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Role of gonadotropin-releasing hormone in the ovarian cells Kang, Sung Keun 2000

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ROLE OF GONADOTROPIN-RELEASING HORMONE IN THE OVARIAN CELLS by Sung Keun Kang D.V.M. , Seoul National University, 1993 M.Sc, Seoul National University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPY in THE FACULTY OF GRADUATE STUDIES Reproductive & Developmental Sciences Program Department of Obstetrics & Gynecology University of British Columbia We accept this thesis as confirming to the required standard The University of British Columbia July 2000 © Sung Keun Kang, 2000 UBC Special Collections - Thesis Authorisation Form Page 1 of 1 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department o The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 8/2/00 ABSTRACT Considering the extrapituitary roles for gonadotropin-releasing hormone (GnRH) in reproductive tissues, the present study investigated role of GnRH in the ovarian cells. The GnRH and GnRH receptor (GnRHR) mRNA were expressed and autoregulated in a biphasic mariner by a GnRH agonist (GnRH-a) in human ovarian surface epithelium (hOSE) cells and an ovarian cancer cell line, OVCAR-3. The GnRH-a had a receptor-mediated growth inhibitory effect in both cell types. The growth inhibitory effect was associated with apoptosis in OVCAR-3 cells. In addition, 17(3-estradiol induced a significant down-regulation of GnRH mRNA in OVCAR-3, but not in hOSE cells and of GnRHR mRNA in both hOSE and OVCAR-3 cells. Pre- or co-treatment with 17p-estradiol significantly attenuated the growth inhibitory effect of the GnRH-a in OVCAR-3 cells, but not in hOSE cells. These results strongly support the presence of functional autocrine GnJAH7GnRHR loop and potential interaction with estrogen/estrogen receptor. The GnRH-a stimulated M A P K activation in human granulosa-luteal cells (hGLCs) via a PKC-dependent pathway, which mediated the inhibitory effect of GnRH-a in progesterone secretion. The GnRH-a also stimulated M A P K activation in OVCAR-3 cells, normal placenta-derived cells (IEVT) and placental carcinoma (JEG-3) cells. The GnRH-induced M A P K activation mediated the growth inhibitory effect of GnRH-a in OVCAR-3 cells, but not the stimulatory effect of GnRH-a on the hCG mRNA level in JEG-3 cells. These results demonstrated that GnRH stimulated M A P K activation that mediated the cellular functions of GnRH in normal and neoplastic cells of human ovary. ii The present study demonstrates that one mechanism by which cell-specific expression of the human GnRHR is achieved is through the binding of distinct cell-specific regulatory factors to various promoter elements in the 5'-flanking region of the gene. In this study, the second form of GnRH (GnRH-II) and GnRH-I differentially regulated GnRHR and its ligands. Gonadotropins also differentially regulated GnRH-II and GnRH-I mRNA levels. GnRH-II inhibited basal and hCG-stimulated progesterone secretion. The antigonadotropic effect of GnRH-II was mediated through down-regulating gonadotropin receptors without affecting cAMP levels. In summary, on the basis of the expression, differential regulation and/or functional roles of GnRH, our studies strongly support the notion that an intrinsic GnRH axis plays an important role in regulating normal and malignant ovarian cell functions. iii TABLE OF CONTENTS ABSTRACT ii T A B L E OF CONTENTS iv LIST OF T A B L E viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xii PUBLICATION LIST xv ACKNOWLEDGEMENTS xviii PART 1. LITERATURE REVIEW 1. INTRODUCTION 1 2. M O L E C U L A R STRUCTURE OF GnRH AND ITS RECEPTOR 3 2.1 Cloning and molecular structure of GnRH 3 2.2 Cloning and molecular structure of GnRHR 7 3. DISTRIBUTIONS A N D PHYSIOLOGICAL ROLE OF GnRH/GnRHR 12 3.1 Central nervous system and pituitary 12 3.2 Extrapituitary 13 3.2.1 Intra- ovarian and -testicular action 13 3.2.2 Placenta-endometrium-embryo 16 3.2.3 Reproductive tumors 18 3.2.4 Clinical applications 18 4. REGULATION OF GnRH A N D ITS RECEPTOR 19 4.1 Regulation of hypothalamic GnRH 19 4.2 Regulation of pituitary GnRHR 21 4.3 Intraovarian regulation of GnRH and GriRIIR 22 5. ACTIVATION OF GnRH RECEPTOR AND SIGNAL TRANSDUCTION 23 5.1 Receptor-G protein coupling 25 5.2 Inositol phosphate, calcium and D A G 26 5.3 Protein kinase C 27 5.4 Role of PLD signaling pathway 28 5.5 cAMP/cGMP signaling 29 iv 5.6 Role of PLA2 signaling pathway 29 5.7 Mitogen-activated protein kinases 30 5.8 Expression of early response genes 35 6. N O R M A L OVARIAN SURFACE EPITHELIUM (OSE) A N D OVARIAN CANCER 36 6.1 Biology of normal ovarian surface epithelium 36 6.2 Endocrine, paracrine and autocrine factors in OSE and ovarian cancer 37 6.2.1 Gonadotropin-releasing hormone 39 6.2.2 Steroids 41 6.2.3 Gonadotropins 44 6.2.4 Epidermal growth factor and transforming growth factor-a 45 6.2.5 Transforming growth factor-P family 46 6.2.6 Other growth factors and cytokines 48 HYPOTHESIS 50 SPECIFIC OBJECTIVES 50 PART 2. G E N E R A L MATERIALS AND METHODS 51 PART 3. THE R O L E OF GONADOTROPIN-RELEASING HORMONE AS A N AUTOCRINE GROWTH FACTOR IN H U M A N OVARIAN SURFACE EPITHELIUM 3.1 INTRODUCTION 67 3.2 MATERIALS A N D METHODS 68 3.3 RESULTS 71 3.4 DISCUSSION 81 PART 4. AUTOCRINE ROLE OF GONADOTROPIN-RELEASING HORMONE AND ITS RECEPTOR IN OVARIAN CANCER C E L L GROWTH 4.1 INTRODUCTION 84 v 4.2 MATERIALS A N D METHODS 4.3 RESULTS 4.4 DISCUSSION 85 88 97 PART 5. ESTRADIOL REGULATES GONADOTROPIN-RELEASING HORMONE (GnRH) AND ITS RECEPTOR GENE EXPRESSION A N D M O D U L A T E S THE GROWTH INHIBITORY EFFECTS OF GnRH IN H U M A N OVARIAN EPITHELIAL A N D OVARIAN CANCER CELLS 5.1 INTRODUCTION 99 5.2 MATERIALS A N D METHODS 100 5.3 RESULTS 102 5.4 DISCUSSION 113 PART 6. GONADOTROPIN-RELEASING HORMONE ACTIVATES MITOGEN-ACTIVATED PROTEIN KINASE IN H U M A N OVARIAN AND P L A C E N T A L CELLS 6.1 INTRODUCTION 117 6.2 MATERIALS A N D METHODS 118 6.3 RESULTS 120 6.4 DISCUSSION 132 PART 7. STIMULATION OF MITOGEN-ACTIVATED PROTEIN KINASE B Y GONADOTROPIN-RELEASING HORMONE IN H U M A N G R A N U L O S A - L U T E A L CELLS: ITS INTRACELLULA SIGNALING PATHWAYS 7.1 INTRODUCTION 137 7.2 MATERIALS A N D METHODS 138 7.3 RESULTS 141 vi 7.4 DISCUSSION 155 PART 8. DIFFERENTIAL EXPRESSION OF H U M A N GONADOTROPIN-RELEASING HORMONE RECEPTOR GENE IN PITUITARY A N D OVARIAN CELLS 8.1 INTRODUCTION 158 8.2 MATERIALS A N D METHODS 159 8.3 RESULTS 165 8.4 DISCUSSION 176 PART 9 DIFFERENTIAL H O R M O N A L REGULATION OF TWO FORMS OF GONADOTROPIN-RELEASING HORMONE MESSENGER RIBONUCLEIC ACID (mRNA) IN CULTURED H U M A N GRANULOSA-LUTEAL CELLS 9.1 INTRODUCTION 179 9.2 MATERIALS A N D METHODS 180 9.3 RESULTS 182 9.4 DISCUSSION 195 PART 10. S U M M A R Y A N D FUTURE STUDIES 10.1 S U M M A R Y 199 10.2 FUTURE STUDIES 204 BIBLIOGRAPHY 206 vii LIST OF TABLES Table 1. Primary amino acid sequence of known GnRH structures: Comparison with mammalian GnRH viii LIST OF FIGURES Figure 1. The hypothalamo-pituitary-gonadal axis 2 Figure 2. Schematic representation of the human GnRH-I and GnRH-II genes 6 Figure 3. Schematic representation of the human GnRHR gene and cDNA 9 Figure 4. The diagramatic structure of mammalian GnRHR 10 Figure 5. Signal transduction mechanism for GnRHR 24 Figure 6. Schematic representation of M A P K cascades 32 Figure 7. The potential interaction of several endocrine hormones and growth factors 38 Figure 8. A standard curve for the protein assay 61 Figure 9. A standard curve for the progesterone assay 64 Figure 10. A standard curve for the cAMP assay 66 Figure 11. Detection of GnRH mRNA by RT-PCR amplification 73 Figure 12. Detection of GnRHR mRNA by RT-PCR amplification 74 Figure 13. Validation of semi-quantitative RT-PCR for GnRH (A) and p-actin (B) in hOSE cells 76 Figure 14. Construction of native (target) and mutant (competitive) cDNA and validation of competitive RT-PCR for GnRHR transcript in hOSE cells 77 Figure 15. Effect of (D-Ala6)-GnRH on GnRH mRNA (A) and GnRHR mRNA (B) in hOSE cells 79 Figure 16. Effect of (D-Ala 6)-GnRH and cotreatment of antide on growth of hOSE cells 80 Figure 17. A standard curve for DNA assay 87 Figure 18. Validation of semi-quantitative RT-PCR for GnRH (A) and p-actin (B) in OVCAR-3 cells 89 Figure 19. Validation of competitive RT-PCR for GnRHR transcript in OVCAR-3 cells 90 Figure 20. Effect of (D-Ala6)-GnRH on GnRH mRNA and GnRHR mRNA in OVCAR-3 cells 92 Figure 21. Effect of the (D-Ala6)-GnRH (A) and antide (B) on growth of OVCAR-3 cells 93 Figure 22. Effect of continuous treatment of the (D-Ala6)-GnRH and antide on GnRHR mRNA levels 95 Figure 23. Induction of DNA fragmentation in OVCAR-3 cells by the GnRH analogs 96 Figure 24. Detection of E R a and ERp mRNA by RT-PCR amplification 104 Figure 25. Detection of E R a and ERp protein by immunoblot analysis 105 Figure 26. Effect of 17p-estradiol on GnRH mRNA in OVCAR-3 (A) and hOSE cells (B) 107 Figure 27. Effect of 17p-estradiol on GnRHR mRNA in OVCAR-3 (A) and hOSE cells (B) 108 Figure 28. Effect of 17p-estradiol and tamoxifen co-treatment on GnRH and GnRHR mRNA OVCAR-3 (A and B) and hOSE cells (C) 109 Figure 29. Effect of 17p-estradiol on the growth of OVCAR-3 (A) and hOSE cells (B) 111 Figure 30. Effect of 17p-estradiol treatment on the growth inhibitory effect of GnRH agonist in OVCAR-3 (A) and hOSE cells (B) 112 Figure 31. The effect of GnRH on M A P K activation in hGLCs 121 ix Figure 32. The effect of GnRH on M A P K activation in OVCAR-3 cells 122 Figure 33. The effect of GnRH on M A P K activation in aT3 -1 cells 123 Figure 34. The effect of GnRH on M A P K activation in ffiVT cells 125 Figure 35. The effect of GnRH on M A P K activation in JEG-3 cells 126 Figure 36. The effects of GnRH and PD98059 on progesterone secretion in hGLCs 128 Figure 37. The effects of GnRH and PD98059 on OVCAR-3 cell growth 129 Figure 38. The effect of GnRH and PD98059 treatment on phCG mRNA in JEG-3 cells 131 Figure 39. A time- and dose-dependent effect of GnRH on M A P K activation in hGLCs 142 Figure 40. The effects of antide and PD98059 on GnRH-induced M A P K activation in hGLCs 143 Figure 41A and B. The effects of PMA, GnRH, and PKC inhibitor GF109203X on M A P K activation 145 Figure 41C. The effects of PMA, GnRH, and PKC inhibitor GF109203X on M A P K activation 146 Figure 42. The effects of 8-Br-cAMP and PTX on the M A P K activation 147 Figure 43. The effects of C T X and PTX pretreatment on GnRH-induced M A P K activation 149 Figure 44A and B. The effects of forskolin, hCG, and GnRH on intracellular cAMP accumulation 150 Figure 44C. The effects of forskolin, hCG, and GnRH on intracellular cAMP accumulation 151 Figure 45. The effects of GnRH and PD98059 on Elk-1 phosphorylation 153 Figure 46. The effect of GnRH on c-fos mRNA levels 154 Figure 47. A standard curve for P-galactosidase assay 163 Figure 48. Different expression levels of GnRHR mRNA in pituitary tissues and primary culture of ovarian carcinomas 167 Figure 49. Different expression levels of GnRHR mRNA in aT3-l and OVCAR-3 cells 168 Figure 50. Construction a series of 3 '-deletion clones 169 Figure 51. Functional analysis of the human GnRHR promoter by transient transfection assays 170 Figure 52A. EMSAs performed with PR1 (-771 to -557) 172 Figure 52B. Competitive EMSAs performed with PR1 (-771 to -557) 173 Figure 53A. EMSAs performed with the PR2 (-13 51 to -1022) 174 Figure 53B. Competitive EMSAs performed with the PR2 (-1351 to -1022) 175 Figure 54. Validation of semi-quantitative RT-PCR for GnRH-JJ (A), FSHR (B) andLHR(C) 184 Figure 55. Homologous regulation of GnRH-II and GnRH-I mRNA 185 Figure 56. Homologous regulation of GnRHR mRNA 186 Figure 57. The effect of FSH on GnRH-II (A), GnRH-I mRNA (B) and progesterone secretion (C) 188 Figure 58. The effect of hCG on GnRH-II (A), GnRH-I mRNA (B) and progesterone secretion (C) 189 Figure 59. The effect of GnRH-II and GnRH-I on basal and hCG-stimulated x progesterone secretion 190 Figure 60. The effect of GnRH-JJ (A), GnRH-H-a (B) or GnRH-I-a (C) on FSFTR mRNA 192 Figure 61. The effect of GnRH-II (A), GnRH-H-a (B) or GnRH-I-a (C) on LHR mRNA 193 Figure 62. Effects of GnRH-II on basal and hCG-stimulated intracellular cAMP accumulation 194 xi LIST OF ABBREVIATIONS A A Arachidonic acid A N O V A Analysis of variance A R Androgen receptor ATP Adenosine 5'-triphosphate bp Base pairs C Celcius C a 2 + Calcium cAMP Cyclic adenosine monophosphate cDNA Complementary deoxyribonucleic acid cGMP Cyclic guanosine monophosphate cGnRH-n Chicken JT gonadotropin-releasing hormone sGnRH Salmon gonadotropin-releasing hormone Ci Curie cpm Counts per minute C T X Cholera toxin D A G Diacylglycerol DDT Dithiothreitol DEPC Diethylpyrocarbonate DHT 5a-dihydrotestosterone dl.dC Polydeoxyinosinic acid/polydeoxycytidylic acid dNTP Deoxynucleoside triphosphate DNA Deoxyribonucleic acid DNase Deoxyribonuclease EDTA Ethylene diaminetetraacetic acid E2 17P-estradiol EGF Epidermal growth factor ELISA Enzyme-linked immunosorbant assay EMSA Electrophoretic gel mobility shift assays ER Endoplasmic reticulum E R a / p Estrogen receptor a/p ERE Estrogen response element ERGs Early (primary) response genes ERK1/2 Extracellular signal-regulated kinase 1/2 Fas L Fas ligand FBS Fetal bovine serum FSH Follicle stimulating hormone g Acceleration of gravity GAP GnRH-associated peptide GDP Guanosine diphosphate GLB Gel loading buffer GnRH Gonadotropin-releasing hormone GnRH-a Gonadotropin-releasing hormone agonist GnRH-H-a Gonadotropin-releasing hormone-U agonist GnRHR Gonadotropin-releasing hormone receptor xii G-protein GTP-binding protein GPCR G-protein coupled receptors GTP Guanosine triphosphate h Hour FffiSS Hank's balanced salt solution hCG Human chorionic gonadotropin HGF Hepatocyte growth factor hGLCs Human granulosa-luteal cells IGF Insulin-like growth factor IGFBP Insulin-like growth factor binding protein IP Inositol phosphate D?3 Inositol 1, 4, 5-triphosphate IU International unit TVF In vitro Fertilization JNK/SAPK c-jun terminal kinase/stress-activated protein kinases Kb Kilobase kDa Kilodaltons L H Luteinizing hormone LPA Lysophosphatidic acid Micro M A P K Mitogen-activated protein kinase MAPKKs (=MKK) M A P K kinases MAPKKKs M A P K K kinases M A P K K K K s M A P K K K kinases MEK1/2 M A P K / E R K kinase 1/2 mGnRH Mammalian gonadotropin-releasing hormone ml Mililiters min Minutes MMP Matrix metalloproteinases mRNA Messenger ribonucleic acid M W Molecular weight n (as in nM) Nano OSE Ovarian surface epithelium p (as in pM) Pico P4 Progesterone PAGE Polyacrylamide gel electrophoresis PBS Phosphatase buffered saline PBS-G Phosphate buffered saline-gelatin PCR Polymerase chain reaction PEt Phosphatidylethanol PGF2a Prostaglandin F2a PI Phosphatidylinositol PIP Phosphatidylinositol 4-phosphate PIP2 Phosphatidylinositol 4, 5-phosphate PIP3 Phosphatidylinositol 3,4,5-triphosphate PKA Protein kinase A xiii PKC Protein kinase C aPKC Atypical protein kinase C cPKC Conventional protein kinase C nPKC Novel protein kinase C PLA Phospholipase A PLC Phospholipase C PLD Phospholipase D PMA Phorbol 12-myristate 13-acetate PMSF Phenylmethylsulfonyl fluoride POA Preoptic area PR Progesterone receptor PTP Phosphotyrosine phosphatase PTX Pertussis toxin RIA Radioimmunoassay rpm Revoultions per min RT Room temperature RT-PCR Reverse transcription polymerase chain reaction sec Seconds SD Standard deviation SDS Sodium dodecyl sulphate Taq Thermus aquaticus, source of a DNA polymerase TCF Ternary complex factor T E Tris-EDTA T E M E D N, N, N' , N'-tetramethylethlenediamine TGF-a Transforming growth factor-a TGF-p Transforming growth factor-P TIMP Tissue inhibitor of metalloproteinase T M Transmembrane Tris Tris(hydroxy methyl) aminomethane Txf Tamoxifen U V ultraviolet v/v Volume per volume w/v Weight per volume xiv PUBLICATION LIST REFEREED PAPERS 1. Kang SK, Choi K - C , Cheng KW, Nathwani PS, Auersperg N, Leung PCK 2000 Role of gonadotropin-releasing hormone as an autocrine growth factor in human ovarian surface epithelium. Endocrinology 141: 72-80 2. Kang SK, Cheng KW, Ngan ESW, Chow B K C , Choi K - C , Leung PCK 2000 Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. Mol Cell Endocrinol 162:157-66 3. Kang SK, Cheng KW, Nathwani PS, Choi K - C , Leung PCK 2000 Autocrine role of gonadotropin-releasing hormone (GnRH) and its receptor in ovarian cancer cell growth. Endocrine, In press 4. Kang SK, Tai CJ, Cheng KW, Leung PCK 2000 Gonadotropin-releasing hormone activates mitogen-activated protein kinase in human ovarian and placental cells. Mol Cell Endocrinol, In press 5. Kang SK, Tai CJ, Nathwani PS, Choi K - C , Leung PCK 2000 Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone in human granulosa-luteal cells. Endocrinology, Submitted 6. Kang SK, Tai CJ, Nathwani PS, Leung PCK 2000 Differential hormonal regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid (mRNA) in cultured human granulosa-luteal cells. Endocrinology, Submitted 7. Kang SK, Choi K - C , Tai CJ, Auersperg N, Leung PCK 2000 Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and modulates the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. J Clin Endcrinol Metab, Submitted 8. Nathwani PS, Kang SK, Cheng KW, Choi K - C , Leung PCK 2000 Regulation of gonadotropin-releasing hormone (GnRH) and its receptor gene expression by 17p-estradiol in cultured human granulosa-luteal cells. Endocrinology 141: 1754-1763 9. Tai CJ, Kang SK, Cheng KW, Choi K - C , Nathwani PS, Leung PCK 2000 Expression and regulation of purinergic receptor (P2UR) in human granulosa-luteal cells. J Clin Endocrinol Metab 85: 1519-1597 10. Cheng KW, Ngan ESW, Kang SK, Chow B K C , Leung PCK 2000 Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein 1. Endocrinology, In press xv 11. Tai C J, Kang SK, Leung PCK 2000 The role of protein kinase C in regulating ATP-evoked cytosolic calcium oscillations in cultured human granulosa-luteal cells. J Clin Endocrinol Metab, In press 12. Auersperg N, Wong AST, Choi K - C , Kang SK, Leung PCK 2000 Ovarian surface epithelium: biology, endocrinology and pathology. Endocr Rev, In press 13. Leung PCK, Kang SK 2000 Extrapituitary gonadotropin-releasing hormone (GnRH) receptor. Hormone Frontier in Gynecology, In press 14. Tai CJ, Kang SK, Choi K - C , Leung PCK 2000 Prostaglandin F2a activates mitogen-activated protein kinase in human granulosa-luteal cells. J Clin Endocrinol Metab, Submitted 15. Choi K C , Kang SK, Nathwani PS, Cheng KW, Auersperg N, Leung PCK 2000 Differential expression of activin/Inhibin subunit and activin receptor mRNAs in normal and neoplastic ovarian surface epithelium (OSE). Mol Cell Endocrinol, Submitted 16. Tai CJ, Kang SK, CR Tzeng, Leung PCK 2000 ATP activates mitogen-activated protein kinases in human granulosa-luteal cells. Endocrinology, Submitted 17. Choi K C , Kang SK, Tai CJ, Auersperg N, Leung PCK 2000 The regulation of apoptosis by activin and TGF-b in early neoplastic and tumorigenic ovarian surface epithelium (OSE). Submitted 18. Choi K C , Kang SK, Tai CJ, Auersperg N, Leung PCK 2000 Estradiol up-regulates anti-apoptotic bcl-2 mRNA and protein in tumorigenic ovarian surface epithelium (OSE). Submitted 19. Nathwani PS, Kang SK, Cheng KW, Cheung AP, Leung PCK 2000 RU486 regulates gonadotropin-releasing hormone receptor messenger ribonucleic acid levels in cultured human granulosa-luteal cells. Manuscript in preparation 20. Nathwani PS, Cheng KW, Kang SK, Leung PCK 2000 Expression and function of chicken gonadotropin-releasing hormone II (cGnRH-II) mRNA in human ovarian and placental cells. Manuscript in preparation ABSTRACT AND PRESENTATION 1. Kang SK, Cheng KW, Choi K - C , Leung PCK. Combinations of cell-specific factors may explain the differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. The Meeting for Health Research of the Medical Research Council of Canada, 1999 xvi 2. Cheng KW, Kang SK, Tai CJ, Nathwani PS, Leung PCK. P-2, Transcriptional regulation of human gonadotropin-releasing hormone receptor (GrtflHR) in human placental cells. 32n d Annual meeting of society for the study of reproduction, 1999 3. Nathwani PS, Kang SK, Cheng KW, Leung PCK. P-56, Estradiol regulates gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acid (mRNA) levels in human granulosa-luteal cells. 32n d Annual meeting of society for the study of reproduction, 1999 4. Tai CJ, Kang SK, Cheng KW, Choi K - C , Nathwani PS, Leung PCK. Expression of purinergic receptor (P2UR) in human granulosa-luteal cells. ASRM/CFAS '99 Annual Meeting Program, S-47,1999 5. Choi K - C , Kang SK, Cheng KW, Nathwani PS, Tai CJ, Auersperg N, Leung PCK. The expression levels of activin/inhibin subunits and activin receptors: Autocrine function of activin in ovarian surface epithelium and ovarian cancer. Health Sciences Student Research Forum Poster Presentation, 1999 6. Kang SK, Cheng KW, Leung PCK. Characterization of the 5'-Flanking region of the human Gonadotropin-Realeasing hormone receptor gene. Health Sciences Student Research Forum Poster Presentation, 1997 7. Choi K - C , Kang SK, Tai CJ, Auersperg N, Leung PCK. Endocrine influences on normal and neoplastic ovarian surface epithelium (OSE) cell growth. A S R M '2000 Annual Meeting Program, 2000 8. Tai CJ, Kang SK, Choi K - C , Leung PCK. Prostaglandin F2a activates mitogen-activated protein kinase in human granulosa-luteal cells. A S R M '2000 Annual Meeting Program, 2000 xvii ACKNOWLEDGEMENTS My gratitude and deep appreciation goes to my supervisor, Dr. Peter C.K. Leung for his supervision and resources throughout my studies. His advice and guidance have proven valuable to my research and future career pursuits. I would like to extend my sincere gratitude to my supervisory committee members, Drs. Young S Moon, Nelly Auersperg, Calvin D. Roskelley, Monique A. Bertrand for their direction and criticisms. I also wish to express my gratitude to the British Columbia Research Institute for Child and Family Health who provided financial support during my studies. I offer my appreciation to my colleagues K-C. Choi, K.W. Cheng, P.S. Nathwani, C J . Tai, M . Woo, and members of Dr. Auersperg's lab for encouragement and instructive suggestions. Grateful acknowledgement must be extended in particular to Choi's family, my fiancee and her family for their encouragement, support, and love throughout my studies. This thesis is dedicated to my family who have always encouraged me to follow my dreams and who were my source of strength and inspiration. Mom and sisters, this is for you. xviii PARTI Literature Review 1. Introduction The concerted hormonal regulation at the hypothalamic, pituitary and gonadal level plays an important role in the control of the sexual maturation and reproductive functions. Gonadotropin-releasing hormone (GnRH) functions as a key neural regulator of the hormonal cascade (Braden and Conn, 1993; Conn, 1994). Synthesized in the hypothalamic neurons, GnRH is secreted into the hypothalmo-hypophyseal portal circulation in a pulsatile manner. Through the portal circulation, it travels to the anterior lobe of the pituitary gland, where it binds to specific high-affinity receptors on the membrane of gonadotrophs and stimulates the release of the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Braden and Conn, 1993; Conn, 1994; Leung and Steele, 1992). Numerous factors including GnRH, gonadotropins short feedback loop, gonadal steroids and peptide hormones have been shown to regulate the synthesis and release of GnRH and GnRH receptor (GrJUHR) (Fig. 1) (Braden and Conn, 1993; Kalra and Kalra, 1983). In addition to the hypothalamic-pituitary axis, GnRH and its receptor have been detected in other reproductive tissues including the gonads, placenta and tumors arising from these tissues (Chatzaki et al., 1996; Imer et al., 1995; Lin et al., 1995; Olofsson et al., 1995; Peng et al., 1994; Qayum et al., 1990; Yin et al., 1998). Although the functional role of GnRH has not been clearly established, GnRH has been suggested to be an autocrine/paracrine regulator of reproductive functions and tumor growth. 