no difference Figure 436. Periosteal Perimeter (mm). There w ere no significant differences with PGE2 treatment in dwarf and Lewis rats 0 .1 .2 .3 .4 .5 .6 .7 .8Marrow Area (mm2) Dwarf Lewis VEH PGE2 0 1 2 3 4 5 6 7 8Periosteal Perimeter (mm) Dwarf Lewis VEH PGE2

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161 Figure 437. Endocortical Perimeter (mm). PGE2trea t ed dwarf rats showed a significantly increased value compared to their vehicle treated controls (P=0.0273) wh ereas PGE2treated Lewis rats showed no difference Figure 438. Cortical Width ( m). PGE2treated Lewis rats showed a significantly decreased mean value compared to their vehicle treated controls (P=0.0049) wh ereas PGE2treated dwarf rats showed no difference 0 .5 1 1.5 2 2.5 3 3.5Endocortical Perimeter (mm) Dwarf Lewis VEH PGE2 0 100 200 300 400 500 600 700 800Cortical Width (um) Dwarf Lewis VEH PGE2

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162 Figure 439. Periosteal Mineralizing S urface (%). There w ere no sign ificant differences with PGE2 treatment in dwarf and Lewis rats Figure 440. Periosteal Mineral Apposition Rate (um/d). PGE2 treatment significantly increased the mean value in dwarf rats (P<0.0001), but not in Lewis rats 0 20 40 60 80 100 120Periosteal Mineralizing Surface (%) Dwarf Lewis VEH PGE2 0 .5 1 1.5 2 2.5 3 3.5Periosteal MAR (um/d) Dwarf Lewis VEH PGE2

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163 Figure 441. Periosteal Bone Formation Rate (102um3/um2/d). No significant differences were observed in dwarf and Lewis rats with PGE2 treatment Figure 442. Endocortical Mineralizing Surface (%). PGE2 treatment did not induce a significant effect in dwarf a nd Lewis rats 0 50 100 150 200 250 300 350Periosteal BFR/BS (10-2um3/um2/d) Dwarf Lewis VEH PGE2 0 20 40 60 80 100 120Endocortical Mineralizing Surface (%) Dwarf Lewis VEH PGE2

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164 Figure 443. Endocortical Mineral Apposition Rate (um/d). There was a significant increase in PGE2treated dwarf rats compared to their vehicle treated controls ( P<0.0001), but not in PGE2treated Lewis rats Figure 444. Endocortical Bone Formati on Rate (102um3/um2/d). PGE2treated dwarf rats exhibited a significant increase (P=0.0033) but no t PGE2treated Lewis rats 0 .25 .5 .75 1 1.25 1.5 1.75 2 2.25Endocortical MAR (um/d) Dwarf Lewis VEH PGE2 0 20 40 60 80 100 120 140 160 180Endocortical BFR/BS (10-2um3/um2/d) Dwarf Lewis VEH PGE2

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165 Figure 445. Biomechanical Load (N). PGE2treated dwarf and Lewis rats did not show significant differences compared to their vehicle treated controls Figure 446. Biomechanical stress (N*mm2). There was a significant increase in PGE2treated Lewis rats (P<0.0005) but not in PGE2treated dwarf rats 0 50 100 150 200 250 300Load (N) Dwarf Lewis VEH PGE2 0 5 10 15 20 25 30 35 40Stress (N*mm2) Dwarf Lewis VEH PGE2

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166 Figure 447. Biomechanical Stiffness (N/mm). No significant differe nce s were observed with PGE2 treatment in both dwarf and Lewis rats 0 200 400 600 800 1000 1200Stiffness (N/mm) Dwarf Lewis VEH PGE2

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167 CHAPTER 5 CHANGES IN GENE EXPR ESSION RELATED TO BO NE FORMATION AND BONE RESORPTION IN PTH AND PGE2TREAT ED RATS Introduction The polymerase chain reaction (PCR) has proven to be a rel iable and powerful method to amplify a targeted DNA molecule and quantify tissuespecific gene expression. Basically genes are transcribed by enzymes, such as RNA polymerases, thereby forming RNA. The characterization of RNA is almost always related to transcription (i.e. gene expression). The sequence of the RNA molecules correlates with the DNA from which they are derived (Livak and Schmittgen, 2001, Farrell Jr, 2005) U ltimately, all cell and tissue functio ns are governed by gene expression; in the skeleton, b one formation and resorption are under strict control of gene activation and suppression in response to physiological stimuli, hormones and growth factors, including IGF I and PTH. I nsulin L ike Growth Factor I (I GF I IGF1, Somatomedin C) According to the Gene NICBI (National Center for Biotechnology Information) database, the IGF1 gene, chromosome location 12q23.2 ( Homo sapiens ), encodes the protein IGF I, similar in structure to insulin, and is involv ed in mediating growth and development In the skeleton, s ystemic IGF I, regulated by growth hormone (GH ), has an important role in linear growth and peak bone acquisition, and local IGF I is involved in bone turnover T he literature is rich in studies demonstrating the effects of global, partial or tissuespecific deletion of the IGF I gene (Powell Braxton et al., 1993, Yakar et al., 1999, Rosen et al., 2004, He et al., 2006) on skeletal development and bone meta bolism. The role of IGF I as potential mediator for the bone anabolic effects of PTH has also been scrutinized in several in vivo and in vitro studies, since IGF I gene

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168 expression was found to be upregulated in bone cell cultures and in bone tissue from P TH treated rats (Linkhart et al., 1989, Dempster et al., 1993, Gunness, 1995, Bikle et al., 2002) Several other studies showed that PGE2 upregulates gene expression for IGF I in bone cells, increasing IGF I trans cripts by 2.2 fold (McCarthy et al., 1991) and that IGF I mRNA expression was correlated with osteogenesis (Harada et al., 1995) However, the molecular and cellular mechanisms underlying the skeletal effects of PTH and PGE2, and the role IGF I as a potential mediator are not completely clarified. To better understand the remarkable differen ces between the above mentioned studies and our previous findings with PTH and PGE2 treatment in IGF I deficient dwarf rats, we evaluated the changes in the abundance of IGF I and six other genes involved in bone metabolism. Collagen Type I The organic mas s of bone matrix is composed of about 90% collagen type I the most abundant protein in vertebrates providing the structural framework of the skeleton. It is also responsible for bone shape and its biomechanical properties such as resistance to pressure, torsion, and tension (Mark, 2006) In humans collagen type I is composed by two 1chains and one 2 chain, that are coded by different genes Col1a1 and Col1a2 which are very similar in structure. Mutation in either of these genes causes a genetic disease called osteogenesis imperfecta, or brittle bone disease, characterized by a d ecrease in bone mass, enhanced fragility and multiple fractures. Type I collagen synthesis can be modified by hormones, cytokines, vitamins, and growth factors, including IGF I (Bou Gharios and Crombrugghe, 2008) Type I collagen is synthesized by osteoblasts, and its gene expression is therefore indicative of bone formation.