1 Hypothalamic-P ituitary -Gonadal axis o 0 CD hypothalamus GnRH C^itui tary^> FSH/LH steroids /peptides Gonads Figure 1. The hypothalamo-pituitary-gonadal axis. Synthesized in the hypothalamic neurons, GnRH stimulates the synthesis and release of L H and FSH in the pituitary. In the gonads, gonadotropins stimulate follicular development, spermatogenesis and steroidogenesis. In turn, gonadal steroids and peptide hormones regulate hypothalamic and pituitary function in both positive and negative feedback mechanism. 2 2. Molecular structure of GnRH and GnRH receptor 2.1 Cloning and molecular structure of GnRH GnRH was first isolated and sequenced from mammals (see reviewed by Sherwood et al., 1993; 1997). At present, there are 13 distinct forms of GnRH (Table 1) (Carolsfield et al., 2000). All known forms consist of 10 amino acids in length, with a pyroglutamyl-modified amino-terminus, an amidated carboxy-terminus, and conserved amino acids in position 1, 4, 9, 10 (Carolsfield et al., 2000; Powell et al., 1994; Sherwood et al., 1997). The most common structural variations among the different forms of GnRH reside in amino acids 5-8 (Table 1). In fish, amphibians, reptiles, and birds, there are two or more forms of GnRH within the brain of single species (Sherwood et al., 1993; White et al., 1995). The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGriRH, now here designated as GnRH-I). However, it has been recently demonstrated that two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) are present in brain extracts from adult stumptail and rhesus monkeys (Lescheid et al., 1997). In rhesus monkey, administration of cGnRH-II significantly increased plasma L H levels during the luteal phase, demonstrating that the second form of GnRH has biological function in the mammal (Lescheid et al., 1997). Subsequently, the cDNA encoding GnRH-II was isolated from monkey (Terasawa et al., 2000; Urbanski et al., 2000) and a gene encoding second form of GnRH was cloned from human (White et al., 1998). Structurally, the genes that encode for the different forms of GnRH are very similar (Fig. 2). In humans, the gene for GnRH-I is located on chromosome 8, whereas the gene for GnRH-II is located on chromosome 20 (White et al., 1998). The gene for GnRH-II is much smaller in size (2.1 kb) than GnRH-I (5.1 kb) (Hayflick et al., 1989). Genes for GnRH exist as part of a larger 3 precursor gene (see reviewed by Sherwood et al., 1993). The precursor cDNA consists of GnRH that is extended at the N-terminus by a signal peptide and at the C-terminus by a Gly-Lys-Arg sequence, characteristic of an enzymatic amidation and precursor processing site, followed by a GnRH-associated peptide (GAP) (Sherwood et al., 1993; 1997; White et al., 1998). The GnRH genes consist of four exons and three introns (Radovick et al., 1990; Sherwood et al., 1993). In the GnRH-I gene, the first exon encodes the 5'-untranslated regions. The second exon encodes the signal peptide, GnRH, the enzymatic amidation and precursor processing site, and the first 11 amino acids of GAP. The third exon encodes GAP amino acids 12-43 and the fourth exon encodes the remaining amino acids and the 3'-untranslated region. GAP has been shown to co-exist with GnRH in hypothalamic neurons and is thought to be involved in the correct processing and packaging of the hormone (Merchenthaler et al., 1989; Sar et al., 1987; Sherwood et al., 1993; 1997). Distinct distributions of neurons containing both forms of GnRH in the brain have been demonstrated. The majority of GnRH-I-synthesizing neurons are localized in the preoptic area (POA) and adjacent sites in the rostal portion of hypothalamus (Sherwood et al., 1993; Lescheid et al., 1997). By in situ hybridization, it was shown that GnRH-II mRNA is expressed in the primate midbrain, hippocampus and discrete nuclei of the hypothalamus (Urbanski et al., 2000). Using double-label histochemistry, studies have shown that GnRH-I and GnRH-II are expressed by two separate populations of cells in the rhesus macaque hypothalamus (Latimer et al., 2000). Interestingly, GnRH-II is expressed at significantly higher levels outside the brain, especially in the kidney (up to 30-fold), bone marrow (up to 4-fold), and prostate (up to 4-fold) in the human (White et al., 1998). The unique location and differential expression levels of GnRH-II within and outside the brain in a single species including human suggests that it may have distinct functions from those of GnRH-I. 4 Table 1. Primary amino acid sequence of known GnRH structures: Comparison with mammalian GnRH G n R H Amino acid sequence 1 2 3 4 5 6 7 8 9 10 Mammal pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Chicken-II pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Glv-NHo Chicken-I pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 Salmon pGlu-His-Trp-Ser-Tyr-Glv-Trp-Leu-Pro-Glv-NH, Herring pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Glv-NH. Catfish pGlu-His-Trp-Ser-His-Glv-Leu-Asn-Pro-Glv-NH, Seabream pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2 Dogfish pGlu-His-Trp-Ser-His-Glv-Trp-Leu-Pro-Glv-NHo Guinea pig pGlu-Tvr-Trp-Ser-Tvr-Glv-Val-Arg-Pro-Glv-NH, Tunicate-I pGlu-His-Trp-Ser-Asp-Tvr-Phe-Lvs-Pro-Glv-NH, Tunicate-II pGlu-His-Trp-Ser-Leu-Cvs-His-Ala-Pro-Glv-NH, Lamprey-I pGlu-His-Tvr-Ser-Leu-Glu-TrD-Lvs-Pro-Glv-NH, Lamprey-III pGlu-His-Trp-Ser-His-Asp-Trp-Lvs-Pro-Glv-NHo Adapted from Carolsfeld et a l , 2000 5 hGnRH-I hGnRH-II E x o n I E x o n II E x o n III E x o n I V 5'-Untranslated region GnRH sequence 3'-Untranslated region Figure 2. Schematic representation of the human GnRH-I and GnRH-II genes (not to scale). Structurally, the genes that encode for two forms of human GnRH are very similar. Genes consist of four exons and three introns and encode a larger precursor. The mature decapetide is translated from the second exon. Adapted from White et al., 1998 6 2.2 Cloning and molecular structure of GnRHR The GnRHR gene has first been cloned in mice (Reinhart et al., 1992; Tsutsumi et al., 1992) and subsequently cloned in rats (Eidne et al., 1992; Kaiser et al., 1992; Karkar et al., 1994; Perrin et al., 1993), sheep (Brooks et al., 1993), bovine (Kakar et al., 1993), and human (Chi et al., 1993; Fan et al., 1994; Kakar et al., 1992; Kakar, 1997). GnRHR complementary DNA (cDNA) encodes a 327 amino acid protein in the mouse and rat. The human and sheep receptor contain one more amino acid, a lysine residue in the second extracellular loop, and encode a 328 amino acid protein (Chi et al., 1993; Eidne et al., 1992; Fan et al., 1994; Kakar et al., 1992; 1994; Kakar, 1997; Perrin et al., 1993; Reinhart et al., 1992; Tsutsumi et al., 1992). Southern blot analysis revealed that the GnRHR appears to be encoded from a single gene (Fan et al., 1994; Kaiser et al., 1997; Stojikovic et al., 1994). The GnRHR consists of three exons, two introns and putative seven transmembrane (TM) domains, which are characteristic of the family of G-protein coupled receptors (GPCR) (Figs. 3 and 4). In contrast to typical GPCR, GnRHR lacks the typical intracellular carboxyl terminus (Fan et al., 1994; Kaiser et al., 1997; Kakar, 1997; Stojikovic et al., 1994) (Fig. 4). Studies on solubilized native GnRHR gave an approximate molecular weight (wt) of 50,000-60,000 (Clayton, 1989). Considering that the calculated molecular wt of the receptor protein is 37,684, these results indicate that the receptor undergoes posttranslational modifications. Potential N-linked glycosylation sites are present in the extracellular domain, whereas potential sites for phosphorylation by cAMP-dependent protein kinases and protein kinase C (PKC) are located within first and third cytoplasmic domains (Fan et al., 1994; Kaiser et al., 1997; Kakar, 1997; Stojikovic et al., 1994). A highly conserved sequence, i.e. Asp-Arg-Tyr (DRY) triplet in many other GPCR (Probst et al., 1992) is replaced by Asp-Arg-Ser (DRS) in the GnRHR (Fan et al., 1994; Kaiser et al., 1997; 7 Kakar, 1997; Stqjikovic et al., 1994) (Fig. 4). Another highly unusual feature of the GnRHR is the exchange of conserved aspartate (D) and asparagine (N) residues (Fan et al., 1994; Kaiser et al., 1997; Kakar, 1997; Stojikovic et al., 1994). An aspartate residue in the second T M domain, which is highly conserved and is essential for normal agonist binding and G protein coupling in many GPCRs, is replaced by asparagine87 in the GnRHR. Also, there is a reverse substitution in the seventh T M domain, in which a highly conserved asparagine318 is replaced by asparatate residue. The most unique structural feature of the mammalian GnRHR among GPCRs is the absence of a carboxy-terminal cytoplasmic tail, a region that has been implicated in coupling to G proteins in GPCRs (Fan et al., 1994; Kaiser et al., 1997; Kakar, 1997; Stojikovic et al., 1994) (Fig. 4). However, the nonmammalian GnRHRs such as the African catfish, goldfish and chicken contain an intracellular C terminus with phosphorylation consensus sites and Cys residues (Blomenrohr et al., 1997; Tensen et al., 1997; Troskie et al., 1997), raising the questions of the evolutionary and physiological significance of the absence of the intracellular C-tail in mammalian GnRHR. 8 cDNA T M I II in IV V VI VII Figure 3. Schematic representation of the human GnRHR gene and cDNA. The GnRHR gene is encoded from a single gene and consists of three exons, two introns and putative seven TM domains, which are characteristic of the family of GPCR. The human GnRHR cDNA encodes a 328 amino acid protein. 9 Extracelluar Membrane C O O H Intracelluar Asp-Arg-Ser Figure 4. The diagramatic structure of mammalian GnRHR. In many GPCR, a highly conserved sequence in the second cytoplasmic domain, i.e. Asp-Arg-Tyr (DRY) triplet has been changed to Asp-Arg-Ser (DRS) in GnRHR. Another highly unusual feature of the GnRHR is the exchange of conserved aspartate (D) and asparagine (N) residues that are present in the second and seventh T M domains, respectively. The most unique structural feature of the mammalian GnRHR among GPCRs is the absence of a carboxy-terminal cytoplasmic tail, a region that has been implicated in coupling to G proteins in GPCRs. 10 Recently, two GnRHR subtypes (Type I and Type II) in brain and pituitary from the gold fish (Carassius auratus) have been reported (Illing et al., 1999). The goldfish GnRHRs share lower identity with mammalian GnRHR (43%) and have distinct ligand selectivity. Both subtypes are expressed in the goldfish pituitary, and each has a unique pattern of expression in the goldfish brain, ovary and liver as revealed by in situ hybridization studies. In addition, a partial clone of the type II GnRHR has been demonstrated in the Xenopus laevis (Troskie et al., 1998). The 5'-flanking region of the mouse GnRHR gene has been cloned and characterized (Albraracin et al., 1994; Clay et al., 1995). A major transcriptional start site was defined at 62 nucleotides upstream of the translational start site (Albraracin et al., 1994). The gene does not seem to use a T A T A box for transcription. In transfection assays with a series of deletion clones, the DNA element between -500 and -400 relative to translational start site was shown to be important to the mouse GriRHR gene expression in the aT3-l pituitary tumor cell line (Clay et al., 1995). The basal activity of mouse GnRHR promoter was dependent on the steroidogenic factor-1 (SF-1) binding sites, activator protein-1 (AP-1) binding site and GnRHR activating sequence (Duval et al., 1996; 1997). The study has demonstrated that transcriptional regulation of mouse GnRHR gene is mediated in part by two repressor elements and by the cyclic AMP response element (CRE) (Maya-Nunez and Conn, 1999). The promoter region of the rat GnRHR has been cloned and partially characterized (Reinhart et al., 1997). Like in the mouse promoter, a major transcriptional site was identified at 103 nucleotides upstream of the translational start site. Consistent with the mouse promoter, the absence of typical T A T A and C A A T sequences was found in the promoter region of the rat GnRHR. The 5'-flanking region of the human GnRHR has been cloned and characterized (Fan et al., 1994; 1995; Kakar, 1997). Sequencing analysis demonstrated that it consists of five consensus T A T A boxes within the 700-nucleotide region 11 and multiple transcription initiation sites in association with these T A T A boxes (Fan et al., 1995). The binding sites for putative cis-acting elements were identified in the 5'-flanking region of the human GnRHR. A putative CRE and progesterone response element (GRE/PRE) were identified. Consensus binding sites for some transcription factors, including PEA-3, AP-1 and Pit-1, were also identified (Fan et al., 1994; 1995; Kakar, 1997). Recently, it has been demonstrated that SF-1, by interacting with a gonadotroph specific element (GSE) motif within the first exon of the human GnRHR gene, is largely responsible for its gonadotroph-specific expression (Ngan et al., 1999). 3. DISTRIBUTION AND PHYSIOLOGICAL ROLE OF GnRH/GnRHR 3.1. Central nervous system and pituitary Distribution The majority of GnRH-synthesizing neurons are localized in the POA and adjacent sites in the rostal portion of hypothalamus. GnRH mRNA and peptide have also been shown in cultured rat pituitary cells and immortalized pituitary gonadotroph (aT3-l) cells, suggesting the presence of an autonomous or paracrine GnRH system at the pituitary level (Krsmanovic et al., 2000). Autoradiographic localization of brain GnRHR showed specific binding in hippocampus, lateral septal nucleus, anterior cingulate cortex, subiculum, and entorhinal cortex (Millan et al., 1985). High affinity binding sites for GnRH have been demonstrated in the hypothalamic GT1 neurons (Krsmanovic et al., 1993). GnRHRs have been characterized in the pituitaries of several species, as well as in immortalized ocT3-l gonadotropes (Kaiser et al., 1997; Naor Z et al., 1980; 12 Stojilkovic et al., 1994; Wormald et al., 1985). Among the pituitary cell types, GnRH binding sites in mammals are located exclusively in the gonadotrophs (Hyde et al., 1982). GnRH binds to bihormonal gonadotrophs (expressing L H and FSH), as well as to monohormonal cells (expressing only L H or FSH) (Childs, 1984; Naor and Child, 1986). Physiological Role In terms of binding to its receptor, hypothalamic GnRH stimulates the release of the gonadotropins, L H and FSH (Braden and Conn, 1993; Conn, 1994). The gonadotropins, in turn, control the functions of gonads in a systemic manner. The gonadotropins are synthesized and secreted into the systemic circulation and travel to the gonads, where they bind to specific receptors to modulate steroids synthesis and gametogenesis. In females, FSH stimulates follicular development and L H induces ovulation, formation of corpus luteum (CL) and oocyte meiosis. In males, FSH stimulates spermatogenesis and L H stimulates androgen synthesis in the testes. 3.2. Extrapituitary Although the functions of GnRH in these tissues are not well understood, the presence of GnRH and its receptor suggeste an autocrine and/or paracrine role of GnRH in regulating reproductive functions 3.2.1. Intra- ovarian and -testicular action 13 Distribution Local expression of GnRH and its receptor mRNA was demonstrated in the ovary and testis of humans (Dong et al., 1993), monkeys (Dong et al., 1996), and rats (Bahk et al., 1995; Oikawa et al., 1990). In situ hybridization studies localized GnRH mRNA primarily in granulosa cells of primary, secondary, and tertiary follicles in the ovary (Clayton et al., 1992; Whitelaw et al., 1995). Low affinity and high capacity binding sites for GnRH in the ovary have been demonstrated using radiolabeled ligand binding studies (Bramely et al., 1986; Clayton et al., 1979). The presence of mRNA for GnRHR has also been identified in the rat ovary and in human granulosa-luteal cells (hGLCs) using reverse/transcriptase-polymerase chain reaction (RT-PCR) amplification (Minaretzis et al., 1995; Olofsson et al., 1995; Peng et al., 1994). In male testis, GnRH is present in Sertoli cells (Bahk et al., 1995), whereas its receptors are expressed in Leydig cells (Bourne et al., 1980; Clayton et al., 1980). Sequence analysis of the rat and human ovarian GnRHR revealed that they have a sequence identical to that found in the pituitary (Olofsson et al., 1995; Peng et al., 1994). Physiological Role In the ovary, GnRH has been involved in a variety of both inhibitory and stimulatory responses, affecting follicular and luteal function and ovulation. There is evidence for a role of GnRH in atresia (Billing et al., 1994; Hsueh et al., 1984). During the follicular phase, GnRHR expression is high in atretic rat follicles (Whitelaw et al., 1995). In vitro culture was used to show that GnRH inhibited DNA synthesis (Saragueta et al., 1997) or induced apoptosis in rat granulosa cells (Billing et al., 1994). 14 During the periovulatory period, GnRH induced transcription of several genes involved in the follicular rupture and oocyte maturation (Ny et al., 1987; Natraj and Richard, 1993; Wong and Richards, 1992). These genes include plasminogen activator (Ny et al., 1987), prostaglandin endoperoxide synthase type 2 (Wong and Richards, 1992), and the progesterone receptor (Natraj and Richard, 1993). There are reports to suggest GnRH is involved in the process of luteinzation and luteolysis. For example, GnRH induced remodeling of the extracellular matrix by inducing structural luteolysis in superovulated rats through stimulating the expression of matrix metalloproteinase (MMP)-2 and membrane type 1-MMP in developed C L , which degraded collagens type TV, type I and III, respectively (Goto et al., 1999). During early pregnancy in the rat, GnRH suppressed serum progesterone levels, and increased the degree of DNA fragmentation in the C L (Sridaran et al., 1998; 1999a). Similarly, GnRH induced increases in the number of apoptotic human granulosa cells obtained during oocyte retrieval for in vitro fertilization (Zhao et al., 2000). It has been demonstrated that GnRH modulates both basal and gonadotropin-stimulated steroidogenesis (Clayton et al., 1979; Olofsson et al., 1995; Peng et al., 1994) in the ovary. The inhibitory actions of GnRH on steroidogenesis involve suppression of receptors for FSH and L H (Piquette et al., 1991; Tilly et al., 1992), gonadotropin-induced cAMP levels (Knecht et al., 1985; Richard, 1994) or steroidogenic enzyme activity such as peripheral-type benzodiazepine receptor, steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, and 3(3-hydroxy-steroid dehydrogenase (Sridaran et al., 1999a; 1999b). However, some groups reported a stimulatory (Olsson et al., 1990; Parinnaud et al., 1988) or no effect (Casper et al., 1984) of GnRH on progesterone production in hGLCs. In testis, GnRH exerted a direct stimulatory effect on basal steroidogenesis, and an inhibitory effect on gonadotropin-stimulated androgen biosynthesis (Hsueh et al., 1981). 15 There are reports to suggest GnRH is involved in the process of fertilization. GnRH and GnRH agonists have been shown to increase cleavage rate of bovine oocyte (Funston et al., 1995). Moreover, GnRH enhanced sperm-zona pellucida binding ability, which is completely blocked by cotreatment with GnRH antagonist (Morales, 1998). During the luteal, not the follicular phase of the menstrual cycle, both GnRH mRNA and protein have been demonstrated in the human fallopian tube, where spermatozoa and oocytes are deposited to form zygotes (Casan et al., 2000). 