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169 Osteocalcin Osteocalci n is a bone specific protein, secreted by osteoblasts, that participates in bone mineralization and calcium homeostasis. It is also known as bone gammacarboxyglutamic acidcontaining protein (BGLAP), encoded by the BGLAP or OC gene, and accounts for up t o 20% of noncollagenous protein in bone, with an affinity for bone mineral. However, when the affinity for hydroxyapatite is reduced in its uncarboxylated form, the osteocalcin molecule can enter the systemic circulation more easily and function as a horm one, regulating glucose metabolism, energy expenditure and fat mass (Lee et al., 2007, Clemens and Karsenty, 2011) Recent studies, in vitro and in vivo revealed that osteocalcin increases the number of pancreati c cells, insulin secretion and sensitivity, and release of adiponectin by fat cells (Lee et al., 2007, Ferron et al., 2008, Yoshikawa et al., 2011) The osteocalcin gene is not expressed in nonosseus cells, or ev en in osteoprogenitor cells I ts transcription is controlled by the runt related transcription factor 2 (Runx2), following osteoblast differentiation (Stein et al., 2008) Due to its specificity, osteocalcin is commonly used as a biomarker for bone formation. Osterix Osterix (Osx) is a transcriptional factor required for osteoblast differentiation and bone formation. When first identified by Nakashima in 2002 as a novel zinc finger containing transcription factor, o sterix was thought to be specifically expressed in developing bone cells, and required for osteoblast differentiation during embryonic development. No bone formation occurred when Osx expression was deleted in mice, which died at birth. M esenchymal cells did not deposit bone matrix and cells in the periosteum did not differentiate into osteoblasts (Nakashima et al., 2002) However,

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170 Zhou (2010) demonstrated that o sterix is also required for bone growth and homeostasis in the postnatal period, by deleting Osx in mice at several different point times postnatally. It was also noticed that the inactivation of Osx caused severe disruption in the morphology, maturation and function of osteocytes as well as accumulation of unresorbed calcified cartilage be low the growth plate (Zhou et al., 2010) RANKL The receptor activator of nuclear factor kappa ligand (RANKL) is also known as tumor necrosis factor related activationinduced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF). RANKL is a transmembrane ligand expressed in osteoblasts and bone marrow cell s. It binds to RANK (receptor activator of nuclear factor kappa which is expressed in osteoclast progenitor cells to induce osteoclastogenesis in the concomitant presence of M CSF (macrophage colony stimulating factor) (Asagiri and Takayanagi, 2007) In addition to osteoclast differentiation, RANKL is also essential t o induce osteoclast activation and, consequently bone resorption. RANK is present on the cell surface of mature osteoclasts, and in response to activation by its ligand, the osteoclast goes through structural changes that initiate bone resorption. Chang es occurring in the osteoclasts actin cytoskeleton are followed by formation of a sealed compartment adjacent to the bone surface, with release of lytic enzymes and consequent erosion o f the underlying bone (Boyle et al., 2003) Therefore, RANKL is the most important cytokine involved in osteoclast differentiation and activation, being essential for bone resorption. R A NKL is encode by the T nfss11 gene and RANK by the T nfss11a gene Mice with deletion of

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171 either of these genes exhibit severe osteopetrosis and def e ct ive tooth eruption due to a complete lack of osteoclas ts In osteopetrosis, the defect i n bone turnover due to the lack of osteoclastogenesis results in skeletal fragility despite an increase in bone mass, growth impairment, and hematopoietic insufficiency (N akashima et al., 2012) The genetic ablation of RANK also leads to defects i n immune system cells (B and T cells), demonstrating an additional role for RANKL in lymph node formation (Dougall et al., 1999) Osteo protegerin (OPG) Osteoprotegerin was first described in 1999 as a novel glycoprotein, and a member of the TNF (tumor necrosis factor) superfamily that regulate s bone resorption (Simonet et al., 1997) OPG particip ates in bone metabolism by competing with RANKL for binding to RANK, as it shows a strong homology to RANK receptors expressed in several tissues, including osteoblast lineage cells in bone. OPG acts as a decoy receptor for RANKL and therefore inhibits RA NKL mediated osteoclastogenesis and the survival of preexisting osteoclasts (Lian and Stein, 2006) The balance between RANKL and OPG determines osteoclast formati on and bone resorption activity OPG is encoded by T nfss11b gene and its deletion in OPG / mice results in an excess of RANKL activity with spontaneous fractures and vertebral deformities, although skeletal growth is not impair ed (Feige, 2001) Sclerostin Sclerostin is a glycoprotein encode d by the SOST gene and a potent negative regulator of bone formation (Keller and Kneissel, 2005) Sclerosteosis is an autosomal recessive condition characterized by progressive and generalized osteosclerosis, with enlargement of the jaw, thickening of the skull, sometimes causing cranial nerve

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172 entrapment and eventually potential loss of smell and hearing gigantism and syndactyly (Whyte, 2006, Canalis et al., 2007, Bezooijen et al., 2008) Although it is a rare bone disorder, sclerosteosis generated considerable interest due to the resulting phenotype that indicated a n imbalance in bone metabolism in favor of bone formation. T he bone formed was of overall good quality, with increased bone volume and bone mineral density without occurrence of pathological fractures (Bezooijen et al., 2008) In 2001, sclerosteosis was related to the deactivating mutation in the gene encoding scl erostin (SOST) and five mutations of the SOST gene have been identified to date (Whyte, 2006) Sclerostin is a member of the DAN (differential screening selected gene aberrant neuroblastoma) th at shares the ability to antagonize bone morphogenetic proteins (BMPs) Sclerostin is also reported to antagonize Wnt signaling (mammalian homologue of drosophila gene wingless, that induces differentiation o f boneforming cells), binding to the receptors LRP (lipoprotein receptor related protein) 5 and 6 (Canalis et al., 2007, Bezooijen et al., 2008) The inhibition of sclerostin production and/or activity by m onoclonal antibodies against sclerostin, preventing its binding to Wnt receptors can enhance Wnt signaling and increase bone mass in rodents and nonhuman primates (Warmington et al., 2005) Sclerostin antibody treatment has been evaluated in animal models, and it was reported to increase bone formation, bone mass and strength in a rat model for postmenopausal osteoporosis (Li et al., 2009) and increase bone healing in rats with metaphyseal fractures (Agholme et al., 2011) Its clinical applicability must be confirmed to avoid unwanted nonskel etal effects. Sclerostin is synthesized and secreted primarily by osteocytes. SOST is considered a target gene for the bone anabolic actions of PTH Its osteoblastogenic effects appear to