3.2.2. Placenta-endometrium-embryo Distribution Immunoreactive GnRH has been shown in the cytotrophoblast, villus stroma, and syncytiotrophoblast (Miyake A et al., 1982; Seppala et al., 1980). The total human placental content of immunoreactive GnRH progressively increases during the first 24 weeks of gestation and remains relatively constant in the third trimester, as measured by radioimmunoassay (RIA) (Siler-Khodr and Khodr, 1978). In contrast, using solution hybridization/ribonuclease protection and in situ hybridization assays, it has been demonstrated that GnRH mRNA levels remain constant throughout gestation (Kelly et al., 1991). Low affinity binding sites for GnRH in human placenta were identified using radiolabeled ligand binding studies (Currie et al., 1981; Iwashita et al., 1986). GnRHR mRNA was detected in both syncytiotrophoblasts and cytotrophoblasts from placentas at all ages except term placentas, and is regulated dynamically during pregnancy (Lin et al., 1995). The GnRHR mRNA signals are abundant at 6 weeks of gestation, peak at 9 weeks, and decline in the second trimester. By term, the signals have fallen below the sensitivity 16 of detection by in situ hybridization. GnRH and its receptor mRNA is expressed in freshly isolated endometrial epithelial and stromal cells (Casan et al., 1998; Emons et al., 1993b; Pahwa et al., 1991; Raga et al., 1998a). It has been shown that immunoreactive GnRH was produced and secreted in vitro by cultured rhesus monkey embryo (Seshagri et al., 1994), mouse embryo (Raga et al., 1998b), and human embryo (Casan et al., 1999; Raga et al., 1998c). Physiological Role In the placenta, GnRH stimulates the secretion of human chorionic gonadotropin (hCG) via a receptor-mediated event (Ertl et al., 1993; Currie et al., 1993; Kim et al., 1987; Lin et al., 1995; Prager et al., 1992; Siler-Khodr et al., 1986). The responsiveness of placental explants to GnRH stimulation in hCG secretion is parallel with the changing intensity of GnRHR gene expression (i.e. first trimester placentas are more sensitive to GnRH stimulation than term placentas) (Lin et al., 1995). Tissue inhibitor of metalloproteinase-1 (TIMP-1) and -3 mRNA expression in cultured endometrial stromal cells and protein secretion into the medium were significantly decreased by a GnRH agonist (Raga et al., 1999b). These results provided evidence for a role of GnRH in the implantation process by remodeling endometrium. Recent evidence suggests a role for GnRH in embryo development. Preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH agonist and was significantly decreased by GnRH antagonist (Raga et al., 1999a). It has been demonstrated that infertile woman undergoing in vitro fertilization and embryo transfer had a significantly higher pregnancy and implantation rate if the administration of GnRH agonist was maintained during the early stages of embryonic development and implantation (Rega et al., 1998c). 17 3.2.3. Reproductive tumors Distribution GnRH and its receptor have been identified in human ovarian carcinoma (Emons et al., 1992a; 1993a; Imai et al., 1994a; 1994c; Irmer et al., 1995; Yin et al., 1998), breast tumor tissues (Harris et al., 1989; Harris et al., 1991), endometrial carcinoma (Chatzaki et al., 1996; Emons et al., 1989; 1993b; Imai et al., 1994b), as well as prostate tumors (Qayum et al., 1990). Using the molecular cloning technique, Kakar and colleagues have reported that a nucleotide sequence of the GnRHR in human ovarian tumors was identical to that of the GnRHR found in the human pituitary gland (Kakar et al., 1994). Physiological Role GnRH has been suggested to be an autocrine/paracrine regulator of tumor growth. Since bioactive GnRH analogues have been developed, GnRH and its synthetic analogues have recently been used and proven to be efficient in treating the sex steroid-responsive cancers such as ovarian cancer (see the section 6 for more details), breast cancer, prostate cancer, uterine endometrial cancer (Emons et al., 1989; 1992a; 1993a; 1993b; 1996; Gallagner et al., 1991; Harris et al., 1989; 1991; Klijn, 1984; Qayum et al., 1990; Shally et al., 1990). 3.2.4. Clinical applications The ability of GnRH to stimulate (at pulsatile low doses) or suppress (at high doses) reproductive functions has been clinically applied for various purposes. These include ovulation 18 induction and spermatogenesis, contraception, and for treatment of precocious puberty, and endometriosis (Hazum and Conn, 1988). 4. Regulation of GnRH and its receptor 4.1 Regulation of hypothalamic GnRH In the hypothalamus, numerous hormones regulate the synthesis and release of GnRH either directly, involving GnRH neurons or indirectly, involving neurons that communicate with GnRH neurons through synapses, gap junctions, electrical coupling or diffusible substances (Cavarretta et al., 1999; Kalra and Kalra, 1983; Lackey et al., 1999; Terasawa, 1998; Wetsel et al., 1996; Witkin, 1999). These include GnRH itself, neuropeptide Y, neuroamines (dopamine, norepinephrine, epinephrine), y-aminobutyl acid, glutamate, nitric oxide, endogenous opioid peptides, growth factors and steroid hormones. Mechanisms for GnRH regulation in the hypothalamo-pituitary gonadal axis include ultrashort, short and long feedback loops. Several lines of evidence support that GnRH neurons have an endogenous GnRH pulse-generating mechanism. In vitro, hypothalamic GT-1 cells release GnRH in a pulsatile manner (Krsmanovic et al., 1992; Martinez et al., 1992). GnRH has been shown to regulate its own synthesis in a biphasic manner in immortalized GT1-7 neurons (Krsmanovic et al., 1993; 1994). In vivo, administration of a GnRH agonist into the cerebroventricule decreased GnRH and GnRHR mRNA expression in a dose- and time-dependent manner (Han et al., 1999). The presence of a short loop feedback mechanism in mammal, whereby L H acts back on the hypothalamus to modulate GnRH release, has long been hypothesized, but experimental evidence varies among different species. In rats, long term L H microimplants positioned directly in the median 19 eminence suppressed circulating L H and GnRH concentrations (Corbin, 1966; David et al., 1966). In addition, short term administration of L H into the third ventricle of the rat inhibited endogenous L H secretion and this effect occurs through a neural site of action (Conway and McCann, 1990). In the pig, intravenous administration of hCG inhibited the L H surge (Ziecik et al., 1988). In hypothalamic GT1-7 neurons, L H through its receptor inhibits GnRH release (Lei and Rao, 1994). These results support existence of a short loop mechanism. In contrast, studies in monkey, human and sheep have failed to demonstrate an operative short feedback system (Kesner et al., 1986; Ordog et al., 1997; Skinner et al., 1997). Physiological studies have indicated that estradiol can increase, decrease, or not change GnRH gene expression from the hypothalamus. During the rat estrous cycle, GnRH mRNA levels in the hypothalamus were inversely correlated to plasma estrogen levels (Zoeller and Young, 1988b). In contrast, others have documented that estrogen increases GnRH gene expression in the rat (Park et al., 1990), which may lead to the gonadotropin surge prior to ovulation. Estradiol given over 7 days increased GnRH mRNA in ovariectomized rats, whereas Zoeller et al. (1988a) found that 2 days of estradiol treatment to ovariectomized rats significantly inhibited GnRH mRNA expression. Differences found in these studies may be explained by different effects of gonadal steroids on hypothalamic GnRH release depending upon the duration and dosage of hormone exposure, the method used to measure GnRH secretion, and perhaps species differences. 5oc-dihydrotestosterone (DHT) suppressed GnRH-induced L H release from anterior pituitary cell cultures (Drouin and Labrie, 1976) and GT1-7 hypothalamic neurons (Belsham et al., 1998). The luteal phase elevation in progesterone inhibits pulsatile GnRH and L H secretion, preventing L H surges in response to estrogen (Dierschke et al., 1973; Goodman and Karsch, 1980; Karsch et al., 1987; Kasa-Vubu et al., 1992). 20 4.2 Regulation of pituitary GnRHR It has been demonstrated that GnRHR levels in the pituitary glands vary during many physiological conditions (Bauer-Dantoin et al., 1995b; Braden and Conn, 1993; Conn, 1994). The number of pituitary GnRHRs varies with developmental stages, estrous cycle, pregnancy, and lactation. During the estrous cycle of rats, GnRHR number is greatest prior to the onset of the preovulatory gonadotropin surges (Bauer-Dantoin et al., 1995b). These high levels were sustained throughout the duration of gonadotropin surges for several hours, followed by a marked decrease at metestrus, and a subsequent slight increase during the day of estrus. In contrast, during pregnancy and lactation, lower levels of GnRHR have been observed than during the estrous cycle (Clayton et al., 1980b; Marian et al., 1981). In lactating rats, pituitary GnRHR mRNA levels were reduced by 60% as compared to diestrous rats, while the removal of the sucking stimulus reestablished GnRHR mRNA expression to the diestrous levels. The expression of the GnRHR in the pituitary is tightly regulated by its cognate ligand, gonadal steroids, and peptide hormones such as activin and inhibin (Braden and Conn, 1993; Conn, 1994). The ability of GnRH to regulate the expression of its own receptor in the pituitary has been well documented. Stimulation with physiological concentrations of GnRH in vitro resulted in a biphasic partem of change in GnRHR numbers (Conn, 1994). Initially, a down-regulation of GnRHR associated with desensitization of the gonadotropes to GnRH is observed, followed by up-regulation. In the ovariectomized rats, estrogen replacement induced a transient fall in the pituitary GnRHR number (Barkan et al., 1983) and mRNA (Kaiser et al., 1993). Testosterone replacement in orchidectomized male rats resulted in a decreased GnRHR mRNA level (Kaier et al., 1993). However, in the absence of an intact hypothalamic-pituitary connection, treatment with estradiol has been shown to increase GnRHR levels (Gregg and Nett, 21 1989). In ovine pituitary cultures, estrogen increased and progesterone decreased the number of GnRHR (Laws SC et al., 1990a; 1990b; Gregg et al., 1990; Menon et al., 1985). Gonadal peptide hormone, inhibin, has also been shown to increase GnRHR number in ovine pituitary cell cultures (Laws SC et al., 1990b; Gregg et al., 1991). Combination treatment of inhibin and estradiol increased GnRHR mRNA level to a higher extent than with each treatment alone. In contrast, inhibin has been shown to decrease the number of GnRHRs in rat pituitary cell culture. Activin increases GnRHR number (Braden and Conn, 1992) and regulates the receptor mRNA level at the transcriptional level (Fernandez-Vazquez et al., 1996). The observed discrepancies in regulation of the GnRHR in the pituitary may indicate that regulation of the GnRHR is complex and this process may occur through different pathways in different species and/or various physiological conditions. 4.3. Intraovarian Regulation of GnRH and GnRHR Unlike in the hypothalamus and pituitary, regulation of GnRH and its receptor in the ovary is poorly understood. In the rat ovary, in situ hybridization analysis revealed that GnRHR gene expression was dependent on the degree of follicular development and the estrous cycle stage. The GnRHR expression was greatest in the granulosa cells from Graafian and atretic follicles, with lower levels of expression present in preantral and small antral follicles and C L (Bauer-Danton and Jameson, 1995a). The GnRHR mRNA levels in atretic follicles increased by 3-fold on the day of proestrus coincident with the preovulatory gonadotropin surges, while levels of GnRHR gene expression in C L significantly increased between the morning of metestrus and the afternoon of proestrus (Bauer-Danton and Jameson, 1995a). Interestingly, GnRHR expression in the rat ovary correlated with the expression of pituitary GnRHR. The highest level of receptor 22 expression was observed in proestrus, just prior to the gonadotropin surge and these levels were maintained throughout the gonadotropin-surge, followed by a decline in metestrus (Bauer-Danton and Jameson, 1995a). In preovulatory rat granulosa cells, GnRH induced an increase in the receptor levels in a dose-dependent manner, whereas L H decreased in GnRHR mRNA levels (Olofsson et al., 1995). In the hypophysectomized rats, administration of FSH or human menopausal gonadotropin altered the distribution of GnRHR mRNA (White et al., 1995). In the rat, estradiol treatment stimulated GnRHR gene expression in granulosa cells from growing and atretic follicles (Kogo et al., 1999). However, little is known in the primate ovary. Recently, GnRH and its receptor have been demonstrated in cultured hGLCs (Peng et al., 1994). Treatment with GnRH induced a biphasic effect on GnRH and GnRHR mRNA expression in these cells. Human C G inhibited GnRHR gene expression in hGLCs (Peng et al., 1994). In hGLCs, estrogen induced a dose- and time-dependent decrease in GnRH mRNA. In contrast, a biphasic effect on GnRHR mRNA expression with time was observed in response to estrogen (Nathwani et al., 2000). 5. Activation of GnRHR and signal transduction Coupled to a G-protein, GnRHR activates multiple signaling pathways, including phosphoinositol turnover, release of intracellular calcium, influx of extracellular calcium, and activation of PKC (Fig. 5). 23 5.1 Receptor-G protein coupling The G a subunit can be divided into two subgroups: pertussis toxin (PTX) sensitive and PTX insensitive. Those that are categorized under PTX sensitive include G ti (t = transducin), Ga, Gn (i = inhibitory), G i 2 , Gi 3 , G 0 , GguSt, G z and have cGMP-phosphodiesterase, K + and C a 2 + channels, and Ca mobilizing receptors as their possible effectors. PTX insensitive G proteins include G s (s=stimulatory), G0if (olfactory), G q , Gn, Gn, G ) 3 , G i 4 and Gi 5 / i 6 , and have Bl - and B2-phospholipase C (PLC) and adenylate cyclase as their effectors (Bourne et al., 1991; Downes and Gautam, 1999). The B subunit is tightly bound to the y subunit, which forms a functional unit. The five B subtypes (Bl, B2, B3, P4, and P5) and 11 y subunit types are identified (Downes and Gautam, 1999). Activation of signal transduction pathways through the plasma membrane receptors is dependent on factors such as the cell type, receptor number, G protein activity and availability, receptor/G-protein affinity, and compartmentalization of signal transduction machinery (Ashkenazi et al., 1987; Stanislaus et al., 1998a; Zhu et al., 1994). Altered combinations of these factors may result in cell-specific activation of markedly different intracellular signaling pathways. It is hypothesized that GnRH is coupled to multiple G-proteins. Studies have demonstrated that the activation of PLC via the GnRHR is mediated through the PTX insensitive G protein (G q/n a) in the pituitary, ovary, testis, mouse gonadotroph ctT3-l, and rat somatolactotroph GGH3 cells (Hsieh and Martin, 1992; Janovick and Conn, 1994; Kaiser et al., 1997; Leung and Steele, 1992; Shah et al., 1994; Stojilkovic et al., 1994). Studies with cholera toxin (CTX; G s a activator) provided evidence for coupling of GnRHR to G s a in rat pituitary cell cultures (Janovick and Conn, 1993), although GnRHR-stimulated cAMP release has not been demonstrated in these cells to date (Conn et al., 1979). However, the GnRHR was capable of activating adenylyl cyclase and releasing cAMP in insect cells transfected with 25 GnRHR and in the stable cell line GGH 31' cells (Delahaye et al., 1997; Kuphal et al , 1994). Recently, it has been shown that activation of GnRHR resulted in time- and dose-dependent palmitoylation (a marker for G protein activation) of G q /n a , G; a and G s a in rat pituitary cell cultures (Stanislaus et al., 1998b). There is evidence that the GnRHR is coupled to Gj a in reproductive tract tumors (Imai et al., 1996b; Limonta et al., 1999). In prostate tumor cells, the GnRHR is coupled to Gj a protein, leading to inhibition of cAMP accumulation, which may mediate the growth inhibitory action of GnRH (Limonta et al., 1999). The coupling of GnRHR to multiple G proteins suggests that the same kind of GPCR may exert distinct cellular functions via differential signal transduction pathways in the target tissues or cells. The binding of GnRH to a pocket formed by the transmembrane regions of the GnRHR induces conformational changes in the receptor. These changes cause the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) from the G protein a subunit and the dissociation of the Py heterodimer, which in turn activates the P-forms of PLC (Spiegel et al., 1992; Stojilkovic et al., 1994). Activation of PLC results in the cleavage of membrane phospholipids into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4, 5-phosphate (PIP2) into inositol 1, 4, 5-triphosphate (IP3) and diacylglycerol (DAG) (Spiegel et al., 1992; Stojilkovic et al., 1994). 5.2 Inositol phosphate, calcium and DAG IP3 is the second messenger molecule in regulating calcium mobilization and mediates both an early and sustained response to GnRH (Naor et al., 1995; Stojilkovic et al., 1994; Stojilkovic and Cart, 1995;). Activation of GnRHR in the pituitary induces a quick rise in intracellular Ca 2 + , followed by a smaller but sustained level (Naor et al., 1995; Stojilkovic and Catt, 1995). The 26 initial and coincident spike phase of the [Ca2+]j is independent of extracellular Ca 2 + , and reflective of C a 2 + released from intracellular stores, as a result of IP3 mediated C a 2 + release from the endoplasmic reticulum (ER). The later sustained peak represents the entry of C a 2 + through voltage sensitive and insensitive C a 2 + channels (Virmani et al., 1990). Calcium binds to C a 2 + binding proteins such as calmodulin and PKC, which cause protein phosphorylation or dephosphorylation, thereby determining cellular response (Noar et al., 1995; Stojilkovic and Catt, 1995). Studies have shown that GnRH-induced L H release is associated with increase in intracellular C a 2 + levels, and the L H release could be inhibited by blocking extracellular C a 2 + (Conn et al., 1983; Conn et al., 1987; Stem and Conn, 1981). The partem of Ca2+mobilization in rat granulosa cells differs from that observed in pituitary cells, as these cells show a rapid transient rise with no sustained phase or oscillations (Leung and Steele, 1992; Wang et al., 1992). In hGLCs, GnRH has been shown to increase in [Ca2+]i, which is abolished by co-treatment with GnRH antagonist (Hori et al., 1998). In addition to IP3, D A G functions as a second messenger for GnRH action through a direct activation of P K C (Naor et al., 1995; Stojilkovic and Catt, 1995). It is suggested that D A G is generated in sequential phases, initially by PLC and later by phospholipase D (PLD) (Naor et al., 1995; Stojilkovic and Catt, 1995). Structural difference in D A G from PLC and PLD may permit selective and/or coordinated activation of the various PKC subspecies, which may lead to cell specific functions of GnRH. 5.3 Protein kinase C The PKC family of proteins is single polypeptides consisting of a regulatory and a kinase domain (Maretelli et al., 1999; Naor et al., 1995). The various PKC subspecies can be classified into conventional PKCs (cPKC: a, Bl, BE, and y), novel PKCs (nPKC: 5, s, n, p, and 0) and atypical PKCs (aPKCs: X, 1) (Maretelli et al., 1999; Naor et al., 1995). The rapid activation of 27 phosphoinositide turnover which supplies C a 2 + and early D A G may activate cPKC, while later, PLC and/or PLD generated D A G might be utilized for activation of nPKC (Naor et al., 1995; Stojilkovic and Cart, 1995). In addition, arachidonate and oleate are known to activate cPKCs at relatively low concentrations, which is independent of C a 2 + and D A G (Naor et al., 1995). aPKCs might be activated by phosphatidylinositol 3,4,5-triphosphate (PIP3). Al l of the second messengers generated by CmRHR activation converge to activate PKC, which in turn regulates the gonadotropin secretion in the pituitary (Kaiser et al., 1997; Naor et al., 1995; Stojilkovic and Catt, 1995). 5.4 Role of PLD signaling pathway In aT3-l gonadotrophs, pituitary cell cultures, GnRH neurons and ovarian granulosa cells, GnRH stimulated an increase in phosphatidylethanol (PEt) production in the presence of low concentrations of ethanol, which is a marker of PLD activation (Liscovitch and Amsterdam, 1989; Naor et al., 1995; Netiv et al., 1991; Zheng et al., 1994). Several lines of evidence indicate that PKC mediates the activation of PLD in GnRH-stimulated pituitary cells (Cesnjaj et al., 1995; Zhen et al., 1995). The production of both D A G and PEt was increased by treatment with phorbol 12-myristate 13-acetate (PMA), and this action was not mediated by PKC activation of PLC-P, as no increase in IP3 production was observed. In addition, GnRH- and PMA-induced elevations of D A G and PEt production were attenuated or abolished in PKC-depleted cells, while GnRH-induced inositol phosphate production was not affected. Also, the PLC inhibitors, U73122 and neomycin, had no effect on PMA-induced PEt formation, while these inhibitors reduced not only the GnRH-induced IP3 response but also the agonist-induced PEt response. The possible role of PLD pathway in GnRH-stimulated exocytosis was investigated in pituitary 28 gonadotrophs using ethanol and propranolol as specific blockers of this pathway (Cesnjaj et al., 1995). These results suggest that the signaling molecules generated by PLD are not essential for agonist-induced exocytosis in pituitary cells (Cesnjaj et al., 1995). In cultured granulosa cells, a similar progesterone production was observed by activation of PLC or treatment with GnRH agonist, suggesting physiological relevance of PLD in GnRH action (Liskovitch and Amsterdam, 1989). This notion was further supported by finding that the stimulatory action of GnRH on steroidogenesis was mimicked by the addition of PA, a product of PLD activity. However, the mechanism by which PLD metabolites modulate cellular constituents involved in steroidogenesis remains to be examined. 5.5 cAMP/cGMP signaling Studies have shown that under certain conditions, GnRH action may be mediated via a cyclic nucleotide-dependent pathway. GnRH has been shown to induce a slight increase in both cAMP and cGMP (Naor, 1990; Sundberg et al., 1976). However, other groups have shown that cyclic nucleotides are not involved in GnRH-stimulated L H release, suggesting that cAMP may be not a second messenger for GnRH (Conn et al., 1979). More detailed studies involving cyclic nucleotides may be necessary to define the role of cyclic nucleotides as a second messenger for GnRH action. 