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173 result from the inhibitory effects of PTH on sclerostin synthesis by osteocytes (Keller and Kneissel, 2005, Bellido, 2006) Materials and M ethods Animal Models and Experimental Design Dwarf rats express significantly decreased IGF I serum levels compared to their back ground strain, Lewis rats, representing a major reduction of 60 % in circulating IGF I. Therefore, the dwarf rat is a reliable model to study the physiological changes seen in IGF I deficiency. In addition, GH/IGF I deficiency in the dwarf rat has profound negative effects on bone growth, accumulation of bone mass and osteoblast activity in both cortical and cancellous bone. For this reason, it is an adequate animal model to study the osteopenic effects caused by I GF I deficiency. Since IGF I may mediate the skeletal effects of bone anabolic agents, the dwarf rat is a lso a promising animal model for studies of these interactions. Female Lewis (background strain) and dwarf rats were obtained from Harlan Laboratories (UK). These animals were distributed in 6 experimental groups, as follows: 1. Female VEH treated Lewi s rats (N=13). 2. Female VEH treated Dwarf rats ( N =13) 3. Female PTH treated Lewis rats ( N = 7) 4. Female PTH treated Dwarf rats (N=10) 5. Female PGE2treated Lewis rats (N=7) 6. Female PGE2treated Dwarf rats (N=7) They were housed two per cage, with relative humidit y, air quality, illumination (12h light/12h dark cycles), and temperature controlled according to the criteria established by the Institute for Laboratory Animal Research and the National Research

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174 Council (2011) The protocol for us e of rats in this research project was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida. At 9 weeks of age, the rats were injected subcutaneously, daily for 2 weeks with vehicle hPTH 1 34 at a dose of 50 g/kg body weight or PGE2 at a dose of 3 mg/kg body weight according to their experimental groups. At the end of the treatment period all rats were euthanized by exsanguination under ketamine/xylazine anesthesia RNA E xtraction and cDNA S ynthesis RNA integrity is critical for all gene expression analyses and to reliably use real time RT PCR, high quality and undegraded RNA is required Bone t issue Distal f emora were selected in order to obtain greater RNA yields due to the size of the bones and the higher proportion of trabecular to cortical bone (Li et al., 2008) The bone samples were collected a t the time of euthanasia, dissected free of surrounding soft tissue snap frozen in liquid nitrogen and stored at 80 C until used for RNA extraction with Trizo l (Invitrogen, Carlsbad, CA) according to the manufacturers instructions adapted for bone tissue. Bone is a mineralized tissue, containing an abundant matrix rich in degradative enzymes, which makes the isolation of nondegraded RNA free from inhibitors a major challenge in the analysis of gene expression in the skeleton (Ireland, 2003) For these reasons, before the Trizol extraction, bone samples were pulverized using a 6750 Freezer Mill and Auto Extractor 6814 (Spex CertiPrep, Metuchen, NJ) (Figure 5 1 ) to guarantee adequate cellular disruption, as RNA is isolated from t he nucleus or cytoplasm by direct cell lysis (Farrell Jr, 2005) This resulted in good quality pellets of large size and easily visible after the extrac tion protocol (Figure 52 ).

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175 Following RNA extraction, the concentrations were determined with a Nanodrop spectrophotometer (ND 1000, ThermoFisher, Wilmington DE), and based on these values, all samples were treated with an RNase free DNase kit ( Qiagen RNea sy Plus), to remove any residual genomic DNA. The concentrations were re evaluated for quantity control, the n sent to the Interdisciplinary Center for Biotechnology Research at the University of Florida (ICBR) for quality control The integrity of the RN A was measured by capillary electrophoresis using an Agilent Bioanalyzer, 2100 model that provides an improved method to visualize nucleic acids, as software generates gel like images, an electropherogram, and displays sample concentration and ribosomal r atio, as shown in one of our reports in Figure 53. If the obtained RNA integrity number (RIN) did not reveal good RNA quality, the sample was excluded from further analysis. The assessment of RN A integrity is a critical step in obtaining reliable gene expression data The RNA samples were converted to cDNA with a High Capacity cDNA Archive kit, following the manufacturers protocol (Applied Biosystems, Foster City, CA). Once synthesi zed, the cDNA was stored at 20 C until quantitative real time PCR ( qR T PCR) was performed. Hepatic t issue Liver samples were homogenized with Trizol ( Invitrogen, Carlsbad, CA) with a TissueLyser LT ( Qiagen ) machine that speeds the extraction process for soft tissue samples RNA extraction was then performed following the same protocol as for bone samples, with the same quantity and quality control, and converted to cDNA. Quantitative R eal T ime PCR Primers and probes for each gene were designed from the corresponding murine ( Rattus norvegicus ) mRNA in the NCBI Reference Sequence (National Center for

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176 Biotechnology Information Gene Database), with Primer Express software (Applied Biosystems), as seen in Figures 54 to 5 10. The optimization and efficiency of the primers and probes were performed for all seven genes before re al time PCR reactions. The abundance of 18S rRNA (Applied Biosystems), used as endogenous control, was also measured for each sample. The relative expression of all seven genes, IGF I, Collagen Type I, Osteoc alcin, Osterix, RANKL, OPG, and Sclerostin were determined by qRT PCR using FAM Taqman probes and primers, and Taqman RT PCR master mix (Applied Biosystems, Foster City, CA). All samples were run in triplicate for each gene and for 18S rRNA. Each plate was set up to include sample unknowns from all experimental groups (vehicle, PTH and PGE2treated rats) for a single gene, plus notemplate controls from each primer/probe pairs. For liver samples, only the IGF I gene was evaluated in order to compare the abundance of IGF I synthesized in the liver to the local IGF I produced in bone tissue, using the same endogenous control (18S rRNA). Calculations and Statistical A nalysis In each PCR assay, the cycle threshold (Ct) value was obtained in triplicate for each sample, for each gene, and in all groups. Relative mRNA expression of each gene was calculated by determining t mean Ct for each gene and the mean Ct for 18S rRNA mRNA from the same sample. Values for by ANOVA ( analysis of variance) followed by a multiple comparison test (Scheffe post h oc analysis, StatView SAS Software Institute Inc.) to compare the effects of PTH and PGE2 on expression of each gene. Differences were considered significant at P<0.05. Data are depicted in graphs as the mean fold change in mRNA relative to the control group (vehicle treatedrats). The f old change

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177 was calculated using the 2method, as described by Livak (20011), the sample (Livak and Schmittgen, 2001) Results IGF I Bone There was no significant difference in IGF I gene expression in local bone tissue between dwarf and Lewis rats (P=0.9956). The expression of IGF I was significantly decreased by PGE2 treatme nt in dwarf ( P<0.0001) and Lewis rats (P<0.0001). PTH treatment induced a slight decrease in the expression of IGF I in dwarf rats (P<0.0506) but no significant difference was observed in Lewis rats (P=0. 0946), as seen in Figures 5 11 and 512. Liver IGF I expression in the liver was not significantly different between vehicletreated dwarf and Lewis rats (P=0.9994), and between PGE2treated (P=0.1646) and PTH treated dwarf rats (P=0.9912) compared to their vehicle treated controls. PGE2 treatment induced a significant increase in liver IGF I expression in Lewis rats (P<0.0115) and there was a strong trend for an increase in PTH treated Lewis rats (P<0.0562), as seen in Figure 513 and 514 Collagen Type I There was no significant difference in collagen type I gene expression when comparing vehicletreated dwarf and Lewis rats (P=0.9966). Only PGE2 treatment induced a significant decrease in the expression of collagen type I in both dwarf and