5.6 Role of PLA2 signaling pathway Arachidonic acid (AA) can be generated from phospholipids by the separate or concerted actions of PLC, PLD, and PLA (phospholipase A), all of which have been shown to be activated by GnRH (Naor, 1991; Naor et al., 1995). Studies have demonstrated that specific 5-29 lipoxygenase inhibitors reduced the L H response, suggesting role of A A and its lipoxygenase metabolites in GnRH-induced action in the pituitary (Dan-Cohen et al., 1992). In the ovary, exogenous A A and PLA2 activator enhanced basal production of progesterone (Wang et al., 1989; Wang and Leung, 1988). In the testis, A A , like GnRH, directly stimulates basal steroidogenesis, while GnRH agonist-induced testosterone formation was completely blocked by the PLA2 inhibitors chloroquine and quinacrine (Didolkar and Sundaram, 1989; Lin, 1985). 5.7 Mitogen-activated protein kinases The mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases that are activated in response to a diverse array of extracellular stimuli, and mediate signal transduction from the cell surface to the nucleus (Davis, 1994). Extracellular signal-regulated kinases (ERK1 and ERK2), c-jun terminal kinase/stress-activated protein kinases (JNK7SAPK), and p38 are three of the best characterized M A P K family members (Cobb and Goldsmith, 1995; Fanger, 1999) (Fig. 6). Other M A P K family members, ERK3, 4, and 5, four p38-like kinases, and p57 M A P K have been cloned, but biological roles are not well understood (Fanger, 1999). In most cells, ERK1 and/or ERK2 are activated by mitogenic stimuli, whereas JNK/SAPK and p38 are activated in response to stress such as heat shock, osmotic shock, cytokines, protein synthesis inhibitors, antioxidants, UV, and DNA-damaging agent (Garrington and Johnson, 1999; Robinson and Cobb, 1997). Prolonged activation and nuclear retention of ERKs has been also implicated in neuronal cell differentiation (Robinson and Cobb, 1997). M A P K family members are directly regulated by kinases known as M A P K kinases (MAPKKs) which activate the MAPKs by phosphorylating on tyrosine and threonine residues (Fanger, 1999; Robinson and Cobb, 1997). Currently, seven different MAPKKs have been cloned and characterized (Fanger, 30 1997). The first MAPKKs cloned were M A P K / E R K kinase 1 and 2 (MEK1/2) which specifically activate ERKs. MKK3 and 6 specifically activate p38, whereas MKK5 stimulates the phosphorylation of ERK5. MKK4 (also referred as INK kinase or SEK-1) and MKK7 are known to activate INK. The MAPKKs are activated by a rapidly expanding group of kinases called M A P K K kinases (MAPKKKs), which activate the MAPKKs by phosphorylating on serine and threonine residues (Fanger, 1999; Robinson and Cobb, 1997). These include Raf-1, A-Raf, B-Raf, MAPK/ERK kinase 1-4 (MEKK1-4), apoptosis-stimulating kinase-1 (ASK-1), and mixed lineage kinase-3 (MLK-3). The MAPKKKs may be activated by kinases known as M A P K K K kinases (MAPKKKKs), one of which is p21-activated kinase (PAK). In addition, low molecular weight GTP-binding (LMWG) proteins regulate the activity of MAPKKKs and M A P K K K K s (Fanger, 1999). There are several different families of L M W G proteins, two of which include the Ras (N-Ras, K-Ras, and H-Ras) and Rho (Rac 1, 2 and 3, Cdc42 and RhoA, B and C) families. Activated MAPKs have shown to phosphorylate various downstream effectors, exerting their specific functions (Fig. 5). For example, activated ERK1/2 phosphorylate ternary complex factor (TCF) proteins such as Elk-1 and SAP-1, which form transcriptional complexes with serum response factor (SRF) in the promoter region of early response genes (e.g. c-fos, egr-1, junB) and thereby regulate their expression (Wasylyk et al., 1998) (Fig. 6). 31 Growth factor Stress, differentiation, growth factor Stress i r X s " A t M K K K Raf-1, A-raf B-Raf, MEKK1-4 ASK-1 MLK-3 P A K r r M K K MEK1 MEK2 M K K 4 , M K K 7 M K K 3 , M K K 6 r r r MAPK E R K l ERK2 JNK p38 r r r p90rsk, S6 kinase, Elk-l ,Etsl ,c-myc Sap-a, STATS c-Jun, ATF-2, Elk-l,p53 ATF-2, Elk-1 Max Growth, Growth, differentiation Cytokine differentiation survival, apoptosis production, apoptosis Figure 6. Schematic representation of M A P K cascades (Adapted from Garrington et al., 1999). M K K K s are activated in response to a variety of extracellular signals, including growth factors, differentiation factors, and stress signals. The activated M K K K s then activate one or several specific MKKs. Once activated, MAPKs can phosphorylate transcription factors, other kinases, and other regulatory factors, controlling cellular responses including growth, differentiation, apoptosis, and cell survival. 32 The best understood means of activating the M A P K pathway is receptor tyrosine kinase (Cobb and Goldsmith, 1995). The M A P K cascade can also be activated by G protein-coupled receptors (Cobb and Goldsmith, 1995). Diverse mechanisms have been proposed for M A P K activation by ligands operating via GPCRs (Bisen et al., 1996; Crespo et al., 1994; Gupta et al., 1992; Hordijk et al., 1994; Kasuya et al., 1994; Ohmichi et al., 1994; Pang et al., 1993). G q -linked receptors, such as bombesin receptors, are thought to mediate activation of M A P K almost entirely via PKC (Pang et al., 1993), or in the case of TRH, activation is mediated partly by PKC and also by a Ras-dependent pathway (Ohmichi et al., 1994). Thrombin and lysophosphatidic acid (LPA) stimulate M A P K via Gj in a PTX-sensitive, PKC-independent pathway (Gupta et al., 1992; Hordijk et al., 1994a), whereas endothelin-1 acts in a PTX-sensitive and PKC-dependent mechanism (Kasuya et al., 1994). The agents acting via muscarinic receptors ml and m2 stimulate M A P K apparently via the Py-subunits of G protein in a Ras-dependent, PKC-independent process (Crespo et al., 1994). The cell specific role of G s in regulation of M A P K activation has been reported. In some cells, such as fibroblasts, rat adipocytes, human arterial smooth muscle cells, and NIH 3T3 cells, increased cAMP attenuates activation of M A P K (Hordijk et al., 1994b; Sevetson et al., 1993; Wu et al., 1993), resulted in reduced mitogenic responsiveness to epidermal growth factor (EGF) and LPA (Hordijk et al., 1994b). In contrast, elevation of intracellular cAMP is a potent mitogenic signal for a number of cell types, including Swiss 3T3 cell, thyroid epithelial cells, and the somatotrope cells of the anterior pituitary (Withers, 1997). As discussed, GnRHR is a family member of G-protein coupled receptors and shown to activate multiple signal transduction pathways. Studies have demonstrated that GnRH activates the ERK1/2 cascade in the aT3-l gonadotroph cell line, pituitary organ culture and whole 33 pituitary in vivo (Haisenleder et al., 1998; Michell et al., 1994; Sundaresan et al., 1996). GnRH induced a rapid and sustained activation of ERK1/2 in a mouse gonadotroph-derived aT3-cell line (Sundaresan et al., 1996). This activation of M A P K was mediated via PKC-, but not the ras-dependent pathway (Sundaresan et al., 1996). In aT3-l cells, other studies have demonstrated that GnRH stimulates mainly the ERK1 isoform in a PKC-, C a 2 + - and protein tyrosine kinase-dependent pathway (Reiss et al., 1997). PKC- and PKA-dependent activation of ERK 1/2 by GnRH has been demonstrated in GGH3 cells (Han and Conn, 1999). Pituitary M A P K responses to GnRH have been shown to be dependent on the pattern of GnRH signals (Haisenleder et al., 1998). Continuous exposure of pituitary cells to GnRH stimulated ERK1/2 activity for up to 2 h, whereas pulsatile GnRH sustained ERK 1/2 activation for over 8 h. GnRH-induced E R K 1/2 activation in rat pituitary cell cultures was reversed by treatment with a M E K inhibitor, PD98059, indicating that M E K is an immediate upstream activator of ERK1/2 (Haisenleder et al., 1998). There is evidence to report that GnRH-induced ERK1/2 activation participates in the regulation of the basal transcription machinery in the gonadotroph. Activation of the ERK 1/2 pathway by GnRH contributes to the transcriptional regulation of the glycoprotein a-subunit gene via activation of Ets transcription factors (Roberson et al., 1995). Haisenleder et al. (1998) demonstrated that GnRH-induced ERK 1/2 activation regulates glycoprotein a-subunit, FSHp and GnRHR gene expression in cultured rat pituitary cells. Interestingly, the expression of LHP gene is not correlated with ERK activation, suggesting involvement of other pathways for LHP gene expression such as C a 2 + elevation and PKC activation (Haisenleder et al., 1998). It has also been demonstrated that GnRH activates other MAPKs, JNK and p38 in aT3-l cells. Levi et al. (1998) demonstrated GnRH-induced JNK activation was greater but occurred more slowly than that of ERK. GnRH activation of the JNK cascade is dependent on the low 34 molecular weight GTP-binding protein, Cdc42, and appears to be activated independently of diacylglycerol-dependent PKC isozymes, which differentiate GnRH modulation of the ERK and INK cascades. This suggests that the signaling machinery that is involved in the activation of JNK is different from that leading to ERK activation. Activation of the p38 kinase by GnRH is shown to be dependent on activation of PKC (Roberson et al., 1999). Studies have suggested that GnRH-induced p38 M A P K activation may selectively contribute to the regulation of c-fos protooncogene expression, but not c-jun or the glycoprotein hormone a-subunit gene (Roberson etal., 1999). 5.8 Expression of early response genes By responding to second messengers generated by an extracellular signal, early response genes (ERGs) function as transcriptional regulators to control downstream expression of late genes, resulting in changes in cellular functions. In accordance with the role of GnRH in such cellular functions, activation of GnRHR in pituitary cells, aT3-l gonadotroph, and GT1-7 neurons leads to increased expression of c-fos, c-jun, and junB in a time and a dose-related fashion (Cesnjaj et al., 1993; 1994). GnRH-induced expression of ERGs was PKC-dependent as GnRH-induced expression of ERGs was reduced by PKC-depletion and treatment with a PKC inhibitor (Cesnjaj et al., 1993; 1994). Activation of C a 2 + entry by the calcium channel agonist, Bay K 8644, and by high K+-induced depolarization, also stimulated a dose-dependent and transient expression of ERGs (Cesnjaj et al., 1994). In addition, GnRH-induced c-fos expression in pituitary cells was reduced by ethanol or propanolol treatment, suggesting the involvement of the PLD pathway (Cesnjaj et al., 1995). 35 6. Normal Ovarian Surface Epithelium (OSE) and Ovarian Cancer 6.1. Biology of Normal OSE The ovarian surface epithelium (OSE) is the modified pelvic mesothelium that covers the ovary. It comprises only a minute fraction of the total ovarian mass, but it is thought to be the source of human ovarian epithelial carcinomas. Embryologically, OSE originates in the urogenital coelomic epithelium that also gives rise to the Mullerian ducts, which is the source of the epithelia of the oviducts, endometrium, and uterine cervix (Auersperg et al., 1998; Pansky, 1982). The OSE is developmentally related to the underlying stromal fibroblasts. Evidence of the developmental relationship of OSE to stromal fibroblasts persists in culture under which it produces not only epithelial (laminin and collagen type IV) but also mesenchymal (collagen types I and III) components of extracellular matrix, and converts from an epithelial to mesenchymal morphology in response to a variety of environmental cues including three-dimensional culture using a sponge matrix (Auersperg et al., 1994; Dyck et al., 1996; Kruk et al., 1994). Unlike carcinomas in most other organs, where epithelial cells become less differentiated in the course of neoplastic progression than the epithelium from which they arise, ovarian carcinomas are frequently highly differentiated compared to OSE (Auersperg et al., 1997; 1998). With neoplastic progression, the tendency of ovarian surface epithelial cells to express mesenchymal characteristics diminishes and the cells become increasingly committed to an epithelial phenotype (Auersperg et al., 1994; 1995; Dyck et al., 1996; Maines-Bandiera and Auersperg, 1997). Studies have demonstrated that there is an increased commitment of an epithelial phenotype in the OSE from the ovaries of women with strong histories of hereditary breast/ovarian cancer (Auersperg et al., 1996; 1998). It has been demonstrated that the OSE has 36 the capacity to remodel the ovarian cortex through synthetic, physical, and proteolytic functions that influence the repair process, as it proliferates and migrates over the ovulatory site (Kruk et al., 1994; Kruk and Auersperg, 1992; Osterholzer et al., 1985). These functions vary with the reproductive cycle and thus are likely hormone dependent (Nicosia et al., 1991; Nicosia and Nicosia, 1988; Osterholzer et al., 1985). There is evidence that apoptosis, which is induced by prostaglandins (Ackerman and Murdoch, 1993; Murdoch, 1995) and perhaps mediated by the Fas antigen (Baldwin et al., 1999; Quirk et al., 1997), is responsible for this cyclic, localized loss of OSE at the ovulatory sites. 6.2. Endocrine, Paracrine and Autocrine Factors in OSE and Ovarian Cancer Ovarian cancer is the fourth to fifth most common cause of death from gynecologic malignancy among North American women. It is believed to originate from the ovarian surface epithelium, which account for 80-90% of all ovarian cancers (Dietl and Marzusch, 1993). Etiological studies have demonstrated that the risk of epithelial ovarian cancer is decreased by factors that block ovulation such as oral contraceptives, breast-feeding, and pregnancy during the menstrual cycle (Hamilton, 1992; Shoham, 1994). Increasing evidence strongly suggests that multiple factors including peptide hormones, sex steroids, growth factors and cytokines have been implicated as stimulatory or inhibitory growth regulators in normal OSE and ovarian cancers. These regulators appear to exert their actions through specific receptors in an endocrine, paracrine or autocrine manner. The potential interaction of several of these endocrine hormones and growth factors are also suggested (Fig. 7) 37 6.2.1. Gonadotropin-releasing hormone. GnRHR were detected in about 80% of human ovarian epithelial tumors and in numerous ovarian cancer cell lines such as EFO-21, EFO-27, and OV-1063 (Emons et al., 1993a; 1997; Miyazaki et al., 1997). GnRH and its analogues have been shown to be efficient in treatment of tumors of ovary. In vivo, long acting GnRH agonists are thought to act by desensitizing or down-regulating the GnRHR in the pituitary, resulting in a subsequent decline in gonadotropins levels which serve as tumor growth factors. The suppression of endogenous L H and FSH secretion by GnRH-agonist treatment results in growth inhibition of heterotransplanted ovarian cancers in animal models (Peterson, 1994). In vitro, GnRH and its analogs have been shown to inhibit the growth of a number of GnRHR-bearing ovarian cancer cell lines. For instance, Emons et al. (1994a) reported a time- and dose-dependent inhibition on the growth of two ovarian 6 cancer cell lines, EFO-21 and EFO-27 by the GnRH agonist, [D-Trp ]LHRH. In other studies, growth inhibition of the ovarian cancer cell line, OVCAR-3, was observed by the administration 6 of GnRH agonists such as [D-Trp ]LHRH and Lupron-SR (Mortel et al., 1986; Peterson et al., 1994). Another GnRH agonist, buserlin, suppressed FSH-induced proliferation of DMBA-OC-1 cell line (Maruuchi et al., 1998). Interestingly, an antagonistic analog of GnRH, SB75, also dose-dependently inhibited the proliferation of OV-1063 cells as indicated by the reduction in cell number and DNA synthesis (Yano et al., 1994). In a clinical trial, the combined treatment with 6 the GnRH agonist, [D-Trp ]LHRH and cisplatin has been shown to improve the positive outcome as compared to patients on chemotherapy alone (Medl et al., 1993). To improve the therapeutic efficiency of GnRH analogs against cancer cells and reduce cytotoxicity against normal cells, targeted chemotherapy based on the GnRHR has been developed recently (reviewed in Schally and Nagy, 1999). Targeted cytotoxic peptide conjugates consist of a peptide that binds to 39 receptors in tumors and a cytotoxic chemical. Cytotoxic analogs of GnRH, AN-152 in which a cytotoxic chemical, doxorubicin (DOX) is linked to a peptide, [D-Lys 6]GnRH, and AN-207 which consists of 2-pyrrolino-DOX (AN-201) coupled to the same peptide, have been developed. Preliminary studies have demonstrated that these cytotoxic analogs of GnRH showed high-affinity binding for GnRHR in tumor cells and were less toxic and more effective than their respective radicals in inhibiting the growth of GnRHR positive human ovarian, mammary or prostatic cancer cells (Kahan et al., 1999; Szepeshazi et al., 1992). AN-152 given intraperitoneally was more effective and less toxic than equimolar doses of DOX in reducing the growth of GnRHR positive OV-1063 human ovarian cancers in nude mice (Miyazaki et al., 1997). In the same study, AN-152 did not inhibit the growth of GnRHR negative UCI-107 human ovarian carcinoma, indicating a targeted cytotoxic effect of the GnRH-conjugate. In a recent study, another cytotoxic analog of GnRH (AN-207) also inhibited the growth of ovarian tumor cells, OV-1063, in nude mice with less toxicity than equimolar doses of its radical 2-pyrrolino-DOX (AN201) (Miyazaki et al., 1999). AN-152 and AN-207 have also been shown to inhibit the growth of estrogen-independent M X T mouse mammalian tumor cells (Szepeshazi et al., 1997) and PC-82 human prostate cancer cells in nude mice (Koppan et al., 1999). The exact mechanism underlying the growth inhibitory effect of GnRH analogs remains to be elucidated. At the ovarian GnRHR level, the putative endogenous ligand may stimulate the proliferation of the cells through the receptor, which might be down-regulated by continuous treatment with a potent GnRH agonist. The finding that continuous treatment with GnRH agonists, which is thought to induce receptor down-regulation, inhibited ovarian cancer cell growth, and that this effect was abolished by co-treatment with a specific GnRH antagonist, corroborated this view (Thomson et al., 1991). Alternatively, the ovarian GnRHR might mediate 40 direct antiproliferative effects of GnRH analogs. However, this notion is not corroborated by the observation that both antagonistic and agonistic analogs have been reported to induce growth inhibition of ovarian cancer cells (Yano et al., 1994). Recently, it has been suggested that the well established GnRHR signaling mechanism mediated by PLC and PKC is likely not involved in the antiproliferative effects of GnRH in tumor cells (Emons et al., 1998). Rather, GnRH binding in cancer cells could activate a down-stream phosphotyrosine phosphatase (PTP) in GnRHR-bearing tumors, thereby counteracting the effects of growth factors that function through receptor tyrosine kinase (Imai et al., 1996a; Lee et al., 1996). It has been reported that analogues of GnRH reverse the growth stimulatory effect of EGF and insulin-like growth factor (IGF) in cancer cells including carcinomas of the ovary (Emons et al., 1996; Hershkovitz et al., 1993; Marelli et al., 1999), possibly by down regulating their receptor numbers and/or mRNA levels. At the ovarian cell level, it has been demonstrated that GnRH analogs reduce cell proliferation by increasing the portion of cells in the resting phase, Go-Gi (Thomson et al., 1991) and inducing cell death or apoptosis (Motomura, 1998; Sridaran et al., 1998). Treatment of ovarian cancer cells with GnRH analogues may induce apoptosis mediated by the Fas ligand-Fas system (Imai et al., 1998a; 1998b), which has been shown to trigger apoptosis in a variety of cell types (Nagata and Goldstein, 1995). Recently, it has been demonstrated that a GnRH analogue may modulate ovarian cancer cell growth by inhibiting telomerase activity without altering the RNA component of telomerase expression (Ohta et al., 1998). 6.2.2. Steroids Both epidemiological and experimental observations have implicated sex steroids in the pathogenesis and growth regulation of carcinomas arising from the ovary (Chien et al., 1994; 41 Galtier-Dercure et al., 1992; Garg et al., 1998; Hoover et al., 1977; Langon et al., 1994; Rodriguez et al., 1995; Silva et al., 1998; 1999; Young et al., 1985). Estrogens taken as oral contraceptives during premenopausal years are protective, but when used in postmenopausal years as hormone replacement therapy, may increase the risk of ovarian cancer (Clinton and Hua, 1997; Garg et al., 1998; Rao and Slotman, 1991; Risch, 1998; Rodriguez et al., 1995). Breast-feeding, which appears to offer protection in a number of studies (Liu et al., 1983), is associated with reduced serum concentrations of estradiol. In experimental animal models, ovarian epithelial tumors were induced by estrogen and testosterone stimulation in guinea pigs (Silva et al., 1997; 1998) and exogenous estrogen stimulated the growth of several ER-positive ovarian carcinoma cell lines in vitro (Chien et al., 1994; Galtier-Dercure et al., 1992; Langon et al., 1994). Other ovarian steroids such as androstenedione, testosterone and progestins have also been implicated as risk factors for ovarian cancer (Clinton and Hua, 1997; Rao and Slotman, 1991; Risch, 1998). In patients with ovarian cancer, elevated plasma levels of 17(3-estradiol, estrone, progesterone, 20oc-hydroxyprogesterone, dehydroepiandrosterone sulfate, androstenedione, and testosterone have been observed and shown to correlate with tumor volume (Backstrom et al., 1983; Mahlck et al., 1985; 1986a; 1986b; 1988). Elevated levels of sex steroid hormones are thought be produced by ovarian tumor cells. This notion is supported by the increased levels of sex steroids in the ovarian vein draining the tumor-bearing ovary, as compared with the contralateral ovarian vein and the peripheral blood (Aiman et al., 1986; Heinonen et al., 1986; Kitayama and Nakano, 1990). In addition, in vitro ovarian epithelial tumor cell cultures have been shown to produce steroids (Ridderheim et al., 1993; Wimalsasena etal., 1991). 