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178 Lewis rats There were no significant differences caused by PTH treatment in dwarf (P=0.9628) and Lewis rats (P=0.9811), as seen in Figures 515 and 516. Osteocalcin The expression of osteocalcin was affected by PGE2 treatment, showing a significant decrease in both dwarf (P<0.0001) and Lewis rats (P<0.0004), but PTH treatment induced a significant decrease only in dwarf rats (P <0.0058), with no effect in Lewis rats ( P=0.9998), as seen in Figures 517 and 518 Osterix Osterix gene expression was significantly decreased in PGE2treated dwarf (P<0.0001) and Lewis rats (P<0.011), wh ereas PTH treatment did not induce any significant changes in dwarf (P=0.999) or Lewis rats (P=0.7968), as seen in Figures 519 and 520. RANKL There was a statistically significant decrease in RANKL gene expression in PGE2treated dwarf (P <0.0001) PGE2treated Lewis (P<0.0001), and PTH treated Lewis rats (P<0.005), but no effect i n PTH treated dwarf rats (P=0.1235), as seen in Figures 521 and 522. OPG There was a statist ically significant decrease in OPG gene expression in PGE2treated dwarf (P<0.0001) and Lewis rats (P<0.0001), but no significant changes w ere induced by PTH treatment in dwarf ( P=0.2787) and Lewis rats (P=0.0603), as seen in Figures 523 and 524.

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179 Sclerostin G ene expression for sclerostin was affected by both treatments i n dwarf rats, with statistically significant decreases for PGE2 (P<0.0004) and PTH (P<0.001) In Lewis rats, gene expression for sclerostin was significantly decreased by PGE2 treatment, but no significant change w as observed with PTH treatment (P =0.2032) as seen in Figure 5 25 and 526. Discussion Surprisingly, o ur findings did not reveal a significant difference in the IGF I gene expression in the liver, nor in the local bone tissue of vehicletreated dwarf and Lewis rats. These results contrast strong ly with the decreased serum levels observed in dwarf rats, regardless if treated with vehicle, PTH or PGE2. We speculate that if RNA transcription is not affected, as indicated by our findings, the production of IGF I can be altered at the translati onal l evel, with a decrease in protein synthesis, and consequently in serum levels Similarly, in other studies, protein levels did not reflect changes in RNA abundance (Wood and Giroux, 2003) Flyvberg et al. also use d dwarf rats to study the effects o f isolated growth hormone and IGF I deficiency on diabetic renal hypertrophy, and observed that despite transient kidney IGF I accumulation, no increase was found in kidney mRNA during the first 4 days of the onset of diabetes, indicating that local IGF I synthesis at the transcriptional level was unchanged (Flyvbjerg et al., 1992) In another study with GH deficient (dw/dw) rats, hIGF I treatment was associated with increased longitudinal growth, weight gain, and increased plasma IGF I levels, but not in hepatic IGF I mRNA expression, when c ompar ed to saline control treatment. Insulin treatment, however, increased the hepatic IGF I mRNA expression, with a reduction in plasma IGF I levels (Butler et al., 1996) However, m ale Wistar rats

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180 hypophysectomized on postnatal day 26, showed markedly reduced serum GH and IGF I levels compared to intact controls, as w ell as lower IGF I mRNA levels in the liver (Domene et al., 1993) IGFBP 3 mRNA levels were also reduced in hypophysectomized animals Considering that h epatic IGFBP 3 and ALS synthesis are stimulat ed by GH (Molina, 2006a) and that they form the ternary complex that represents 80% of the circulating IGF I we hypothesize that if the IGFBP 3 production is decreased at any level it could also affect the bioavailability of IGF I in the circulation and tissues as well. IGFBPs and ALS were not evaluated in our experiments It is also relevant that the dwarf rats not only presented lower levels of serum IGF I, but marked phenotypic changes in bone structure and metabolism were detected by histomorphometric and pQ CT analyses in our previous experiments. RANKL was significantly decreased in PGE2treated dwarf and Lewis rats, or showed a trend for a decrease in both groups of PTH treated rats while OPG gene expression was decreased by only PGE2 treatment in dwarf and Lewis rats. Although unclear, since PTH is considered to participate in the regulation of RANKL and OPG (Huang et al., 2004) these findings did not lead us to emphasize the involvement of these two main regulat ors of bone resorption on the observed bone anabolic effects, mainly with PT H treatment. Type I collagen, osteocalcin and osterix gene expression were not induced by PTH and PGE2 treatments in a manner consistent with the observed increase in bone formati on However, the decreased expression of the SOST gene in dwarf rats treated with PTH and PGE2 is consistent with studies that relate this gene to the anabolic effects of PTH through a direct inhibitory effect on osteocyt ic synthesis of sclerostin and it s antagonist ic action on the Wnt signaling pathway (Bellido,

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181 2006, Canalis, 2010, ten Dijke et al., 2008) Keller (2005) demonstrated that PTH strongly suppresses SOST expression in vivo and in vitro, and consider ed SOST a direct target gene for PTH, regulated at the transcription level (Keller and Kneissel, 2005) Mice with overexpression of the SOST gene were also found to have a blunted response to PTH induced bone gai n (Kramer et al., 2010) Interestingly, the dwarf rats, deficient in IGF I showed a significant decrease in SOST gene expression with PTH treatment while Lewis rats, with normal IGF I levels did not This finding contrasts with several studies in vitro and in vivo that postulate a role for IGF I as an essential mediator for the stimulatory effects of PTH on bone formation (Canalis et al., 1989, Dempster et al., 1993, Gunness, 1995, Bikle et al., 2002) Conclusions The major findings were: 1. G ene expression for IGF I in bone tissue did not show a significant difference between IGF I deficient dwarf rats and their background strain, Lewis rats 2. Although dwarf rats show lower levels of serum IGF I, gene expression for IGF I in the liver was not significant ly different compared to Lewis rats. 3. In dwarf rats, PTH treatment did not change the abundance of IGF I mRNA in bone or liver, nor the expression of genes related to bone formation : collagen type I, oste ocalcin, and osterix Genes related to bone resorption, RANKL and OPG, also did not show any significant differences in PTH treated dwarf rats 4. PGE2 treatment induced significant decrease s in the gene expression for IGF I in bone (liver IGF I decreased only in Lewis rats) and all the genes involved in bone formation (collagen type I, osteocalcin, and osterix) and bone resorption (RANKL and OPG). 5. SOST/ sclerostin gene expression was downregulated in dwarf rats treated with PTH and PGE2, despite their low IGF I serum levels

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182 A B Figure 51 Extractor (A) and f reezer mill (B) used to pulverize bone tissue. Photos courtesy of Dr. Ana Cristina F. Bassit Figure 52 RNA pellets obtained from bone samples Photo courtesy of Dr. Ana Cristina F. Bassit