42 The classical estrogen receptor (ER), now referred to as ERa, and the progesterone receptor (PR) were found in <50% of ovarian tumors, whereas androgen receptor (AR) was detected in the majority of cases reported (> 80%) (Clinton and Hua, 1997; Rao and Slotman, 1991; Risch, 1998). Receptors for estrogen (ERa), progesterone and androgen were found at the mRNA and/or protein level in rat OSE cells (Adams and Auersperg, 1983) and human OSE cells (Karlan et al., 1995). Also, the expression of a second isoform of estrogen receptor (ERP) has been reported in normal and malignant ovarian cells in primary cultures or cell lines (Brandenberger et al., 1998;Lauetal., 1999). The exact mechanism of action of steroid hormones in normal and ovarian cancer remains unclear. Induction of c-myc oncoprotein has been shown to mediate the mitogenic response to growth stimuli. Depending on the levels of estrogen receptor, up-regulation of c-myc protein by estrogen has been shown to mediate estrogen-induced ovarian cancer cell growth (Chien et al., 1994). It has been demonstrated that estrogen and prolactin stimulate proliferation of ovarian and breast carcinoma cells and concurrently upregulate BRCA1 mRNA and protein (Favy et al., 1999; Romagnolo et al., 1998). In normal OSE, exogenous estrogen did not affect growth, suggesting indirect effects. It has been demonstrated that estrogen interacts with other growth factors in the normal OSE and ovarian cancer cells. Estrogen has been shown to modulate levels of hepatocyte growth factor (HGF) (Liu et al., 1994) and EGF both of which stimulate OSE growth (see below). In the ovarian cancer cell line, PE01, the estrogen-mediated growth stimulatory effects were reversed by treatment with an EGF receptor-targeted antibody (Simpson et al., 1998). In addition, estrogen induced a significant increase in TGF-a protein concentration in media and regulated EGF receptor (EGFR) expression in those cells. These results suggest that estrogen may act through increasing production of transforming growth factor (TGF)- a and 43 regulation of the EGFR. Estrogen produced a concentration-related potentiation in the growth response to IGF-1 and EGF under conditions where the growth responses to EGF and IGF-1 were submaximal (Wimalasena et al., 1993). Estrogen has been shown to exert its enhancement of EGF- and IGF-1-mediated growth through increased binding affinity for EGFR and IGF-1 receptor (IGFR) number (Wimalasena et al., 1993). In other studies, estrogen caused a marked decrease in insulin-like growth factor binding protein-3 (IGFBP-3) mRNA, but increased IGFBP-5 mRNA levels, suggesting that IGFBP expression can be regulated in estrogen-responsive ovarian cancer by E2 (Krywicki et al., 1993). In ovarian carcinoma cells, estrogen and progesterone markedly influence the steady state levels of mRNA for the HGF receptor Met (Moghul et al., 1994) and DHT downregulated the expression of mRNA for the TGFp receptors (TGFpRs) (Evangelou et al., 2000). 6.2.3. Gonadotropins Epidemiological studies indirectly indicated the involvement of gonadotropins in ovarian tumorigenesis. An increased occurrence of ovarian cancer with exposure to high levels of gonadotropins during menopause or infertility therapy, as well as a reduced risk of ovarian cancer associated with multiple pregnancy and contraceptive use (Rao and Slotman, 1991; Shoham, 1994; Shushan et al., 1996; Whittemore et al., 1992). Receptors for FSH and L H were found to be present in normal OSE and half of ovarian tumors (Konishi et al., 1999; Mandai et al., 1997), further supporting the role of gonadotropins in ovarian cancer. Clinically, administration of human menopausal gonadotropin for ovulation induction may increase the risk of epithelial ovarian tumors (Shushan et al., 1996). It has been shown that gonadotropins increase cell proliferation of normal OSE of several species in vivo and in vitro (Davies et al., 44 1999; Osterholzer et al., 1985). In addition, FSH, L H and hCG increased human ovarian cancer cell growth in a dose- and time-dependent manner (Kurbacher et al., 1995; Wimalasena et al., 1992). The mechanism by which gonadotropins increase ovarian cancer cell growth is unclear. It has been shown that hCG induced estradiol production in a dose dependent manner, whereas FSH had no such effect in primary cultures of epithelial ovarian cancer (Wimalasena et al., 1991). The combined treatment of hCG with estradiol may regulate the growth response of epithelial ovarian cancer cells through IGF-1 and EGF pathway (Wimalasena et al., 1993). Human C G treatment has been demonstrated to suppress cisplatin-induced apoptosis by 58% in the ovarian carcinoma cell line, OVCAR-3 (Kuroda et al., 1998), suggesting that gonadotropins may play a role in preventing apoptosis. Recently, elevated levels of gonadotropins, as found in menopause or after ovariectomy, have been reported to promote the growth of human ovarian carcinoma by induction of tumor angiogenesis (Schiffenbauer et al, 1997). 6.2.4. Epidermal Growth Factor and Transforming Growth Factor-a EGF and TGF-a stimulate proliferation of normal OSE cells and ovarian tumor cells (Berchuck et al., 1993; Crew et al., 1992; Jindal et al., 1994; Kurachi et al., 1991; Pierro et al., 1996; Rodriguez et al., 1991; Zhou and Leung, 1992;). Amphiregulin, which has homology with EGF, acts as a potent mitogen for normal OSE in tissue culture (Gordon et al., 1994; Johnson et al., 1991). TGF-a has been demonstrated immunohistochemically in human OSE in vivo and in vitro (Jindal et al., 1994). The presence of EGFR has been shown in 33% to 75% of ovarian tumors (Battagila et al., 1989; Bauknecht et al., 1988; 1993; Berchuck et al., 1991; Henzen-Logmans et al., 1992; Kohler et al., 1992; Morishige et al., 1991; Owens et al., 1991) and normal 45 OSE cells (Berchuck et al., 1991). The level of EGF receptor has been demonstrated to be higher in malignant ovarian tumors than in benign tumors or the normal ovary (Berns et al., 1992; Owens and Leake, 1993), implicating its prognostic importance. Of particular interest for ovarian cancer is the HER-2/neu receptor, a 185 kDa transmembrane protein kinase with extensive homology to the EGFR (Aguilar et al, 1999). Though normal OSE cells express EGFR, they express little or no HER-2/neu (Berchuck et al., 1990; Gordon et al., 1994; Hung et al., 1992; Kohler et al., 1992; Owens et al., 1991). However, the amplification and/or over-expression of HER-2/neu, frequently observed in different types of tumors, was seen in 30-70% of human ovarian cancers (Haldane et al., 1990; Slamon et al., 1989). The observations of overexpression of the c-erbB-2 (HER-2/neu) in ovarian tumors have stimulated preclinical investigations targeting growth inhibition of HER-2-expressing ovarian tumor cells as novel cancer therapies (Baselga et al., 1996; Fan and Mendelsohn, 1998). 6.2.5. Transforming Growth Factor-ft family TGF-P inhibited the proliferation of monolayers of normal human ovarian epithelial cells by 40-70%o (Berchuck et al., 1992) and by 95% in primary epithelial ovarian cancer cell cultures obtained directly from ascites (Hurteau et al., 1994). Daniel et al (1989) reported that TGF-P inhibited colony formation of 7 of 9 fresh ovarian cancers in soft agar. In contrast, immortalized epithelial ovarian cancer cell lines are found to be relatively resistant to the growth inhibition of exogenous TGF-P treatment (Berchuck et al., 1990; 1992). These data suggest that TGF-P may act as a growth inhibitor that prevents inappropriate proliferation of normal ovarian surface epithelial cells, while loss of this autocrine inhibitory pathway may lead to cancer development in vivo and/or immortalization of cell in vitro. In addition, TGFP also counteracts the growth-46 stimulatory effect of EGF (Vigne et al., 1994). The molecular mechanisms that mediate the growth inhibitory effect of TGF-P are poorly understood (Massague, 1992). Binding of TGF-P to its receptors initiates a cascade of molecular events that are thought to decrease activity of cyclin-dependent kinase (CIPl/WAFl/p21), resulting in arrest of cell cycle from G l into S phase of DNA synthesis in normal and neoplastic ovarian cells (Massague, 1992). In addition, it has been shown that TGF-P can induce apoptosis in both normal and malignant cells under certain circumstances (Edwards and Bartlett, 1999; Havrilesky et al, 1995; Selvakumuran et al., 1994). It is reported that malignant ovarian cells are more susceptible to apoptosis in response to TGF-P than their normal nontransformed counterparts (Havrilesky et al, 1995). Activin and inhibin are members of the TGF-P superfamily (Mathews, 1994; Woodruff, 1998). Most primary epithelial ovarian tumors (96%) synthesize and secrete activin in vitro and serum levels of activin are frequently elevated in women with epithelial ovarian cancer (Welt et al., 1997). Activin stimulates cell proliferation in several neoplastic OSE cell lines, including OVCAR-3, CaOV-3, CaOV-4, and SW-626 (Di Simone et al., 1996; Fukuda et al., 1998). The proliferative index of normal OSE cells was unaffected following activin treatment, even though these cells also express activin receptors (Welt et al., 1997). Serum inhibin levels are elevated in most postmenopausal women with mucinous cystadenocarcinomas and mucinous borderline cystic types of epithelial ovarian tumors (Healy et al., 1993a; 1993b). a-inhibin has been proposed to be a serum marker for epithelial ovarian cancer in postmenopausal women (Lambert-Messerlian et al., 1997). 47 6.2.6. Other Growth Factors and Cytokines The HGF receptor is a product of the c-met protooncogene that has been demonstrated in normal OSE and ovarian cancer (Di Renzo et al., 1994; Huntsman et al., 1999). The HGF/Met system is considered to be a principal paracrine mediator of normal OSE growth (Parrott and Skinner, 2000; Wong et al., 1999), and is also involved in proliferation and the spread of ovarian tumors (Corps et al., 1997; Jeffers et al., 1996; Ueoka et al., 2000). In addition to the paracrine loop, the presence of the autocrine-loop has been demonstrated in bovine, rat and human OSE, and epithelial ovarian cancer (Parrott and Skinner, 2000; Wolf et al., 1991), but not mouse OSE (Yang and Park, 1995). The level of Met may be regulated by gonadotropin, steroids, certain cytokines and growth factors in vivo (Hess et al., 1999) and in various carcinoma cell lines derived from human tissues such as ovary, breast and endometrium (Moghul et al., 1994). HGF itself has been shown to autoregulate c-met mRNA levels (Boccaccio et al., 1994; Moghul et al., 1994). Basic fibroblast growth factor (bFGF), a member of the FGF family of growth factors (Basilico and Moscatelli, 1992) stimulates the proliferation of rabbit OSE (Pierro et al., 1996) and maintains viability in cultured rat OSE cells. In ovarian cancer cells, bFGF is produced and exerted mitogenic action in an autocrine manner (Crickard et al., 1994; Di Blasio et al., 1995). Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is produced by ovarian tumors (Abu-Jawdeh et al., 1996; Boocock et al., 1995; Olson et al., 1994; Yeo et al., 1993), and stimulates DNA synthesis of vascular endothelium (Olson et al., 1994) in viro. IGF family has been shown to affect the growth and differentiation in normal and neoplastic cells (Daughaday, 1990; LeRoith et al., 1995). Platelet-derived growth factor (PDGF), PDGF-a receptor (PDGFa-R) and PDGF-B receptor (PDGFB-R) are expressed in the normal ovary and ovarian neoplasms (Henriksen et al., 1993). It has been shown that PDGF stimulates proliferation 48 of human OSE cells (Dabrow et al., 1998). Bioactive cytokines produced by human OSE and ovarian carcinomas include macrophage colony-stimulating factor (M-CSF) (Kacinski et al., 1990; Ziltener et al., 1993), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Cimoli et al., 1991; Ziltener et al., 1993), Interleukin-1 (IL-1) and IL-6 (Scambia et al., 1994; Ziltener et al., 1993) and tumor necrosis factor-a (TNF-a) (Balkwill, 1992; Bast et al., 1993; Wu et al., 1992; Wu S et al., 1993). Interestingly, fms (receptor for M-CSF) is expressed by many ovarian cancers but not by benign ovarian tumors (Kacinski et al., 1994) or normal OSE (Berchuck et al., 1993). These cytokines has been related in normal ovarian function and neoplastic progression. 49 Hypothesis An intrinsic GnRHs/GnRHR loop will be present in the ovarian cells and have functional roles via activating multiple signaling pathways. This autocrine system will interact with the estrogen/ER system in the ovary. In terms of regulation of gene expression, functional GnRHR found in the ovary will be different from that in the pituitary. The expression, hormonal regulation and function of GnRH-II in the ovary will further strengthen the notion that GnRH is an autocrine regulator in the ovary. Specific Objectives 1. To examine the expression and regulation of GnRH/GnRHR and its functional role in normal human ovarian surface epithelium. 2. To examine regulation of GnRH/GnRHR and its functional role in neoplastic ovarian cells. 3. To examine the relationship between the GnRH/GnRHR system and estrogen/ER in normal and neoplastic ovarian cells. 4. To examine effect of GnRH in the activation of M A P K and its role in both normal and carcinoma cells of the human ovary and placenta 5. To examine the GnRH-induced activation of M A P K and its intracellular signaling pathway in hGLCs. 6. To examine the molecular mechanism for differential transcriptional regulation of the GnRHR gene in the ovary. 7. To examine the expression and its hormonal regulation and function of GnRH-II in the ovary. 50 PART 2. General Materials and Methods 1. Cell cultures The use of human OSE cells and hGLCs in vitro was approved by the Clinical Screening Committee for Research and Other Studies involving Human Subjects of the University of British Columbia. The human OSE cells were scraped from the ovarian surface during laparoscopics for non-malignant disorders and were cultured as described previously (Kruk and Auersperg, 1992) in medium 199:MCDB 105 (Life Technologies, Inc., Burlington, Canada) containing 10% FBS (Life Technologies, Inc), 100 U/ml penicillin G and 100 ug/ml streptomycin (Life Technologies, Inc) in a humidified atmosphere of 5% C02-95% air and passaged with 0.06% trypsin (1:250)/0.01% E D T A (Life Technologies, Inc) in Mg 2 +/Ca 2 +-free Hanks' balanced salt solution (HBSS, Life Technologies, Inc) when confluent. The human ovarian epithelial carcinoma cell line, OVCAR-3 (kindly provided by Dr. T. C. Hamilton, Fox Chase Cancer Center, Philadelphia, PA), was cultured in medium 199 (Life Technologies, Inc.) supplemented with 10% FBS, 100 U/ml penicillin G and 100 ug/ml streptomycin. Primary ovarian tumors (n = 3) were obtained and cultured as described previously (Crickard et al., 1983). The minced tissues were dissociated in medium 199 containing 4 mg/ml collagenase type m (Roche Molecular Biochemicals, Laval, Canada) and 0.1 mg/ml bovine pancreatic deoxyribonuclease I (Roche Molecular Biochemicals) for 1 h in a humidified atmosphere of 5% C02-95% air. The cells were passed through a fine mesh and centrifuged on Ficoll-Paque (Pharmacia Biotech, Morgan, Canada; 1000 xg) to 51 remove red blood cells. To obtain pure cancer cells and remove fibroblasts and mesothelial cells, cell suspensions were placed in 100 mm plastic culture dishes in 10 ml medium 199 supplemented with 20% FBS for 24 h. The supernatant containing unattached tumor cells was collected and subsequently placed on 100 mm culture dishes. The tumor cells were maintained in the presence of medium 199 supplemented with 20% FBS, 100 U/ml penicillin G, and 100 ug/ml streptomycin. For hGLCs, follicular aspirate was collected during oocyte retrieval from women undergoing In Vitro Fertilization at the University of British Columbia and the Genesis Fertility Center. Human GLCs were prepared as previously described (Peng et al., 1994), with some modifications. The follicular aspirates were centrifuged at 1000 xg and the supernatant was removed. The cell pellet was resuspended in 6 ml of Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 100 U/ml penicillin G and 100 ug/ml streptomycin and layered onto Ficoll-Paque and centrifuged to remove red blood cells. Cells in the interface were retrieved and rinsed twice with D M E M . The cell pellet was resuspended in D M E M supplemented with 10% FBS, 100 U/ml penicillin G and 100 u,g/ml streptomycin at a density of 1 x 105 cells/ml. The cells were seeded in 35 mm culture dishes and cultured. An immortalized extravillous trophoblast cell line, IEVT (kindly provided by Dr. P.K. Lala, University of Western Ontario, Canada) and a chorionic carcinoma cell line, JEG (obtained from American Type Culture Collection, Rockville, Maryland) were cultured in D M E M supplemented with 10% FBS, 100 U/ml penicillin G and 100 ug/ml streptomycin. 52 Mouse gonadotroph ctT3-l cells (kindly provided by Dr. Pamela L Mellon, University of California, San Diego, CA) and human embryonic kidney (HEK)-293 cells (kindly provided by Dr. AJW Hsueh, Standford University, CA) were cultured in D M E M supplemented with 10% FBS, 4.5 g/1 glucose, 100 U/ml penicillin G and 100 ug/ml streptomycin. All cell types were cultured in a humidified atmosphere of 5% C02-95% air. 2. Isolation of total RNA Total RNA was prepared from cultured cells using the RNaid kit (Bio/Can Scientific, Mississauga, Canada) according to the manufacturer's suggested procedure. Cells were washed twice with phosphate buffered saline (PBS=137 mM NaCl, 2.7 mM KC1, 4.3 mM Na 2 HP0 4 , 1.4 mM K H 2 P 0 4 , pH 7.3) and were disrupted in 500 pi of lysis buffer [4 M guanidine thiocynate, 25 mM sodium citrate (pH 7.0), 0.5% N-lauroyl sarcosine, and 0.1 M B-mercaptoethanol]. The cellular lysate was then transferred to a microcentrifuge tube (1.5 ml) and 50 pi of sodium acetate (pH 4.0), 500 pi of DEPC-saturated phenol (pH 6.0) and 100 pi of chlorofornv.isoamyl alcohol (24:1) were added, vortexed and left on ice for 20 min. Following incubation, the tubes were centrifuged (10,000 g; 20 min at 4 C) and supernatant (top phase) was collected without disturbing interphase. A second extraction with 400 pi of chloroform:isoamyl alcohol (24:1) was performed and the top phase again carefully removed and placed in a new microcentrifuge tube. Vortexed RNA Matrix (10 pi) was added to each tube, vortexed for 30 sec and incubated for 15 min at room temperature (RT) with occasional mixing to allow RNA adsorption. Tubes were then centrifuged for 1 min at 10,000 g to pellet the RNA/RNA Matrix complex. The 53 supernatant was removed and the pellet was washed twice with 70% ethanol (500 pi), centrifuged and vacuum-dried. The pellet was resuspended in 15 pi of ribonuclease-free water and incubated for 5 min to elute RNA. The RNA concentration was measured based on absorbance at 260 nm. In order to check the integrity of the RNA, the extracted RNA was run on formaldehyde agarose gel in RNA running buffer (0.02 M MOPS, 8 mM NaOAc, 1 mM EDTA). RNA samples were loaded (2 pg in 10 pi) along with 1 x gel loading buffer (GLB; 50% glycerol, 1 mM EDTA, 0.4% bromophenol blue, 0.4% xylene cyanol), and the gel was run (70 V; 3 h). Staining of the gel with ethidium bromide (40 pg/100 ml gel) revealed two RNA bands (18S and 28S), confirming the integrity of the RNA. 3. Reverse transcription of RNA to cDNA Total RNA was reverse transcribed into first strand cDNA (Amersham Pharmacia Biotech., Oakville, Canada), following the manufacturer's procedure. Total RNA (1 to 2.5 pg in 8 pi) was heated at 65 C for 10 min and centrifuged for 1 min at 10,000 g. DTT (1 pi), oligo-dT (1:25 dilution, 1 pi) and bulk mixture (5 pi) were added and incubated for 1 h at 37 C. After incubation, the tube was boiled for 10 min to inactivate reverse transcriptase and stored at -20 C until use. 4. Southern blot hybridization Ten microliters of PCR products were mixed with 1 pi of lOx GLB and were loaded on a 1.5% agarose gel. The gel was run for 1 h at 100 V in 1 x TBE running buffer (0.09 54 M Tris-borate/0.002 M EDTA, pH 8.0) and stained with ethidium bromide (200 ug/100 ml gel). After running, the gel was denatured in a solution containing 1.5 M NaCl/0.5 M NaOH with agitation for 30 min and neutralized in a solution (1.5 M NaCl/1.0 M Tris (pH 8.0)) for 15 min (2 times). Gels were transferred to a Whatman 3 M M paper wick resting on a support. The wick was placed in a tray containing lOx SSC (lx SSC=0.15 M NaCl/0.015 M Na3citrate- 2H 2 0, pH 7.0) and wetted with lOx SSC. The gel was then transferred on top of the Whatman wick. Nylon membrane (Hybond N, Amersham Pharmacia Biotech.) cut to the same dimensions of each gel was wetted with lOx SSC and placed onto the gel. A dry piece of Whatman 3MM paper, followed by a 8 cm thick stack of paper towels was placed on top of the nylon membrane. A glass plate was placed on top of the paper towels and the apparatus was compressed by the addition of a 0.5 kg weight. This transfer setup was left overnight and dismantled the next day in order to allow for capillary transfer of the denatured DNA from the gel onto the membrane. The membrane was wrapped in Saran Wrap™, and exposed to U V light for 5 min to fix the DNA. cDNA probes were labeled with digoxigenin using DIG DNA labeling kit according to the manufacturer's recommended procedure (Roche Molecular Biochemicals). Template DNA (1 ug) was denatured by heating for 10 min and quickly chilled on ice. The DNA was labeled in 20 ul reaction containing 2 ul of hexanucleotide mix, 2 ul of dNTP mix, 1 pi of Klenow enzyme for 30 h at 37 C. After incubation, the reaction was stopped by adding 2 ul of 0.2 M E D T A (pH 8.0) and labeled DNA was precipitated with 2.5 ul of 4 M LiCl and 75 pi of prechilled 100% ethanol for 30 min at -70 C. The D N A was pelleted by centrifugation (10,000 g, 30 min) and washed with 200 ul of cold 70% ethanol. The DNA pellet was dried under vacuum and dissolved in 50 ul 55 T E (Tris-EDTA = 10 mM Tris, 1 mM EDTA, pH 8.0). Labeling efficiency was determined by comparing signal intensity with DIG-labeled control-DNA provided in the kit. The membrane was prehybridized for 2 h and hybridized with denatured DIG-labeled cDNA probes (boiled for 10 min) in hybridization buffer (50% deionized formamide, 5x SSC, 0.1% w/v N-lauroylsarcosine, 0.02% w/v SDS, "2% blocking reagent) for 20 h at 42 C. The membrane was then washed twice with 2x SSC, 0.1% SDS for 10 min at RT, followed by high stringency washing with O.lx SSC, 0.1% SDS at 68 C. After washing, the membranes were incubated for 30 min in Buffer 2 (1% blocking reagent in buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)) and incubated with anti-DIG-AP conjugate (1:10,000 dilution) for 30 min. The membrane was then washed twice in washing buffer (0.3% (v/v) Tween 20 in buffer 1) and equilibrated for 5 min in buffer 3 (0.1 M Tris-HCl, 0.1 M NaCl, 50 mM MgCl 2 , pH 9.5). The membrane was incubated with CSPD R (1:200 dilution) for 10 min at RT. The membrane was sealed, incubated another 15 min at 37 C and exposed to Kodak Omat X-ray film (Eastman Kodak Co., Rochester, NY). 5. Subcloning and plasmid isolation PCR products isolated from gel were cloned into pCRII vector using the T A Cloning Kit (Invitrogen, San Diego, CA). PCR products were fractionated onto 1.2% agarose gel and purified using the PCR Extraction Kit (Qiagen, Hilden, Germany). The gel slice weighed into a 1.5 ml centrifuge tube and 3 vol of Buffer QX 1 and 20 pi of QIAEX II were added. The tube was incubated at 50 C for 10 min to solublize the agarose and bind the DNA with vortexing every 2 min. After centrifugation (10,000g, 1 min), the pellet 56 (DNA/QIAXEn complex) was washed once with 500 pi of Buffer QX1 and twice with 500 pi of Buffer PE, centrifuged and air-dried. The pellet was resuspended in 30 pi of 10 mM Tris-HGl (pH 8.5) and incubated for 5 min at RT to elute DNA. Purified PCR product (10 ng) was ligated in 10 pi ligation reaction containing 25 ng pCR II vector, lx Ligation buffer, 20 IU T4 DNA Ligase for 16 h at 4 C. The ligated DNA was transformed into competent E. Coli (One Shot™ cells) by heat shock at 42 C for 60 sec. Eight hundred microliter of LB broth (10 g Bacto-tryptone, 5 g Bacto-yeast extract, 5g NaCl in 1000 ml distilled H2O) was added and the mixture was incubated for an additional 1 h at 37 C. The ligation mixes were then plated on LB-ampicillin plates (50 mg/ml LB) supplemented with X-Gal (20 pi of 20 mg stock solution) and IPTG (10 pi of a 100 mM stock solution). Plates were incubated for 20 h at 37 C and positive colonies were identified by the blue (negative)-white (positive) selection. Positive colonies were cultured in 2 ml of LB and plasmid DNA was isolated using the alkaline lysis procedure. Bacteria culture (1.5 ml of LB) was transferred to a microcentrifuge tube and centrifuged for 30 sec. The pellet was resuspended with 200 pi of ice-cold Solution I (50 mM Tris- HC1 (pH 8.0), 10 mM E D T A (pH 8.0), 100 pg/ml RNase A), lysed with 200 pi of Solution 2 (200 mM NaOH, 1% SDS) for 5 min and neutralized with 200 pi of Solution 3 (3.0 M potassium acetate, pH 5.5). The tube was centrifuged (10,000 g, 20 min) and supernatant was transferred to the new tube. The supernatant was extracted with equal volume (500 pi) of phenolxhloroform/isoamylalcohol (25:24:1) and plasmid DNA was precipitated by adding 450 pi of isopropanol. The DNA pellet was washed with 70% ethanol, dried under vacuum, and dissolved in 20 pi of TE. The DNA was stored at -20 C until use. 57 6. Sequence analysis The isolated DNA was sequenced by the dideoxy nucleotide chain termination method using the T7 DNA polymerase sequencing kit (Amersham Pharmacia Biotech.). All sequence analysis was performed using both M l 3 universal forward and reverse primers. Double-strand DNA templates (2 ug in 32 pi) were denatured for 10 min by adding 8 pi of 2 M NaOH in reaction volume of 40 pi. Following incubation, DNA was precipitated with a sodium acetate/ethanol (7 pi of 3 M sodium acetate, pH 4.8, 4 pi of distilled water, and 120 pi 100% ethanol) and collected by centrifugation (10,000 g, 20 min), washed with 70% ethanol, and dried under vacuum. The denatured DNA was resuspended in 10 pi of BbO, combined with 2 pi of primer, and 2 pi of annealing buffer (1 M Tris-HCl, pH 7.6, 100 mM MgCl 2 , 160 mM DTT). The reaction was incubated for 5 min at 65 C and then for 10 min at 37 C. After incubation, the reaction mixture was allowed to cool to RT for 15 min in order to promote annealing. After the annealing reaction, 3 pi of Labeling Mix, 5 pCi of [a- SJdATP (Amersham Pharmacia Biotech.), and 2 pi of diluted T7 DNA polymerase (3 U) were added and incubated for 5 min at RT. Four of 2.5 pi dNTP mix (840 pi M of A, C, G, T mix) were prepared and prewarmed for 5 min before addition of 4.5 pi of the labeling reaction to each mix. After incubation for 5 min at 37 C, reactions were stopped with 5 pi of Stop Solution (0.3% each bromphenol blue and xylene cyanol FF, 10 mM E D T A (pH 7.5), and 97.5% deionized formamide) and boiled for 5 min prior to loading (2.5 pi) onto a sequencing gel. Polyacrylamide 6%/7 M urea sequencing gels were prepared and prerun at 45 W constant power for 50 min prior to sample loading. Samples was loaded and run for 3 to 4 h at 45 W constant power. Subsequently, gels were dried at 80 C for 2 h using a gel dryer (Model 58 583, Biorad Laboratories, Richmond, CA) and subjected to autoradiography at -70 C for 24 h. The sequence of PCR products was sent to GenBank at NCBI (National Center for Biotechnology Information) through the internet (www.ncbi.nlm.nih.gov) to compare the identity with published sequences. 7. [3H]thymidine incorporation assay After treatment, the culture medium was removed, and the cells were washed three times with PBS and precipitated with 0.5 ml 10% trichloroacetic acid for 15 min at 4 C. After methanol washes, the precipitate was solublized in 0.5 ml 1 N sodium hydroxide, and the incorporated radioactivity was measured in a 1217 Rackbeta liquid scintillation counter (LKB Wallac, Turku, Finland). Each experiment was repeated three times with triplicate samples. 8. Immunoblot analysis Cells were washed twice with ice-cold PBS and lysed in 100 pi of in ice-cold RTPA buffer (150 mM NaCl, 1% Nondiet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH, 7.5), and 1 mM PMSF, 10 pg/ml leupeptin, 100 pg/ml aprotinin) and collected. The extracts were placed on ice for 15 min and centrifuged to remove cellular debris. The protein content of the supernatants was determined using a Bradford assay (Bio-Rad Laboratories). Cellular extract (10 pi) was incubated with 25 pi of Reagent A and 200 pi of Reagent B, for 15 min with gentle shaking at room temperature. Absorbance of the samples was measured using an ELISA reader (Model EL311, Bio-Tek Instruments, Vermont, CA) at 630 nm wavelength. A typical standard curve for the protein assay is 59 presented in figure 8. Thirty to Fifty pg of total protein was mixed with lx sample buffer (12.5 mM Tri-HCl, pH 6.8, 2.5% SDS, 0.025% bromophenol blue, 2.5% glycerol, 0.62% 2-mecapthoethanol) and boiled for 10 min. The sample mixture was run on 10% SDS-PAGE gels (acrylamide:bisacrylamide= 29:1) in lx gel running buffer (25 mM Tris/250 mM glycine, pH 8.3/0.1%) SDS) at 30 milliamphares for 3 h and electrotransferred to a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech.) in transfer buffer (39 mM glycine/48 mM Tris/20% methanol) at 100 V for lh (34). The protein molecular marker at the range of 7.2 to 207 kDa (Bio-Rad Laboratories) was loaded along with samples. The membrane was then incubated with blocking solution [5% skim milk, 0.05% Tween 20 in TBS (Tris-buffered saline, pH 7.6: 5 mM Tris base/75 mM sodium chloride)] for 2 h and immunoblotted using primary antibody for 16 h at 4 C with gently shaking. After washing three times with TBS-T (0.1% Tween 20 in TBS) for 15 min, the signals were detected with horseradish peroxidase-conjugated secondary antibody (1:1000 dilution in blocking solution), and visualized using the E C L chemiluminescent system (Amersham Pharmacia Biotech.). The membrane was incubated with equal volume (0.125 ml/cm2 membrane) of Detection Solution 1 and 2 for 5 min at RT, wrapped, and followed by autoradiography. 60 Figure 8. A standard curve for the protein assay. Cellular extract (10 pi) was incubated with 25 pi of Reagent A and 200 pi of Reagent B, for 15 min with gentle shaking at room temperature. Absorbance of the samples was measured using ELISA reader at 630 nm wavelength. 61 9. Northern blot analysis Total RNA (40 pg) was denatured in 50% formamide/2.2 M formaldehyde and incubated at 60 C for 15 min. Samples were mixed with lx GLB and then electrophoresed on 1% denaturing agarose gel (20 mM MOPS, 2.2 M formaldehyde, 8 mM sodium acetate, 1 mM E D T A pH 8.0) at a constant voltage of 70 for 3h. Capillary transfer of RNA to nylon membrane was conducted overnight in lOx SSC, as described in Southern blot analysis. Nylon membranes were then irradiated by U V light for 5 min in order to cross-link the RNA to the membrane. Radioactive labeled cDNA probes were prepared from the Random Labeling Kit (Life Technologies, Inc.), according to the manufacturer's suggested procedure. DNA templates (100 ng) were denatured at 100 C for 5 min and placed on ice. Two pi each of the following dNTP (dCTP, dGTP and dTTP), 15 pi of random priming buffer, 50 pCi of [a 3 2-P]dATP (3000 Ci/mmol, Amersham Pharmacia Biotech.) and 3 U of Klenow fragment were added. After an incubation period of 3 h at 25 C, the reaction was stopped by adding 5 pi of Stop Buffer (0.2 mM Na 2 EDTA, pH 7.5). The labeled DNA was purified using G-50 Sephadex column (Amersham-Pharmacia Biotech.). The membrane was pre-hybridized in standard hybridization solution (50% formamide, 5x SSPE, 5x Denhardt's, 0.5% SDS, 100 pg/ml denatured herring sperm DNA) for 3 h at 42 C. Hybridization with radioactive cDNA probes was performed for 20 h at 42 C. The membrane was washed once with 2x SSC/0.1% SDS at RT and twice with O.lx SSC/0.1% SDS at 65 C for 15 min. After washing, the membrane was exposed to Kodak Omat x-ray film (Eastman Kodak Co.). Alternatively, the membrane was washed twice in 0.2x SSC/0.1%) SDS at 65 C for 30 min and at least twice in 0.2x SSC at RT for 30 min. The stripped membrane was then 62 reprobed with radioactive labeled probe for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Olofsson et al., 1995). 10. RIA for progesterone The progesterone concentration was measured by an established RIA, as previously described (Li et al., 1993). The samples were incubated with 0.05 mg rabbit P4 antisera (kindly provided by Dr. DT Armstrong, University of Western Ontario) and 20,000 cpm tracer ([1,2,7,6,17- Ffjprogesterone; Amersham Pharmacia Biotech.) per tube. The range of standards was from 0.39-25 ng/ml. All assay samples were 200 times diluted with 0.1 M phosphate buffered saline-gelatin (PBS-G) containing 8.8 g/1 Na 2HP04, 5.3 g/1 NaH2PO/f H 2 0 , 0.1% gelatin) to a final assay volume of 300 pi. After 16 h incubation at 4 C, 500 pi of a cold charcoal:dextran solution (0.25:0.025%) w/v in PBS-G) was added to each tube and vortexed gently to separate bound from unbound antigen. After centrifugation, 500 pi of the supernatant was collected and diluted with 3 ml of scintillation cocktail (BDH, Oakville, Canada) and counted in a Wallac 1217 Rack beta-counter. The standards and samples were assayed in triplicate. Intra- and interassay variation coefficients were 5% and 7%, respectively. A typical standard curve for the progesterone assay is presented in figure 9. 63 y = -17.926Ln(x) +72.197 R 2 = 0.9951 10 0 -I 1 1 0.1 1 10 100 Progesterone concentration (ng/ml) Figure 9. A standard curve for the progesterone assay. The standard progesterone (0.39-25 ng/ml) was incubated with 0.05 mg rabbit P4 antisera and 20,000 cpm tracer ([l,2,7,6,17-3H]progesterone) per tube. After 16 h incubation at 4 C, 500 pi of a cold charcoakdextran solution (0.25:0.025% w/v in PBS-G) was added to each tube and vortexed gently to separate bound from unbound antigen. After centrifugation, 500 pi of the supernatant was collected and diluted with 3 ml of scintillation cocktail and counted in a Wallac 1217 Rack beta-counter. The standards were assayed in triplicate. 64 11. RIA for intracellular cAMP After the desired hormone treatment, medium was removed and 500 ul of prechilled 100% ethanol was added to the dishes. After an incubation period of 20 min on ice, ethanol was collected and centrifuged (10,000 g, 20 min) to precipitate cellular debris. The supernatant was transferred to a new tube, dried completely under vacuum and dissolved in 120 pi of Reagent 1 (0.05 M Tris, pH 7.5/4 mM EDTA). Intracellular cAMP levels were measured using a [ 3H]-cAMP assay system (Amersham Pharmacia Biotech), according to the manufacturer's suggested procedure. A typical standard curve for cAMP assay is presented in Fig. 10. The range of standards and sensitivity was from 1-16 pmol/tube (20-320 pmol/ml of extract) and 0.05 pmoles, respectively. The samples (50 pi) were incubated with 100 pl/tube of Binding protein (purified from bovine muscle) and 50 pl/tube of [8-3H]adenosine 3',5'-cyclic phosphate on ice. Preliminary studies were performed to determine a dilution factor to fit the reading to the range of the standard curve. All assay samples were 2.4- to 10-fold diluted with Reagent 1. After 16 h incubation at 4 C, 100 pi of a cold charcoal was added to each tube and vortexed gently to separate bound from unbound antigen. After centrifugation, 200 pi of the supernatant was collected and diluted with 3 ml of scintillation cocktail (BDH) and counted in a Wallac 1217 Rack beta-counter. The standards and samples were assayed in duplicate. 65 y = 2.3291x-2.6543 -2 -\ 1 1 1 1 1 0 2 4 6 8 10 Co/Cx Figure 10. A standard curve for the cAMP assay. The standard cyclic A M P (1-16 pmol/tube) was incubated with 100 ul/tube of Binding protein (purified from bovine muscle) and 50 pl/tube of [8-3H]adenosine 3',5'-cyclic phosphate on ice. After 16 h incubation at 4 C, 100 pi of a cold charcoal was added to each tube and vortexed gently to separate bound from unbound antigen. After centrifugation, 200 pi of the supernatant was collected and diluted with 3 ml of scintillation cocktail and counted in a Wallac 1217 Rack beta-counter. The standards were assayed in duplicate. Co, cpm bound in the absence of unlabelled cyclic AMP; Cx, cpm bound in the presence of standard cyclic AMP 66 PART 3 Role of Gonadotropin-Releasing Hormone as an Autocrine Growth Factor in Human Ovarian Surface Epithelium I. Introduction Considering the integral role of OSE cells in normal ovarian physiology, understanding their growth regulation appears to be very important. Intraovarian regulators such as IGF, TGF, EGF and GnRH act through autocrine/paracrine mechanisms to modulate ovarian functions and growth of ovarian cells (Adashi and Rohan, 1992). In addition to its well documented role in the regulation of gonadotropin synthesis and secretion, GnRH has direct effects on the gonads and reproductive tumors including carcinomas of the ovary (Conn, 1994; Emons et al., 1993a; 1993b; Harries et al., 1991; Leung and Steele, 1992; Ny et al., 1987; Qayum et al., 1990; Wong et al., 1992; Yano et al., 1994). In addition to the functional role, the presence of an autocrine/paracrine regulatory system based on GnRH in normal and malignant reproductive tissues suggests that GnRH and its receptor may be regulated in these extrapituitary tissues similarly to those in the hypothalamus and pituitary. In view of role of GnRH in modulating ovarian functions, GnRH may be implicated in the functions of OSE cell physiology. However, the role of GnRH and its regulation in OSE cells has not been reported. In the present study, to examine the potential role of GnRH as an autocrine regulator in hOSE cells, the expression and homologous regulation of GnRH and its receptor gene weres investigated. Furthermore, to investigate the physiological significance, the direct eceptor-mediated growth regulatory effects of GnRH on hOSE cells were studied. Elucidation of the 67 regulation and function of GnRH and its receptor in the ovarian surface epithelium will contribute to a better understanding of normal ovarian physiology and the role of OSE in ovarian carcinogenesis. II. Materials and Methods Treatments To study the homologous regulation of GnRH and GnRHR mRNA by a GnRH agonist, 2 x 105 hOSE cells (n = 3; passage 2) were cultured for 48 h and treated with the (D-Ala 6)-GnRH (Sigma-Aldrich Corp., Oakville, Canada) at concentrations of 10"7, 10"9 and 10"n M for 24 h. To confirm the specificity of the GnRH agonist, the cells were treated with different concentrations of (D-Ala 6)-GnRH (10~n,10"9or 10"7M) plus the GnRH antagonist (antide; 10"9 or ICT7 M , Sigma-Aldrich Corp.) for 24 h. Cell cultures were also treated with antide alone. The preliminary studies were performed to determine the optimal concentration of antide to block the effect of (D-Ala 6)-GnRH. Control cultures were treated with vehicle. RT-PCR amplification of GnRH and GnRHR mRNA To clone GnRH and GnRHR mRNA, two sets of primers were designed based on the published sequence of human hypothalamic GnRH (Adelman et al., 1986) and pituitary GnRHR cDNA sequence (Kakar et al., 1992), respectively. Primers for GnRH were: sense, 5'-A T T C T A C T G A C T T G G T G C G T G - 3 ' (Fl); and antisense, 5'-G G A A T A T G T G C A A C T T G G T G T - 3 ' (Rl). Primers for GnRHR were: sense, 5'-A A T A T G G C A A A C A G T G C C T C T - 3 ' (P48-2F); and antisense, 5'-68 G G A T A T T T T T C T C T G T G A T T G - 3 ' (P48R). The cDNA (2 ul from 2.5 pg total RNA) was amplified in a 50 pi PCR reaction containing 2.5 units Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mM MgCb, 2 m M deoxy-NTP, and 50 pmol specific primers. PCR amplification was carried out for 33 cycles with denaturing for 1 min at 94 C, annealing for 35 sec at 53 C (for GnRH) or 60 C (GnRHR and B-actin), extension for 90 sec at 72 C, and a final extension for 15 min at 72 C. Primers for B-actin were designed based on published sequences (Ng et al., 1985). PCR for B-actin was performed to rule out the possibility of RNA degradation and was used to control the variation in mRNA concentration in the RT reaction, as previously described (Peng et al., 1993). To confirm the identify of the PCR product, Southern blot analysis was performed. The PCR products were transferred to a nylon membrane and hybridized with digoxigenin-labeled cDNA probe for human GnRH, GnRHR and P-actin. Sequence analysis was performed to further verify the identity of PCR products. Construction of the native (target) and mutant (competitive) cDNA for GnRHR Using internal primers for human GnRHR, a 347 bp fragment of native GnRHR-cDNA (the target) was obtained from PCR amplification of a 760 bp human pituitary cDNA as a template (Fan et al., 1994). The primers employed were: sense, 5'-G T A T G C T G G A G A G T T A C T C T G C A - 3 ' (P44F); and antisense, 5'-G G A T G A T G A A G A G G C A G C T G A A G - 3 ' (P45R). PCR amplification was carried out for 33 cycles with denaturing for 1 min at 94 C, annealing for 35 sec at 60 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C. The PCR product was verified by Southern blot and sequence analysis. After confirmation, the cloned fragment was subcloned into pBSKII (Stratagene, CA) at Sac I and Xho I sites for cloning purposes. To generate a mutant (the 69 competitor) cDNA, the subcloned fragment (1 pg) was digested with Hind III (10 IU) and Sty I (10 IU) for 2 h at 37 C and subsequently self-ligated with T4 DNA ligase (20 IU) for 16 h at 14 C. This step resulted in a cDNA fragment of a 247 bp with a 120 bp deletion compared with the target cDNA but with identical primer amplification sites (P44F and P45R). Quantification of GnRH and GnRHR mRNA To compare different expression levels for GnRH and GnRHR mRNA, semi-quantitative and competitive PCRs were performed, respectively. For GnRH, 1 pi of the first cDNA was amplified with denaturing for 1 min at 94 C, annealing for 35 sec at 53 C, extension for 90 sec at 72 C, and final extension for 15 min at 72 C for 26 cycles. For GnRHR, 2 pi of the first cDNA was coamplified with 0.002 pg of competitor (mutant GnRHR) cDNA. Amplified PCR products were quantified using a computerized visual light densitometer (model 620, Bio-Rad Laboratories) after Southern blot analysis. [ HJthymidine incorporation assay To investigate the role of GnRH in growth regulation of hOSE cells, a [3H]thymidine incorporation assay was performed as previously described (Wang et al., 1996). Human OSE cells (n=3; passage 2) were plated in 24-well plates at 2 x 104 cells/well and cultured for 24 h in 0.5 ml medium 199:MCDB 105. The cells were then incubated with medium containing 1 pCi [3H] thymidine (5.0 Ci/mmol; Amersham Pharmacia Biotech Inc, Canada.), collected after 24 h and served as day 0 controls. On the day of treatment, the cells were treated with different concentrations (10"n, 10"9 or 10 7 M) of the GnRH agonist, (D-Ala 6)-GnRH for 2, 4, and 6 days on a daily basis. In order to block the effect of the GnRH agonist, the cells were treated with the 70 (D-Ala 6)-GnRH (10"7 M ; GnRHa), antide (10"7 M), and (D-Ala 6)-GnRH plus antide at equimolar concentration for 2, 4, and 6 days. Control cultures were treated with vehicle. Prior to the day of collection, the cells were incubated with medium containing the hormone and 1 pCi [3H] thymidine. After 24 h incubation, proliferative index was measured using a [3H]thymidine incorporation assay. Data analysis GnRH mRNA levels were expressed as the ratio of GnRH to B-actin. The amount of GnRHR transcript was calculated from the ratio of the target to competitive cDNA. Expression levels of GnRH and GnRHR mRNA are expressed as the percent change from the control value. Data are shown as the means of three individual experiments with duplicate samples and are presented as the mean ± SD. In the proliferation study, values are expressed as the percentage of growth compared with the control value and are the mean + SD of three individual experiments with triplicate samples. The data were analyzed by one-way A N O V A followed by Tukey's multiple comparison test. P< 0.05 was considered statistically significant. III. Results Expression of GnRH and GnRHR mRNA in hOSE cells Using primer F l and R l (Fig. 11 A), a 380 bp DNA fragment was obtained from the hOSE cells, a primary culture of ovarian carcinoma, and the OVCAR-3 cell line and were validated as GnRH by hybridization with a specific probe for GnRH cDNA (Fig. 1 IB). To investigate the expression of GnRHR mRNA, one set of primers was designed based on the human pituitary 71 GnRHR cDNA sequence (Fig. 12A). As shown in Fig. 12B, the predicted PCR products were obtained in hOSE cells, primary culture of ovarian carcinoma and the OVCAR-3 cell line, and were confirmed as GnRHR by hybridization with a GnRHR cDNA probe. The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed and detected in negative controls (without template and without reverse transcriptase in the RT reaction) by ethidium bromide staining and Southern blot analysis, respectively (Figs. 11B and 12B). In addition, the authenticity of the PCR products was further confirmed, because no PCR products were obtained from human embryonic kidney (HEK-293) cDNA (data not shown). Sequence analysis revealed that GnRH and its receptor have sequences identical to those found in the hypothalamus and the pituitary, respectively (data not shown). 