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183 F igu r e 53. Bioanalyzer results indicating high RNA quality and integrity

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184 Figure 54. IGF I primers and probe sequences obtained with Primer Express 3.0

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185 Figure 55. Collagen Type I primers and probe sequences obtained with Primer Express 3.0

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186 Figu re 5 6. Osteocalcin primers and probe sequences obtained with Primer Express 3.0

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187 Figure 57. Osterix primers and probe sequences obtained with Primer Express 3.0

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188 Figure 58. RANKL primers and probe sequences obtained with Primer Express 3.0

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1 89 Figure 5 9. Osteoprotegerin primers and probe sequences obtained with Primer Express 3.0

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190 Figure 510. Sclerostin primers and probe sequences obtained with Primer Express 3.0

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191 F igure 511. Fold change in bone IGF I expression in dwarf rats (RQ=relative q uantity) Figure 512. Fold change in bone IGF I expression in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in IGF I gene expression in Dwarf rats 0 0.5 1 1.5 RQLEW PTH RQ LEW VEH RQ LEW PGE2Fold change in IGF I gene expression Lewis rats

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192 Figure 513. Fold change in liver IGF I expression in dwarf rats Figure 514. Fold change in liver IGF I expression in Lewis rats 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in Liver IGF I gene expression in Dwarf rats 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold chang e in Liver IGF I gene expression in Lewis rats

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193 Figure 515. Fold change in c ollagen type I gene expression in dwarf rats Figure 516. Fold change in collagen type I gene expression in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in Collagen Type I gene expression in Dwarf rats 0 0.5 1 1.5 RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold change in Collagen Type I gene expression Lewis rats

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194 Figure 517. Fold change in osteocalcin gene expression in dwarf rats Figure 518. Fold change in osteocalcin gene expr ession in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in Osteocalcin gene expression in Dwarf rats 0 0.5 1 1.5 RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold change in Ostteocalcin gene expression Lewis rats

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195 Figure 519. Fold change in osterix gene expression in dwarf rats Figure 520. Fold change in osterix gene expression in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in Osterix gene expression in Dwarf rats 0 0.5 1 1.5 RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold change in Osterix gen expression Lewis rats

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196 Figure 521. Fold change in RANKL gene expression in dwarf rats Figure 522. Fold change in RANKL gene expression in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in RANKL in geneexpression dwarf rats 0 0.5 1 1.5 RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold change in RANKL gene expression Lewis rats

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197 Figure 523. Fold change in OPG gene expression in dwarf rats Figure 524. Fold change in OPG gene expression in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in OPG gene expression in Dwarf rats 0 0.5 1 1.5 RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold change in OPG gene expression Lewis rats

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198 Figure 525. Fold change in sclerostin gene expression in dwarf rats Figure 526. Fold change in s clerostin gene expression in Lewis rats 0 0.5 1 1.5 RQ DW PTH RQ DW VEH RQ DW PGE2Fold change in Sclerostin gene expression in Dwarf rats 0 0.5 1 1.5 RQ LEW PTH RQ LEW VEH RQ LEW PGE2Fold change in Sclerostingene expression Lewis rats

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199 CHAPTER 6 SUMMAR Y AND CONCLUSIONS Summary of Experiment al Findings Study 1 Summary In study 1, we explored the potential use of the dwarf rat as an animal model to stu dy the effects of deficiency in the GH/IGF I axis. The overall growth and development of the dwarf rat were compared to those observed in their background strain, the Lewis rat. Body weight and femur length wer e evaluated throughout the study. IGF I ser um levels were measured and we systematically analyzed changes in the skeleton and bone tissue by histomorphometry, pQCT and biomechanical testing. Our results showed that body weight and femur length were significantly decreased in the dwarf rat, compare d to the Lewis rat. Most importantly, the dwarf rat expressed significantly decreased IGF I serum levels compared to its back ground strain, Lewis rats, representing a major reduction of 60% in circulating IGF I. In addition, the dwarf rat expressed an ev ident osteopenic phenotype, with profound negative effects on bone growth, accumulation of bone mass, and osteoblast activity in both cortical and cancellous bone. Consequently, decreased bone strength was also observed, with significantly lower load to f ailure in dwarf rats For these reasons, we concluded that the dwarf rat is an adequate animal model to study physiological changes seen in IGF I deficiency and the related osteopenic effects on bone structure and metabolism. Since IGF I may mediate the skeletal effects of bone anabolic agents; the dwarf rat is also a promising animal model for studies of these interactions.

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200 Study 2 Summary Based on the results of study 1, the experiments in study 2 were designed using the same methods to test the bone an abolic effects of intermittent PTH treatment in IGF I deficient dwarf rats. We observed that PTH did not affect body weight or serum IGF I levels in either Lewis or dwarf rats. However, PTH stimulated both cancellous and cortical bone formation, and induced highly significant anabolic effects in vertebral and tibial cancellous bone in dwarf rats, despite low circulating levels of IGF I. Study 3 Summary Similarly to study 2, the experiments in study 3 were also based on the methods and results obtained in study 1, but designed to evaluate the effects of PGE2 on bone mass and formation. PGE2 treatment did not increase body weight or serum IGF I levels in either Lewis or dwarf rats, but our results indicated that PGE2 treatment increased bone mass in dwarf r ats, mainly in cancellous bone, despite lower IGF I serum levels. Study 4 Summary In study 4, PCR assays were performed to evaluate changes in the expression of genes involved in bone formation and bone resorption in response to PTH and PGE2 treatments in dwarf and Lewis rats. Liver IGF I gene expression was also evaluated, as an attempt to differentiate the expression of local from systemic IGF I. The results of this study demonstrated that, although dwarf rats showed lower levels of serum IGF I, gene ex pression of IGF I in the liver was not significantly different compared to Lewis rats. Similarly, gene expression of IGF I in bone tissue did not show a significant difference between IGF I deficient rats and their background strain, Lewis rats.

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201 The resul ts obtained in study 4 also demonstrated that PTH treatment did not affect the abundance of IGF I in bone or liver, nor the genes related to bone formation: collagen type I, osteocalcin, and osterix in dwarf rats. Genes related to bone resorption, RANKL a nd OPG, were not affected by PTH treatment either. PGE2 treatment induced a significant decrease in gene expression of IGF I in bone (liver IGF I decreased only in Lewis rats) and all the genes involved in bone formation (collagen type I, osteocalcin, and osterix) and bone resorption (RANKL and OPG). SOST/sclerostin gene expression was downregulated in dwarf rats treated with PTH and PGE2, despite their low IGF I serum levels Discussion In study 1, we explored the potential use of the dwarf rat as an ani mal model to study the effects of deficiency in the GH/IGF I axis. Several animal models have been developed for this purpose, since 1959, and even earlier, when ordinary laboratory rats, by that time merely described as albino rats, were hypophysectomized and treated with growth hormone. Korner (1959) observed that hypophysectomized female albino rats, not only showed a decrease in thei r body weight and organ size, but also a diminished incorporation of amino acids into proteins in hepatic cells in vitro strongly suggesting that protein biosynthesis in vivo was also decreased. It was also demonstrated that these effects could be suppressed with growth hormone treatment (Korner, 1959) Since th ese early studies, many different strains of genetically modified animal models were created to investigate IGF I deficiency. These mutant animals have been unquestionably useful in studies for the understanding of cellular and biochemical pathways involved i n the regulatory role of IGF I, as an endocrine and paracrine hormone, in skeletal development and mineral acquisition. However, among the