72 A Figure 11. Detection of GnRH mRNA by RT-PCR amplification. The locations of the primers (FI and Rl) employed are indicated (A). First strand cDNAs from the hOSE cells (OSE), primary culture of ovarian carcinoma (PCO), and the OVCAR-3 cell line (OV-3) were amplified using one set of PCR primers derived from human hypothalamic GnRH cDNA (21). The expected products were observed on a ethidium bromide-stained gel (B, top panel). The PCR products were transferred onto a nylon membrane and hybridized with a digoxigenin-labelled 380-bp hGnRH cDNA probe (B, bottom panel). The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed and detected in negative controls (without template [Tm(-)] and without reverse transcriptase [RT(-)] in the RT reaction) by ethidium bromide staining and Southern blot analysis. 73 A H i l l I I I I T M I II III IV VI VII VIII P48-2F P48R • < B MW Tm(-) RT(-) OSE OV-3 PCO 1650 bp 850 bp-1003 bp Figure 12. Detection of GnRHR mRNA by RT-PCR amplification. The locations of the primers employed (P48-2F and P48R) are indicated (A). First strand cDNAs from hOSE cells (OSE), primary culture of ovarian carcinoma (PCO), and the OVCAR-3 cell line (OV-3) were amplified using the primers derived from human GnRHR cDNA (22). The expected products were observed on a ethidium bromide-stained gel (B, top panel). The PCR products were transferred onto a nylon membrane and were hybridized with a digoxigenin-labelled 364 bp hGnRHR cDNA probe (B, bottom panel). The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed and detected in negative controls (without template [Tm(-)] and without reverse transcriptase [RT(-)] in the RT reaction) by ethidium bromide staining and Southern blot analysis. 74 Validation of PCR for GnRH and GnRHR transcript To determine the conditions under which PCR amplification for GnRH and p-actin mRNA were in the logarithmic phase, 2.5 pg of total RNA were reverse transcribed, and aliquots (2 pi) were amplified using different numbers of cycles. A linear relationship between PCR products and amplification cycles was observed in both GnRH (Fig. 13A) and p-actin mRNA (Fig 13B). Twenty-six cycles for GnRH and 18 cycles for P-actin were employed for quantification. For competitive PCR for GnRHR, mutant GnRHR cDNA was generated (Fig. 14A). The standard curve for GnRHR was constructed by a coamplification of a fixed amount of competitive cDNA (mutant GnRHR) by adding serial dilutions of the target cDNA (native GnRHR). The amount of target and competitive cDNA added to each PCR are shown in Fig. 14B. As increasing amounts of target cDNA were added, decreased amplification of competitive cDNA was observed. When plotted, a linear relationship was found between the native and mutant GnRHR cDNA (Fig. 14B). To titrate the amount of competitor, a fixed amount of first strand cDNA from hOSE cells (2 pi from 2.5 pg RNA) was coamplified with serial dilutions of competitive cDNA. Increasing the amount of mutant cDNA resulted in decreased amplification of the native GnRHR from the sample cDNA. A similar degree of amplification was observed when 0.002 pg mutant cDNA was added (Fig. 14C). This concentration was employed for competitive PCR for GnRHR transcript. 75 A Cycle Number 23 26 29 32 CL 0 ' , , 1 20 23 26 29 32 35 Cycle Number B 0 ! 1 15 18 21 24 Cycle Number Figure 13. Validation of semi-quantitative RT-PCR for GnRH (A) and B-actin (B) in hOSE cells. Total RNA from hOSE was isolated and reverse transcribed, and aliquots were amplified using different number of PCR cycles as described in Materials and Methods. A linear relationship was observed between PCR products and amplification cycles when plotted. 76 B Amount of mutant 10 Native GnRHR cDNA (pg) 0.0002 0.0008 0.004 0.01 Amount of mutant cDNA (pg) Amount of first cDNA (ul) 347 bp native " GnRHR .227 bp mutant GnRHR -0.8 y = 0.2918Ln(x) + 1.9878 2 _ 0.9844 0.0001 0.001 0.01 Mutant GnRHR (pg) 0.1 Figure 14. Construction of native (target) and mutant (competitive) cDNA and validation of competitive RT-PCR for GnRHR transcript in hOSE cells. To generate mutant (the competitor) cDNA, a 347-bp fragment of native CmRHR-cDNA (the target) was digested with Hind III and Sty I and subsequently self-ligated (A). The standard curve for GnRHR was constructed by a coamplification of a fixed amount of competitive cDNA (mutant GnRHR) and a serial dilution of the target cDNA (native GnRHR) (B). To titrate the amount of competitive cDNA for competitive PCR, a fixed amount of first strand cDNA from hOSE cells (2 pi from 2.5 pg RNA) was coamplified with serial dilutions of competitive cDNA (C). 77 Homologous regulation of GnRH and GnRHR mRNA Treatment of OSE cells with the GnRH agonist induced a biphasic regulation partem for GnRH and GnRHR mRNA levels. High concentrations of (D-Ala 6)-GnRH (IO 7 and 10"9 M) decreased GnRH and GnRHR mRNA levels, whereas a low concentration (10"n M) resulted in an up-regulation of GnRH and its receptor (Fig. 15A and B). To confirm the specificity of the biphasic effect by the GnRH agonist, the cells were treated with different concentrations of the GnRH agonist together with the GnRH antagonist, antide. Treatment with antide abolished the biphasic response in the hOSE cells (Fig. 15B). Antide alone had no effect on GnRHR mRNA levels (Fig. 15B). [ H]thymidine incorporation assay As shown in Fig. 16A, (D-Ala 6)-GnRH inhibited the growth of hOSE cells in a dose-and time- dependent manner. A significant inhibition of proliferation was detected as early as the second day of treatment at concentrations of 10"7 and 10"9 M . At a lower concentration (10"H M), reduction of cell growth on day 2 was insignificant. Inhibition of growth continued through to the fourth day and was further evident in the sixth day of treatment. On day 6, a high concentration of (D-Ala 6)-GnRH (10"7 M) had reduced cell proliferation to 68% of the control value and a low concentration of 10"11 M reduced growth to 76% of the control level. In order to block the effect of the GnRH agonist, the cells were treated with the (D-Ala 6)-GnRH (IO 7 M ; 7 6 GnRHa), antide (10" M), and (D-Ala )-GnRH plus antide at equimolar concentrations for 2, 4, and 6 days. The antiproliferative effect of the GnRH agonist appeared to be receptor mediated as cotreatment with antide abolished effects of the agonist (Fig. 16B). 78 Control 10"u M 10"9M 10"'M (D-Ala )-GnRH Treatment B 175 150 w a) > —* 125 o ^ c 100 75 1 * * ° a: — 50 c O 25 0 Control 10"11 M • No Antide • With Antide M Antide alone 10"yM (D-Ala 6)-GnRH Treatment Figure 15. Effect of (D-Ala 6)-GnRH on GnRH mRNA (A) and GnRHR mRNA (B) in hOSE cells. After a preincubation period of 48 h, hOSE cells were treated with different concentrations of the (p-Ala6)-GnRH for 24 h. To confirm the specificity of the GnRH agonist, cells were incubated with medium containing (D-Ala 6)-GnRH (10"n M) plus antide (10~9 M), (D-Ala6)-GnRH (10"9 M) plus antide (10 7 M), (D-Ala 6)-GnRH (10"7 M) plus antide (10"7 M) for 24 h. Cell cultures also treated with antide alone. Control cultures were treated with vehicle. GnRH and GnRHR mRNA levels were measured by semi-quantitative and competitive RT-PCR, as described in the Materials and Methods, respectively. Data are shown as the means of three individual experiments with duplicate samples and are presented as the mean ± SD. a, P<0.05 vs. control; b, P<0.05 vs. 10"11 M (D-Ala 6)-GnRH. 79 A (D-Ala 6)-GnRH c o cc o o c (D c "E c o o B c o to It 8 | •= 8 -4-* CO 120 100 80 60 40 20 -4—Control l -*—10-"M " A - 10' 9 M - • — 1Q- 7M 0 2 4 6 Time of treatment [Days] —•— Control —•— GnRHa Nm^I II i l I U A Antide —•—GnRHa+ Antide . 0 2 4 6 Time of treatment [Days] Figure 16. Effect of (D-Ala 6)-GnRH and cotreatment of antide on growth of hOSE cells. The cells were treated with different concentrations of the (D-Ala 6)-GnRH (10"n, 10"9 or 10"7 M) for 2, 4, and 6 days (A). In order to block the effect of the GnRH agonist, the cells were treated with the (D-Ala 6)-GnRH (IO"7 M ; GnRHa), antide (10"7 M), and (D-Ala 6)-GnRH plus antide at equimolar concentrations for 2, 4, and 6 days (B). Control cultures were treated with vehicle. Proliferative index was measured using the [3H]thymidine incorporation assay. Data are shown as the means of three individual experiments with triplicate samples and are presented as the mean ± SD. a, P<0.05 vs. control; b, P<0.05 vs GnRHa + antide. 80 IV. Discussion A diverse range of regulatory molecules are known to be involved in the critical processes of normal ovarian function (Adashi and Rohan, 1992). This study has demonstrated for the first time that GnRH might be a novel regulatory agent of OSE cells in the human. Using RT-PCR and Southern blot analysis, it was demonstrated that hOSE cells express GnRH and GnRHR mRNA. Sequence analysis indicated that GnRH and GnRHR in hOSE cells have a nucleotide sequence identical to GnRH and the receptor found in the hypothalamus and pituitary, respectively. Furthermore, the presence of GnRH and its receptor in hOSE cells as well as in primary culture of ovarian carcinomas and the established ovarian cancer cell line, OVCAR-3 indicate that the local regulatory system based on GnRH in hOSE cells is a normal component of the cells, not one that is newly acquired in the course of neoplastic transformation. One of the interesting findings of the present study is the demonstration that GnRH gene expression is regulated by GnRH itself in normal epithelial cells, as in the hypothalamus. It has been demonstrated that GnRH regulates its own synthesis and release through an ultrashort loop feedback mechanism in the rat hypothalamus (Bedran et al., 1985; Depaolo et al., 1987; Valenca et al., 1987). In addition, GnRH has been shown to exert biphasic effects on GnRH secretion from immortalized and normal hypothalamic neurons depending on the concentration and duration of treatment (Krsamanovic et al., 1993; 1994). As well, it has been documented that GnRH-binding sites are both up- and down-regulated by GnRH in the pituitary of various species (Conn, 1994; Kaiser et al., 1997). In general, low doses or pulsatile treatment of GnRH up-regulate its receptor, whereas high doses or continuous treatment down-regulate the receptor numbers. Changes in the GnRHR mRNA level have been explained as at least part of the mechanisms underlying up- and down-regulation of GnRHR receptor numbers. Pulsatile 81 treatment with 10 nM GnRH induced an increase of GnRHR mRNA levels in rat pituitary cells (Kaiser et al., 1993), whereas continuous treatment with the same concentration of GnRH for 48 h decreased levels of the receptor mRNA in cultured sheep pituitary cells (Wu et al., 1994). A biphasic regulation pattern of GnRHR mRNA levels has been reported in human ovarian granulosa-luteal cells (Peng et al., 1994). In the present study, a low dose of GnRH agonist (10 pM) increased GnRHR mRNA levels. In contrast, higher doses (1 and 100 nM) of the GnRH agonist induced a statistically significant decrease in GnRHR mRNA levels. This regulation appears to be receptor mediated as cotreatment with a competitive GnRH antagonist, antide (Legal et al., 1989), blocked the biphasic effect of GnRH. Our results suggest that similar to the pituitary GnRHR, alterations in GnRHR mRNA levels are involved in the regulation of responsiveness to GnRH in hOSE cells. Taken together, these results strongly indicate the presence of an autocrine regulatory system based on GnRH in normal ovarian epithelial cells. GnRH analogs have been used and proven to be efficient in treating GnRHR-bearing tumors including carcinomas of the ovary, breast, and endometrium (Emons et al., 1993a; 1993b; Harries et al., 1991; Qayum et al., 1990; Yano et al., 1994). Using thymidine incorporation assays, the present study demonstrated that a GnRH analogue inhibits growth of the normal ovarian surface epithelial cell in a time- and dose-dependent manner. This growth inhibitory effect of the GnRH agonist appears to be receptor mediated such that cotreatment with antide abolished the effect of the GnRH agonist. The exact mechanism of the growth inhibitory effects of GnRH analogues in hOSE cells at the receptor level is unclear. The putative endogenous ligand may stimulate proliferation of the cells through the receptor, which might be down-regulated by continuous treatment with a potent GnRH agonist. The findings in this study that continuous treatment with the GnRH agonist, which is thought to induce receptor down-82 regulation, inhibits cell growth, and that this effect was abolished by cotreatment with a specific antagonist upholds this view. Alternatively, the receptor might mediate direct antiproliferative effects of GnRH analogs. This notion is supported by the finding in this study that the agonist, not the antagonistic analogue that does not trigger receptor activation, induces growth inhibition of the cells. Recently, it has been demonstrated that GnRH binding in cancer cells could activate a down-stream phosphotyrosine phosphatase in GnRHR-bearing tumors, thereby counteracting the effects of growth factors that function through tyrosine kinase (Imai et al., 1996; Lee et al., 1991). For example, GnRH analogs reverse the growth stimulatory effect of epidermal growth factor and insulin-like growth factor in cancer cells including carcinoma of the ovary (Emons et al., 1996; Hershkovitz et al., 1993; Marelli et al., 1999). Interference by GnRH analogs with growth factor receptors may activate intracellular signaling pathways that partially account for the growth inhibitory effects of GnRH agonist seen in hOSE cells. At the moment, it is not known whether the GnRH analogue in hOSE cells exerts its anti-proliferative effect at the cellular level. It has been demonstrated that GnRH analogs reduce cell proliferation by increasing the portion of cells in the resting phase, Go-Gi (Thomson et al., 1991) or induce cell death or apoptosis in the ovarian cells (Motomura, 1998; Sridaran et al., 1998). 83 PART 4 Autocrine Role of Gonadotropin-Releasing Hormone (GnRH) and Its Receptor in Ovarian Cancer Cell Growth I. Introduction Extrapituitary roles for GnRH in the endocrinology of normal and malignant reproductive tissues have been suggested. This concept is based on the detection of GnRH gene transcripts, synthesis of the GnRH, and the multitude of effects attributed to GnRH receptor-mediated signaling in extrapituitary tissues (Bramley et al., 1986; Harris et al., 1991; Irmer et al., 1994; 1995; Peng et al., 1994). We have recently proposed that GnRH and its receptor may have an autocrine role in human OSE (Kang et al., 2000). In the present study, the possibility of the presence of a GnRH/GnRHR loop was explored in epithelial ovarian tumors that originate from normal OSE. As in the hypothalamic-pituitary axis and normal OSE, homologous regulation of GnRH and its receptor was investigated in human ovarian cancer cell line. Furthermore, to investigate the physiological significance, the direct receptor-mediated growth regulatory effects of GnRH and its possible anti-growth mechanism were studied. Understanding of the regulation of GnRH and its receptor, and of the growth-inhibitory mechanisms of GnRH on ovarian cancer cells, may contribute to better hormonal treatment protocols. 84 II. Materials and Methods Treatments The OVCAR-3 cell line was chosen for this study, because this cell line has been shown to express GnRH and GnRHR (Kang et al., 2000). To study homologous regulation of GnRH and GnRHR mRNA, OVCAR-3 cells were cultured for 48 h and then treated with the (D-Ala 6)-GnRH at concentrations of 10"7, 10"9 and 10"11 M for 24 h. To confirm the specificity of the GnRH agonist, the cells were simultaneously treated with different concentrations of (D-Ala 6)-GnRH (10"H, 10"9 or 10"7 M) plus the GnRH antagonist (antide; 10"9 or IO"7 M) for 24 h. The preliminary studies were performed to determine the optimal concentration of antide to block the effect of (D-Ala 6)-GnRH. Cell cultures were also treated with antide alone. To determine whether the inhibition of OVCAR-3 cell growth was associated with altered GnRHR gene expression, OVCAR-3 cells were incubated in medium containing 10"7M (D-Ala 6)-GnRH for 1-6 days; on a daily basis. Control cultures were treated with vehicle. RT-PCR amplification and quantification GnRH and f3-actin PCRs were performed and quantified, as described in the PART III. For GnRHR, 2 pi of the first cDNA was coamplified with 0.006 pg the competitor cDNA. Cell proliferation assays The growth of OVCAR-3 cells was determined by measuring the DNA content as described previously (Labarca and Paigen, 1980). OVCAR-3 cells were plated in 24-well plates at 3 x 104 cells/well and cultured for 24 h in medium containing 5% FBS. After a preincubation period of 85 24 h, the cells were serum-starved for 16 h and incubated in medium containing 5% FBS and different concentrations of the GnRH agonist, (D-Ala 6)-GnRH (10 7 to 10~n M) for 2, 4, and 6 days added on a daily basis. Control cultures were treated with vehicle. On the day of collection, the cells were washed with PBS three times and trypsinized with 0.5 ml TRTPCK trypsin (2.5 mg/ml, Cooper Biomedical, USA) for 15 min at RT. The cell lysates were diluted with 3 ml PBS and 150 pi (20 pg/ml) of Hoechest 33258 (Sigma-Aldrich Corp.) was added. Amounts of DNA were measured using a DNA spectrophotofluorometer (American Instrument Company, Inc., Maryland, USA) at excitation wavelength 354 nra and emission wavelength 458 nm. A typical standard curve for DNA assay is presented in figure 17. Each experiment was repeated four times with triplicate samples. DNA fragmentation assays OVCAR-3 cells were plated in 60 mm culture dishes at 2 x 105 cells and cultured for 24 h. The cells were then treated with the GnRH analogs for 6 days. At the end of day 5 of the assay, the cells in the positive control plates were transferred to poly-HEMA (Sigma-Aldrich Corp.) coated culture dishes to trigger apoptosis (McGill et al., 1997). Genomic DNA was isolated at the end of day 6. OVCAR-3 cells were lysed with digestion buffer (100 mM NaCl, 10 mM Tris-Cl (pH 8.0), 25 mM E D T A (pH 8.0), 0.5 % SDS, 0.1 pg/ml proteinase K) overnight incubation at 55 C. The lysate was extracted with phenol/chloroform, precipitated with ethanol and resuspended with T E (10 mM Tris, 1 mM EDTA, pH 7.5) buffer. Samples were then RNase A (100 pg/ml) treated for 1 h at 37 C, phenol/chloroform extracted, ethanol precipitated and dissolved in T E (500 ng/pl). The equal amounts of each genomic DNA were fractionated on a 1.5% agarose gel and stained with ethidium bromide. 86 Figure 17. A standard curve for DNA assay. OVCAR-3 cells were washed with PBS and trypsinized with 0.5 ml TRTPCK trypsin for 15 min at room temperature. The cell lysates were diluted with 3 ml PBS and 150 pi (20 ug/ml) of Hoechest 33258 was added. Amounts of DNA were measured using a DNA spectrophotofluorometer at excitation wavelength 354 nm and emission wavelength 458 nm. 87 Data analysis The data were analyzed by A N O V A followed by Tukey's multiple comparison test. P< 0.05 was considered statistically significant. III. Results Validation of PCR PCRs for GnRH and p-actin in OVCAR-3 cells were validated as described in PART III. Like in hOSE cells, twenty-six cycles for GnRH and 18 cycles for P-actin were employed for quantification (Fig. 18). To titrate the amount of the competitor, a fixed amount of first strand cDNA from OVCAR-3 cells (2 pi from 2.5 pg RNA) was coamplified with serial dilutions of competitive cDNA. Increasing the amount of the mutant cDNA resulted in decreased amplification of the native GnRHR from the sample cDNA. A similar degree of amplification was observed when 0.006 pg mutant cDNA was added and this concentration was employed for competitive PCR for GnRHR transcript (Fig. 19). 88 5 T Cycle number 4 23 26 29 32 f mm mmm mm o 1 o -15 18 21 24 Cycle Number Figure 18. Validation of semi-quantitative RT-PCR for GnRH (A) and P-actin (B) in OVCAR-3 cells. Total RNA was isolated, reverse transcribed, and aliquots were amplified using different numbers of cycles as described in the Materials and Methods. A linear relationship was observed between PCR products and amplification cycles when plotted. 89 A Amount of mutant cDNA (pg) 0 . 0 0 0 2 0 . 0 0 0 8 0 . 0 0 8 0 . 0 8 Amount of first cDNA (pi) B 347 bp native GnRHR 227 bp mutant GnRHR y=0.1228Ln(x)+0.6817 0.0001 0.001 0.01 Mutant GnRHR (pg) 0.1 Figure 19. Validation of competitive RT-PCR for GnRHR transcript in OVCAR-3 cells. To titrate the amount of the competitor, a fixed amount of first strand cDNA from OVCAR-3 cells (2 pi from 2.5 pg RNA) was coamplified with serial dilutions of competitive cDNA. Increasing the amount of the mutant cDNA resulted in decreased amplification of the native GnRH-R from the sample cDNA (A). A similar degree of amplification was observed when 0.006 pg mutant cDNA was added (B). 