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202 recently compiled 5 5 different mouse models, only 26 of the created genetic mutations refer to a similar condition i n the context of human IGF I deficiencies (Yakar et al., 2010) Reproducing the characteristics of a disease in a reliable manner is a major requirement when selecting the most adequate anim al model for research. One of our main goals was to evaluate the effects of IGF I deficiency on the skeleton and bone metabolism, but in close similarity to the conditions presented in clinical cases. In this category we consider not only osteoporosis in postmenopausal women, but also other disorders that lead to decreased IGF I levels and osteopenia, including the juvenile onset of osteoporosis, anorexia nervosa and hypothalamic amenorrhea, glucocorticoidinducedosteoporosis, chemotherapy, and alcohol abuse. This was one of the reasons why knockout animals were not selected for our studies, not to mention the poor health status of these animals. Since we also wanted to evaluate how GH/IGF I deficiency would affect bone growth, young rats were used in our studies even though it was clear that their age could be considered a limitation to the applicability of our results to adult osteoporosis and other agerelated conditions in which IGF I levels are decreased. When evaluating the rat as an animal model for osteoporosis studies in the past, many researchers considered that these rodents w ere not suitable due to predominant modeling activity (rather than remodeling) in cancellous bone and the lack of Haversian remodeling in cortical bone. In addition, ost eoporosis in humans causes spontaneous and low impact fractures, whereas the rat does not develop fragility fractures (Jee and Yao, 2001) However, bone remodeling is now known to occur in cancellous bone of adult rats

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203 (Erben, 1996) Furthermore, it has been recently considered that most of the protocols to induce osteopenia and osteoporosis, by hormonal (surgical or pharmaceutical) and dietary interventions, as well as by immobilization can be used in skeletally immature or mature rats if low peak bone mass is achieved (Lelovas et al., 2008) The dwarf rats in study 1 presented evident osteopenic features, with negative effects on bone growth and accumulation of bone mass When considering drug development and approval, guidelines for the FDA and the European Agency for the Evaluation of Medicinal Products (EMEA) require that new potential agents for osteoporosis therapy must be tested in two animal species: rodents (preferably rats) and a nonrodent, large animal model (Thompson et al., 1995, Avouac, 2003, Bagi et al., 2011) Rats reach sexual and skeletal maturity when they are about 2.5 and 10 months old, respectively, although in som e long bones, the epiphyseal plate remains open after 30 months (Jee and Yao, 2001, Lelovas et al., 2008) The female dwarf and Lewis rats chosen for our studies were five week s old at the beginning of the experim ents ; PTH and PGE2 treatments started when they reached 9 weeks of age and they were euthanized at 11 weeks. Therefore, the results obtained can be extrapolated to juvenile patients affected by anorexia nervosa (AN), a condition characterized by severe w eight loss and self induced chronic starvation ass ociated with decreased IGF I serum levels severe bone loss and a twoto three fold increase in fractures (Hofman et al., 2009, Lawson et al., 2010, JacobsonDickm an and Misra, 2010, Misra and Klibanski, 2011) It occurs predominantly in women, affecting 0.2 to 1% of adolescent girls and 1 to 4% of college age young women although the incidence in men has increased in recent years (Jacobson Dickman and Misra, 2010) In a study of AN prevalence, demographic data

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204 of 286 female patients (range 1554 years), the average age was 25 years with 53 patients younger than 18 years (Hofman et al., 2009) AN presents critical negative effects on growth and maturation of the skeleton, and bone mass accrual during a period in life in which peak bone mass should be reached. These residual deficits in bone mass and accrual persist even after achieving weight gain and resuming menstrual function with treatment representing a higher risk of developing osteoporosis later in adulthood (JacobsonDickman and Misra, 2010, Misra and Klibanski, 2011) Malnutrition, and estrogen and androgen deficiency are related to lower IGF I levels and resistance to GH (Gianotti et al., 1998, JacobsonDickman and Misra, 2010) Systemic IGF I levels are markedly decreased in patients with AN compared to normal subjects (Gianotti et al., 1998) as well as the serum levels of acidlabile subunit (ALS) (Fukuda et al., 1999) and IGFBP 3, whereas IGFBP 2 levels are increased (H otta et al., 2000) Nutrition participates in the regulation of IGF I and other anabolic hormones, and low IGF I levels in anorectic patients are correlated with decreased BMD and with negative effects on bone architecture, represented by decreased bone volume, trabecular number, trabecular thickness and increased trabecular separation (Lawson et al., 2010) When comparing the results obtained with PTH and PGE2 treatments in studies 2 and 3, it becomes evident that the effects of PTH on bone mass and formation were superior t o those of PGE2, both in cancellous and cortical bone of dwarf and Lewis rats. In addition, we observed adverse side effects with PGE2 treatment, such as acute abdominal pain, usually related to increased motility in the gastrointestinal and genitourinary tracts. In study 2, t he surprisingly strong bone anabolic effects of PTH in IGF I deficient dwarf rats introduced new questions about the bioavailability and

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205 regulation of IGF I, IGFBPs, and ALS; about their synthesis and action in the local tissues; abo ut how they are influenced, by other hormones, such as PTH; and what mechanisms are involved. It is known that IGF I knockout mice do not respond to PTH treatment (Miyakoshi et al., 2001, Bikle et al., 2002) but it is not understood if when IGF I levels are decreased, instead of abrogated, which levels would be considered as the minimum required to allow the bone anabolic action of PTH. K nockout mice were extensively used to demonstrate that not only IGF I was r equired for the PTH anabolic actions on bone, but also its receptor IRS 1 (Wang et al., 2007) and the t wo other components of the circulating ternary IGF I complex, ALS and IGFBP 3 (Yakar et al., 2006) However, the balance bet ween the systemic and local actions of IGF I is complex, and so is the regulation by six different binding proteins, and the interaction of IGF I with other hormones, notably GH and PTH (Yakar et al., 2010, Bikle an d Wang, 2011) Still, w e could refer to one study, by Miyakoshi (2001), who also tested whether low levels of systemic IGF I would restore the actions of PTH on bone, comparing IGF I knockout mice, IGF I midi mice (an incomplete knockout for IGF I), and w ild type controls. Total BMC and areal BMD, measured by DEXA, and total BMD measured by pQCT, were significantly increased by PTH treatment in IGF I midi mice compared to the control group, whereas there was no significant effect of PTH treatment in IGF I knockout mice. Serum IGF I levels in IGF I midi mice were considered 30% of normal, based on literature reference, but were not measured in th is experiment, and no bone his tologic analysis was performed (Miyakoshi et al., 2001)

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206 Collagen type I gene expression was downregulated in both dwarf and Lewis rats treated with PGE2, and we considered that it could reflect in part the heterogeneity of the results obs erved in pQCT and histomorphometr ic analyses. For example, PGE2 treatment increased trabec ular BMD and BMC only in dwarf rats, but cortical bone parameters were decreased. C ancellous bone volume was increased only in the proximal tibia of dwarf rats, but not in the lumbar vertebra, although there was an increase in the number of trabeculae. T here are numerous growth factors, such as IGFs, BMPs, FGFs, steroid hormones and cytokines involved in the regulation of collagen synthesis and metabolism in bone (Mark, 2006) Despite its bone anabolic effects in in vivo studies, i n many cell culture systems, PGE2 has been shown to affect collagen by increasing its degradation and also to inhibit collagen synthesis, although the receptor and signaling mechanisms for the effect s of PGE2 are not fully explained (Raisz et al., 1993b, Fall et al., 1994) PGE2 stimulates IGF I synthesis in osteoblast cell cultures from rat bone (McCarthy et al., 1991) but i nhibits alpha 1 pr ocollagen gene transcription in osteoblastic cell cultures, in the presence or absence of IGF I (Raisz et al., 1993b) Similarly, PGE2 also inhibits type I collagen gene expression in fibroblasts at the transcriptional level (Go ldring et al., 1996, Riquet et al., 2000) and delays chondrocyte maturation acting on BMP signaling i n murine cell cultures (Clark et al., 2009) PGE2 treatment also decreased osteocalcin gene expression in dwar f and Lewis rats. In cultures of a human osteoblast like cell line MG 63 treated with bovine PTH (134) and PGE2, a significant inhibit ion of osteocalcin secretion in response to the active form of vitamin D3 was observed (Lajeunesse et al., 1991)

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207 PTH treatment did not i nduce any significant difference in the mRNA abundance of collagen type I in dwarf and Lewis rats as w ould be expected, but in vitro results are conflicting. Kream (1980) observed that there was a 50% reduction in collagen synthesis and a 40% decrease in procollagen mRNA levels in fetal rat calvaria cell cultures, an effect detectable after 6h of PTH treatment (Krea m et al., 1980) whereas Thiebaud (1994) found that PTH increased alpha 1collagen mRNA levels in osteoblastic cell cultures (Thiebaud et al., 1994) Osteocalcin is the most abundant noncollagenous protein in bone tissue, synthesized by mature osteoblasts PTH stimulates bone formation mainly by targeting cells of the osteoblast lineage. PTH increas es osteoblast number and activity, decreases osteoblast apoptosis, and increases bone remodeling, favoring bone form ation (Dempster et al., 1993, Jilka et al., 1999, Hodsman et al., 2005) Therefore, the decreased expression of osteocalcin in PTH treated dwarf rats seems to conflict with the dramatic evidence for increased bone formation in these animals. However, other studies of the relationship between osteocalcin gene expression and PTH also show contrasting results. Noda (1988) used rat osteoblast like osteosarcoma cells (ROS17/2.8) and observed that hPTH ( 1 34) increased osteocalcin mRNA levels in a dosedependent manner and this effect would last up to 48h, with a peak at 24h (Noda et al., 1988) Sutherland (1994) also using an osteosarcoma cell line (SaOS 2) found that hPTH ( 1 34) had no significant effects on alkaline phosphatase (another indicator of bone formation, resulting from osteoblast activity) or osteocalcin mRNA levels (Sutherland et al., 1994) It is also intriguing that the lack of osteocalcin, by deleting both OG1 and OG2 genes in mice, l eads to an increase in bone formation (Ducy et al.,

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208 1996) In this striking study, o steocalcin deficient mice (oscm1/oscm1) were reported to be normal at birth, without skeletal malformations, viable and fertile, with no bone structural differences when compared to wild type mice till 6 months of age. At this age, important changes started to develop: increased cortical thickness and density, accompanied by an increased amount of mineralized bone matrix, and increased width of the diaphysis. Cancellous bone was al so increased in the mutant mice compared to their wild type littermates, as was bone strength, evaluated by measurement of failure load as a biomechanical indicator. Histomorphometric analyses of fluorochrome labeling showed an increased bone formation rate, although osteoblast surface and osteoblast number were not increased. On the other hand, there was an increase in osteoclast number and surface, and bone resorption occurred normally when these animals were ovariectomized. Most importantly, this study demonstrated that in osteocalcin null mice, bone formation rate increased without a an increase in osteoblast number, indicating that the osteoblasts were depositing more bone matrix (Ducy et al., 1996) leading t o the hypothesis that the synthesis of osteocalcin by the most mature osteoblasts would slow down the anabolic activity of these cells (Kronenberg, 1997, Yu and Chandrasekhar, 1997) PTH activated the osteocalcin promoter in osteosarcoma cell line (SaOS 2) cultures transiently, as very little induction was seen after 24 and 48h of treatment with PTH analogs (PTH 184, PTH 134, and PTH 131) (Yu and Chandrasekhar, 1997) Y et, we evaluated gene expression for osteocalcin at 14 days of PTH treatment, and we could have possibly exceeded the time for osteocalcin changes at the transcriptional level at that specific time point, although the final effects of increased bone format ion were evident in pQCT,

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209 histomorphometric and biomechanical analyses. We did not evaluate osteocalcin serum levels A comparison of protein levels with gene expression in bone tissue may have allowed us to distinguish between the transcriptional and tr anslational effects of PTH on osteocalcin. PTH treatment did not induce any significant change in RANKL gene expression in dwarf rats, but a decrease was observed in Lewis rats, whereas no significant changes were seen in OPG gene expression in dwarf and Lewis rats. In an attempt to quantify how PTH could induce cha n ges in RANKL and OPG gene expression at different stages of osteoblast differentiation, Huang (2004) made measurements at specific timepoints in mouse primary bone marrow stromal cells. PTH i nduced minimal increases in RANKL gene expression from days 7 to 14, but a significant t difference was observed at day 21 (2 fold) and day 28 (3 fold). OPG gene expression was inhibited with PTH treatment, with a peak on day 14, and continued to show inhibitory effects at days 21 and 28, and these results were related to increased osteoclastogenesis (Huang et al., 2004) Suda (2004) also related PGE2 to the inhibition of OPG gene expression (Suda et al., 2004) and RANKL mRNA levels were significantly increased in osteoporotic primary osteoblast cell cultures (Jurado et al., 2010) Osterix, a transcription factor involved in osteoblast di fferentiation and bone formation, was downregulated by P GE2, but not by PTH treatment. These results were puzzling, as Wang found that PTH (134) stimulated osterix mRNA expressi on in a dosedependent manner, at low concentrations only (Wang et al., 2006) whereas in another study, PTH (1 34) treatment inhibited osterix mRNA levels and downregulated

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210 protein expression in osteosarcoma (UMR) cells and murine calvaria (Hong et al., 2009) PTH and PGE2 treatment significantly decreased sclerostin gene expression in dwarf rats. As menti oned before, PTH inhibits sclerostin expression in vitro and in vivo with some different responses in vivo that could be attributed to variations in time points at which sclerostin expression was analyzed after the last injection of PTH (Keller and Kneissel, 2005, Bellido, 2006) Sclerostin antagonizes Wnt signaling, a key signaling pathway in developmental processes, binding to the low density lipoprotein receptor related protein, LRP5, resulting in decreased bon e formation (Bezooijen et al., 2008) Similar to sclerostin, LRP5 was discovered in studies of two rare diseases, high bone mass syndrome (HBM) and osteoporosis pseudoglioma syndrome (OPPS) HBM is an autosomal dominant condition, characterized by gainof function of LRP5, lead ing to increased bone mass and formation, with a variable symptomatology, ranging from almost no symptoms to jaw enlargement, craniosynostosis, cranial nerve entrapment, and developmental delay OPPS is a low bone mass disorder, wit h occurrence of juvenile onset osteoporosis, fractures, and congenital or infancy onset blindness, related to a loss of function mutation in the LRP5 gene (Balemans et al., 2008, Bezooijen et al., 2008, Ralston, 200 8, Cui et al., 2011) The mechanisms involved in bone mass regulation by LRP5 are not fully understood, but in vitro studies suggest a decreased inhibition of Wnt signaling by the antagonist proteins sclerostin and Dickkopf 1 (DKK1) (Li et al., 2005, Pangrazio et al., 2011, Cui et al., 2011) A complete lack of DKK1 is incompatible with life, but partial loss of function mutation in mice leads to

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211 increased bone mass, whereas mice that overexpress DKK1 in osteoblasts have reduced bone mass (Balemans et al., 2008, Cui et al., 2011) Sclerostin and DKK1 can both bind to LRP5 ; they act independently to antagonize Wnt signaling, a nd compete for binding to this receptor (Balemans et al., 2008) In view of these findings, we consider the effects of PTH treatment in LRP5 knockout mice. If no increase in bone formation occurs, the sclerostin downregulation mechanism of PTH action would be supported. On the other hand, if increased bone mass is demonstrated in LRP5 knockout mice treated with PTH an alternative pathway involving one of the canonical Wnts reported to antagonize sclerostin, such as Wnt10b whose overexpression increases bone m ass in mice, would then be considered (Bennett et al., 2005, Bennett et al., 2007, Bezooijen et al., 2008) PTH has been reported to induce bone anabolic effects in LRP5 knockout mice to the same extent as in wildtype mice (Sawakami et al., 2006, Iwaniec et al., 2007) Therefore, there must be some redundancy in the signaling pathways through which PTH stimulates bone formation. Recently, PGE2 was found to decrease sclero stin expression in osteoblastic cell culture, without affecting DKK1 (Genetos et al., 2011) and in osteoblastic cells concomitantly exposed to mechanical strain (Galea et al., 2011) One of the limitations in the present study, relating mainly to PGE2, was the relatively short period of treatment. In other studies in which PGE2 induced stronger bone anabolic effects, the period of treatment varied from 60 to 180 days of continuous daily injections, with doses between 3 and 6mg/kg body weight (Ito et al., 1993, Jee and Ma, 1997) However, Cui et al. (2001) reported that aged rats (20 months old) treated for only 10 days with the same dose of PGE2 as in the current study (3 mg/kg) exhibited a marked increase in

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212 osteoblast surface which was not observed in PGE2treated dwarf and Lewis rats (Cui et al., 2001) This dis crepancy may be due to the much younger age of the rats (9 weeks old) in the current study. In addition to the relatively short treatment period, we did not have enough groups of animals t o obtain samples at different time points, and target the gene expression window for transcriptional changes in bone tissue. Transient c hanges in mRNA levels may have occurred before the cessation of treatment and euthanasia Conclusions In our studies, PTH showed stronger bone anabolic effects than PGE2, increasing bone mass and formation through downregulation of the sclerostin gene (SOST), even in IGF I deficient rats. Sclerostin is a potent negative regulator of bone formation, and its downregulation may be responsible for the persistent anabolic effects of PTH treatm ent in dwarf rats. Since PTH strongly stimulated bone formation and augmented bone mass in growing, IGF I deficient dwarf rats, this finding suggests that PTH would have anabolic effects in the osteopenic skeleton of juvenile humans with low serum levels of IGF I, such as adolescents with anorexia nervosa. Directions for Future Studies Future studies are needed to compare the effects of PTH and PGE2 treatments in aged and ovariectomized dwarf rats, and examine whether the osteopenic phenotype and responses to treatment would occur in the same manner Further investigations would also be helpful to elucidate the mechanisms involved in t he bone anabolic actions of PTH, such as the previously mentioned use of LRP5 knockout mice, and its e evolution of fractur e healing in relationship to the downregulation of sclerostin. It would

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213 also be interesting to compare the evolution of fracture healing in GH/IGF I deficient animals treated with PTH and PGE2. The expression of a specific gene provides important informat ion as to its biological role and state in cell metabolism ; however, the regulation of protein abundance is not exclusively accomplished by regulation of mRNA (DeRisi et al., 1997) Therefore, we consider that proteomic analysis should be included to evaluate changes at the translational level, in addition to transcriptional mRNA levels.

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233 BIOGRAPHICAL SKETCH Ana Cristina Ferreira Bassit was born in Santos, So Paulo, Brazil. She received her DVM degree from the College of Veterinary Medicine at University of So Paulo i n 1985. In September, 2004 she achieved a m aster s d egree in experimental p hysiopathology, at the College of Medicine University of So Paulo FMUSP, Bra z il. The research subjet was the effect of extracorporeal shock wave therapy on bone healing after femur osteosynthesis with interlocking nails an experimental study in dogs (Canis familiaris) performed at the College of Medicine at the University of S o Paulo, Brazil. In June 2007, she was selected by the J. William Fulbright Scholarship Board (FSB) for a Fulbright Student award in the United States for a Doctoral Program. In August 2007 she officially enrolled in the Graduate Program of the Department of Physiological Sciences at the College of Veterinary Medicine, University of Florida. In M a y, 20 08, she received a Certificate for Outstanding Achievement from the University of Florida International Center. In November 2008, and November 2009, she received the Certificate of Academic Excellence from the University of Florida International Center and the College of Veterinary Medicine. On January 18th, 2011, she received a PhD Degree in Orthopedics College of Medicine University of So Paulo. This activity established an international academic cooperation between the Universities of Florida and S o Paulo, so called Agreement for Thesis under CoOperation between Universities Faculty members of both Universities approved the dissertation with some of the results already obtained from the main project at University of Florida. The r esearch subject was the e ffect of human parathyroid hormone fragment on bone metabolism an experimental study in rats

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234 Her research interests include orthopedics, orthopedic surgery, fracture healing bone growth factors and bone research.