90 Homologous regulation of GnRH and GnRHR mRNA Treatment with the GnRH agonist induced a biphasic regulation pattern for GnRH and GnRHR mRNA levels. High concentrations of (D-Ala6)-GnRH (10"7 and 10"9 M) decreased GnRH and GnRHR mRNA levels, whereas a low concentration (10"n M) resulted in an up-regulation of GnRH and its receptor (Fig. 20, A and B). To confirm the specificity of the biphasic effect by the GnRH agonist, the cells were treated with different concentrations of the GnRH agonist together with GnRH antagonist, antide. Co-treatment with antide abolished the biphasic response in the OVCAR-3 cell line (Fig. 20B). Antide alone had no effects on GnRHR mRNA levels (Fig. 20B). Cell proliferation assays As shown in Fig. 21, the (D-Ala 6)-GnRH inhibited the growth of OVCAR-3 cells in a dose-dependent manner. A significant inhibition of proliferation was detected as early as the second day of treatment at concentrations of 10"7 and 10"9 M . At lower concentrations (10"11 M), reduction of cell growth at day 2 was insignificant. The anti-proliferative effect of the (D-Ala6)-GnRH was also time-dependent as the growth inhibitory effect was increased in time of the treatment. Inhibition of growth continued through to the fourth day and further evident in the 7 ft sixth day of treatment. On day 6, 10" M (D-Ala )-GnRH reduced growth down to 62% of control. 91 Control 1 0 - n M l f J 9 M 1 0 ' 7 M (D-Ala 6)-GnRH Treatment • No Antide Control 1 0 ' n M 10' 9 M 10- 7 M (D-Ala 6)-GnRH Treatment Figure 20. Effect of (D-Ala 6)-GnRH on GnRH mRNA and GnRHR mRNA in OVCAR-3 cells. The cells were cultured and treated with different concentrations of the (D-Ala 6)-GnRH (10~n, 10"9 or 10"7 M) for 24 h (A). To confirm of specificity of the GnRH-a, OVCAR-3 cells were incubated with medium containing (D-Ala 6)-GnRH (10"H M) + antide (10"9 M), (D-Ala 6)-GnRH (10 9 M) + antide (10"7 M), (D-Ala 6)-GnRH (10"7 M) + antide (10"7 M) for 24 h. Cell cultures also treated with antide alone. Control cultures were treated with vehicle. Data are shown as the means of three individual experiments with duplicate samples and are presented as the mean ± SD. a, PO.05 vs. control; b, P<0.05 vs. 10"11 M (D-Ala 6)-GnRH. 92 (D-Ala 6)-GnRH 2 4 6 Time of treatment (day) Figure 21. Effect of the (D-Ala 6)-GnRH on growth of OVCAR-3 cells. The cells were treated with different concentrations of the GnRH agonist, (D-Ala 6)-GnRH for 2, 4, and 6 days on a daily basis. Control cultures were treated with vehicle. The (D-Ala 6)-GnRH inhibited the growth of OVCAR-3 cells in a time and dose-dependent manner. Significant reduction in growth vs. control was found at day 2 for the (D-Ala 6)-GnRH of 10"7 and 10"9 M and at days 4 and 6 for all concentrations, a, P<0.05 vs. control. 93 Down regulation of GnRHR mRNA To determine whether the inhibition of OVCAR-3 cell growth was associated with altered GnRHR gene expression, OVCAR-3 cells were treated with 10"7M (D-Ala 6)-GnRH in a time-dependent manner. As shown in Fig. 22, treating the cells with the (D-Ala 6)-GnRH resulted in a time dependent decrease in GnRHR mRNA levels. Densitometric analysis of the transcript revealed that GnRHR mRNA levels were decreased to 62% of control after 1 day of treatment. The mRNA levels were further down-regulated to 34% of control after 6 days of treatment. DNA fragmentation assays To analyze whether the growth inhibitory effects of the (D-Ala 6)-GnRH are associated with programmed cell death, cells were treated with the GnRH agonist for 6 days and genomic DNA was isolated, fractionated by agarose gel electrophoresis. The cells on polyHEMA-treated culture dishes served as positive controls. As shown in Fig. 23, DNA fragmentation was observed in OVCAR-3 cells treated with the GnRH agonist. To confirm whether the cell death was a receptor-mediated event, cells were treated with the GnRH agonist plus antide. No DNA fragmentation was observed under this condition (Fig. 23). 94 120 Time of treatment (day) Figure 22. Effect of continuous treatment of the (D-Ala 6)-GnRH on GnRHR mRNA levels. The OVCAR-3 cells were treated with a (D-Ala 6)-GnRH (10 7 M) in a time dependent-manner. Densitometric analysis of the transcript revealed that GnRHR mRNA levels were, decreased to 62% of control after 1 day of treatment. By day 6, GnRHR mRNA levels were further down-regulated to 34%) of control after 6 days of treatment, a, P<0.05 vs. control. 95 1 2 3 4 5 6 Figure 23. Induction of DNA fragmentation in OVCAR-3 cells by the GnRH analogs. The cells were treated with the GnRH analogues for 6 days and genomic DNA was isolated, fractionated by agarose gel electrophoresis. Lane 1: DNA molecular weight marker; Lane 2: negative control; Lane 3: antide treatment (IO 7 M); Lane 4: positive control from cells on polyHEMA-treated culture dishes; Lane 5: (D-Ala 6)-GnRH treatment (IO 7 M); Lane 6: treatment with (D-Ala6)-7 7 GnRH (10" M) plus antide (10"' M). DNA fragmentation was observed in cells treated with the GnRH agonist but not antide. Co-treatment with antide abolished effect of the GnRH agonist. 96 IV. Discussion In the present study, for the first time, it was demonstrated that similar to in the hypothalamic-pituitary axis and normal OSE cells, GnRH and GnRHR mRNA are regulated by its homologous ligand in a biphasic manner in ovarian epithelial cancer cell line, OVCAR-3. Low dose of the GnRH agonist (10 pM), increased GnRH and GnRHR mRNA levels. In contrast, higher doses (1 and 100 nM) of the GnRH agonist induced a statistically significant decrease in GnRH and GnRHR mRNA levels in OVCAR-3 cells (Fig. 20). This regulation appears to be receptor mediated as co-treatment with a competitive GnRH antagonist, antide (Leal et al., 1989), blocked the biphasic effect of GnRH. Similar to the hypothalamus, pituitary, and hOSE, these results suggest the presence of an autocrine/paracrine regulatory system based on GnRH in the ovarian tumors. Using in vitro cell proliferation assays, it was demonstrated that the GnRH agonist inhibits growth of the ovarian epithelial cancer cell line, OVCAR-3 in a time dependent manner (Fig. 21). This antiproliferative effect of the GnRH agonist appeared to be associated with altered GnRHR mRNA levels in that continuous treatment with GnRH agonist induced a time-dependent down-regulation of the GnRHR mRNA (Fig. 22). The parallelism in the growth inhibition of OVCAR-3 cell and down-regulation of receptor mRNA suggests a direct correlation between these two parameters. Our results are consistent with the pituitary ccT3-l cell, where inhibition of the cell growth correlated with the down regulation of GnRHR mRNA (Kakar et al., 1997). Agonist-induced decrease in receptor number could occur at the post-translational level, translational level or gene transcriptional level. Down-regulation of GnRHR mRNA levels in OVCAR-3 cells by the GnRH agonist in the present study suggests that loss of receptors can, at 97 least in part, be attributed to decreased receptor mRNA expression. Taken together, these results suggest that inhibition of OVCAR-3 cell growth by continuous treatment of the GnRH agonist is accompanied by down-regulation of GnRHR mRNA levels. The exact mechanism of the growth inhibitory effect of GnRH analogs, however, is still not clear. To elucidate the mechanism of the growth inhibitory effect of GnRH agonist, DNA fragmentation was investigated by agarose gel electrophoresis in cells continuously treated with (D-Ala 6)-GnRH. As shown in Fig. 23, at a high concentration IO"7 M (D-Ala 6)-GnRH but not antide induced DNA fragmentation, which is a hallmark of apoptosis. This effect was abolished by co-treatment with competitive antagonist, antide, suggesting that the action of GnRH agonist is mediated through receptor activation and/or down-regulation. Considering that GnRH analogs induce the expression of immunoactive Fas ligand in Fas/GnRHR bearing tumors (Imai et al., 1998a; 1998b), it is likely that the GnRH analog exerts its antiproliferative action by inducing apoptosis mediated through Fas ligand-Fas system in ovarian cancer cells. 98 PART 5 Estradiol Regulates Gonadotropin-Releasing Hormone (GnRH) And Its Receptor Gene Expression And Modulates The Growth Inhibitory Effects of GnRH In Human Ovarian Surface Epithelial And Ovarian Cancer Cells I. Introduction Estrogen plays a critical role in the events leading to ovulation by modulating GnRH and its receptor levels at the hypothalamus-pituitary level (Braden and Conn, 1993; Conn, 1994). Additionally, other studies have demonstrated the presence of GnRH and its receptor system in the ovary, suggesting the possible regulatory role of estrogen on this system (Kang et al., 2000; Leung and Steele, 1992; Peng et al., 1994). Epidemiological and clinical observations have implicated estrogen in the pathogenesis and growth regulation of carcinomas arising from ovary (Chien et al., 1994; Galtier-Dercure et al., 1992; Langon et al., 1994; Siliva et al., 1997; 1998; Young et al., 1985). Until recently, the classical estrogen receptor (ER, now referred as ERa) was thought to be the only form of nuclear receptor able to bind estrogen, and mediate its hormonal effects in their target tissues. However, the recent cloning of a gene encoding a second type of estrogen receptor (ERf3) in the rat (Kuiper et al., 1996), mouse (Tremblay et al., 1997), and human has prompted further investigations on the estrogen signaling system. Considering that GnRH has recently been proposed to be a potential autocrine regulator in normal OSE and 99 ovarian cancer cells (Kang et al., 2000; PART IV), the relationship between estrogen/ER and the GnRH/GnRHR system was investigated in these cells. In the present study, experiments were designed to investigate (1) the expression of E R a and ERp at the mRNA and protein levels in hOSE and ovarian cancer cells, (2) the regulation of GnRH and its receptor gene by estrogen, and (3) the growth regulatory effect of estrogen. II. Materials and Methods Treatments To study the regulation of GnRH and GnRHR mRNA by 17p-estradiol (Sigma-Aldrich Corp.), 2 x 105 hOSE and OVCAR-3 cells were cultured for 48 h and then treated with 17p-estradiol at concentrations of 10"11, 10"9, and 10"7M for 24 h. To confirm the specificity of 17p-7 7 estradiol, the cells were treated with 17p-estradiol (10" M) plus tamoxifen (10" M , Sigma-Aldrich Corp.) for 24 h. Control cultures were treated with vehicle. The levels of GnRH and GnRHR mRNA were measured by quantitative and competitive RT-PCR, respectively, as described in the PART HI and TV. RT-PCR amplification of ERa and ERp The primers were designed to amplify E R a and ERP based on the published sequences of human E R a and ERp (Green et al., 1986; Mosselman et al, 1996). Primers for E R a were: sense, 5 ' - A T G A C C A T G A C C C T C A A C A C C A A - 3 ' (Fl); and antisense, 5'-CTTGGC A G A T T C C A T A G C C A T A C - 3 ' (Rl). Primers for ERp were: sense, 5'-T A C A G C A T T C C C A G C A A T G T C A C - 3 ' (F2); and antisense, 5'-100 G A A G T G A G C A T C C C T C T T T G A A C - 3 ' (R2). Semi-quantitative PCR amplifications for E R a and ERp were carried out for 30 cycles with denaturing for 1 min at 94 C, annealing for 35 sec at 55 C (for ERa) or 53 C (for ERP), extension for 90 sec at 72 C, and a final extension for 15 min at 72 C. The expression levels of E R a and ERp were normalized against P-actin levels. Immunoblot analysis for ERa and ER$ Fifty pg of total protein from hOSE cells or OVCAR-3 was subjected to immunoblot analysis using a mouse monoclonal antibody for E R a (Santa Cruz Biotech., Santa Cms, CA) (1:1000 dilution) and a goat polyclonal antibody for ERp (Santa Cruz Biotech.) (1:500 dilution), as described in the PART II. f H] thymidine incorporation assay To investigate the role of 17P-estradiol in the growth regulation, hOSE cells (n=3; passage 2) and OVCAR-3 cells were plated in 24-well plates at 2 x 104 cells/well in 0.5 ml medium 199 (phenol red free) supplemented with 2% charcoal-stripped FBS, 100 U/ml penicillin G and 100 pg/ml streptomycin. After a preincubation period of 48 h, the cells were treated with various concentrations (10~8, 10"7or 10"6 M) of 17p-estradiol for 2, 4, and 6 days. In order to investigate the modulatory effect of 17p-estradiol on the growth inhibitory effect of a GnRH agonist, OVCAR-3 cells were pretreated with 17p-estradiol and vehicle for 24 h and then treated with (D-Ala 6)-GnRH (IO"7 M), 17p-estradiol (10'7 M) plus (D-Ala 6)-GnRH (10"7 M) for 2, 4, and 6 days. The same treatment was applied to hOSE cells for 6 days. Control cultures were treated with 101 vehicle. After 24 h incubation, [3H]thymidine incorporation was measured as described in the PART II. Each experiment was repeated three times with triplicate samples. Data analysis The data were analyzed by one-way A N O V A followed by Tukey's multiple comparison test. P< 0.05 was considered statistically significant. III. Results Expression of ERa and ERjd mRNA To investigate the expression of E R a and ERB mRNA in hOSE cells, primary cultures of ovarian carcinoma (PCO), and OVCAR-3 cells, two sets of primers were designed. PCRs for E R a and ER(3 were validated using different numbers of cycles, as described in PART Ul. Thirty cycles for E R a and ERB, and 18 cycles for P-actin were employed for semi-quantitative RT-PCR amplification. A 540 bp for E R a and 279 bp DNA fragment for ERp were obtained from hOSE cells, PCO, and OVCAR-3 cells. These fragments were validated as E R a and ERp by hybridization with a specific probe for E R a and ERp cDNA (Fig. 24A) and sequence analysis (data not shown). The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed and detected in negative controls (without template and without reverse transcriptase in the RT reaction) by ethidium bromide staining and Southern blot analysis, respectively (data not shown). Quantitative analysis of the present study showed different expression levels of E R a and ERp. The expression levels of E R a were 2.5-fold higher in PCO and 1.5-fold higher in OVCAR-3 cells when compared to hOSE cells (Fig. 24B). No 102 significant difference in ERB levels was observed in PCO, whereas a significant increase (3.3-fold) was observed in OVCAR-3 cells when compared hOSE cells (Fig. 24C). Expression of ERa and ERfi protein To investigate the expression of E R a and ERp protein in hOSE and OVCAR-3 cells, immunoblot analysis was performed using a mouse monoclonal antibody for E R a and a goat polyclonal antibody for ERp. As shown in Fig. 25A, E R a protein (68 kDa) and ERp (55 kDa) were observed in both cell types. Quantitative analysis of the present study showed a slight but significant increase in E R a protein in OVCAR-3 cells when compared to hOSE cells. In contrast, a highly significant increase (2.5-fold) in ERp protein was observed in OVCAR-3 cells (Fig.25B). 103 A h O S E P C O O V - 3 hOSE P C O OV-3 Figure 24. Detection of E R a and ERP mRNA by RT-PCR amplification. First strand cDNAs from the hOSE (OSE), primary culture of ovarian carcinoma (PCO), and the OVCAR-3 cell line (OV-3) were amplified using two sets of PCR primers derived from human ERa and ERP cDNA. The PCR products were transferred onto a nylon membrane and hybridized with a digoxigenin-labeled human E R a and ERp cDNA probe (A). Amplified PCR products were quantified using a computerized visual light densitometer after Southern blot analysis (B and C). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, PO.05 vs. hOSE; b, PO.05 vs. OVCAR-3; c, P<0.05 vs. PCO. 104 hOSE OV-3 E R a ER(3 Figure 25. Detection of E R a and ERp protein by immunoblot analysis. Human OSE (hOSE) and OVCAR-3 cells (OV-3) were seeded at a density of 5 x 10 cells in 35 mm culture dishes and cultured for 72 h. Forty pg of total protein was run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane. The membrane was immunoblotted using a mouse monoclonal antibody for E R a and a goat polyclonal antibody for ERp. After washing, the signals were detected with horseradish peroxidase-conjugated secondary antibody, and visualized using the E C L chemiluminescent system, followed by autoradiography (A) and quantification (B). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. hOSE cells. 105 Regulation of GnRH and GnRHR mRNA by 17B-estradiol Treatment with 17(3-estradiol (10"7 M) induced a 38% decrease in GnRH mRNA in OVCAR-3 cells (Fig. 26A). In contrast, no significant down-regulation of GnRH mRNA was observed in hOSE cells (Fig. 26B). Treatment with 17p-estradiol (IO 9 and 10"7 M) resulted in a significant down-regulation of GnRHR mRNA in OVCAR-3 cells. Maximum down-regulation (to 43% of control levels) was observed at IO"7 M (Fig. 27A). In hOSE cells, 17p-estradiol induced a down-regulation of GnRHR mRNA in a dose-dependent manner, with maximum down-regulation (to 52% of control levels) at 10"7 M (Fig. 27B). To confirm the specificity of the effect of 17B-estradiol, the cells were treated with 17p-estradiol together with tamoxifen (10"7M), an estrogen antagonist. Co-treatment with tamoxifen abolished the down-regulation of GnRH in OVCAR-3 cells and GnRH and GnRHR mRNA in both cells (Fig. 28). 106 A 140 120 i 100 a> o < £ z o fc O a: c O B 140 OVCAR -3 0 -11 -9 -7 Concentration of 17P-estradiol (Log M) hOSE 0 -11 -9 -7 Concentration of 17p-estradiol (Log M) Figure 26. Effect of 17p-estradiol on GnRH mRNA in OVCAR-3 (A) and hOSE cells (B). The cells were treated with various concentrations of 17p-estradiol for 24 h. Control cultures were treated with vehicle. Total RNA was extracted, and reverse transcribed into first cDNA. The levels of GnRH mRNA were measured by quantitative RT-PCR. Amplified PCR products were quantified using a computerized visual light densitometer after Southern blot analysis. Data are shown as the means of three individual experiments with duplicate samples, and are presented as the mean ± SD. a, P<0.05 vs. control. 107 A OVCAR -3 120 i 0 -11 -9 -7 Concentration of 17p-estradiol (Log M) Concentration of 17p-estradiol (Log M) Figure 27. Effect of 17p-estradiol on GnRHR mRNA in OVCAR-3 (A) and hOSE cells (B). The cells were treated with various concentrations of 17P-estradiol for 24 h. Control cultures were treated with vehicle. Total RNA was extracted, and reverse transcribed into first cDNA. The levels of GnRHR mRNA were measured by competitive RT-PCR. Amplified PCR products were quantified using a computerized visual light densitometer after Southern blot analysis. Data are shown as the means of three individual experiments with duplicate samples, and are presented as the mean ± SD. a, PO.05 vs. control; b, P<0.05 vs. 10"11 M 17p-estradiol. 108 Control E2 Txf E2 + Txf OVCAR-3 120 -i Figure 28. Effect of 17p-estradiol and tamoxifen co-treatment on GnRH and GnRHR mRNA OVCAR-3 (A and B) and hOSE cells (C). The cells were treated with 17B-estradiol (E2, 10"7 M), tamoxifen (Txf, 10"7 M), 17B-estradiol (10"7 M) plus tamoxifen (10"7 M) for 24 h. Control cultures were treated with vehicle. Total RNA was extracted, and reverse transcribed into first cDNA. The expression levels of GnRH and GnRHR mRNA were measured by quantitative and competitive RT-PCR, respectively. Data are shown as the means of three individual experiments with duplicate samples, and are presented as the mean ± SD. a, P<0.05 vs. control. 109 Effect of 17B-estradiol on the growth of OVCAR-3 and hOSE cells As shown in Fig. 29A, 17(3-estradiol stimulated the growth of OVCAR-3 cells in a dose-and time-dependent manner. A significant increase of proliferation was detected as early as the second day of treatment at concentrations of 10"8 and 10"7 M . At a higher concentration (10"6 M), stimulation of cell growth was significant on days 4 and 6. Stimulation of growth continued through to the fourth day and was further evident in the sixth day of treatment. On day 6, 17p-estradiol (IO 7 M) induced cell proliferation to 195 % of the control value, and 17p-estradiol (10~8 and 10"6 M) stimulated cell growth to 148 % and 139 % of the control level, respectively. In contrast, 17P-estradiol failed to significantly affect the growth of hOSE cells (Fig. 29B). Modulation of growth inhibitory effect of GnRH agonist by 17 B-estradiol As estrogen down-regulated GnRH and GnRHR mRNA levels, we investigated if estrogen treatment antagonizes the growth inhibitory effect of GnRH-a in OVCAR-3 and hOSE cells. The dose and time of GnRH-a treatment was determined as described previously (Kang et al., 2000; PART IV). As shown in Fig. 30A, GnRH-a induced a significant growth inhibition of OVCAR-3 cells as early as the second day of treatment. Inhibition of growth continued through to the fourth day and was further evident in the sixth day of treatment (to 50 % of control level). Pre-treatment with 17p-estradiol for 24 h partially blocked the growth inhibitory effect of GnRH-a at day 2, but failed to block the effect of GnRH-a at day 4 and 6 of treatment. Pre-treatment for 24 h and co-treatment with 17p-estradiol on a daily basis induced a significant attenuation of growth inhibitory effect of GnRH-a throughout the treatment. In contrast, neither pre- nor co-treatment with 17p-estradiol blocked the growth inhibitory effect of GnRH-a in hOSE cells (Fig. 30B). 110 Figure 29. Effect of 176-estradiol on the growth of OVCAR-3 (A) and hOSE cells (B). The OVCAR-3 and hOSE cells were plated at 2 x 104 cells/well and cultured for 24 h. The cells 8 7 f\ were treated with different concentrations of the 17B-estradiol (10" ,10" or 10" M) for 2, 4, and 6 days. Control cultures were treated with vehicle. Prior to the day of collection, the cells were incubated with medium containing the hormone and 1 pCi [3H]thymidine. After 24 h incubation, the cells were collected and the amount of labeled DNA was measured using the [ Ffjthymidine incorporation assay. Data are shown as the means of three individual experiments with triplicate samples, and are presented as the mean ± SD. a, P<0.05 vs. control. I l l A B OVCAR -3 0 2 4 6 Time of treatment [Days] hOSE 0 6 T ime of treatment [Days] Pretreatment "Treatment • Contro l ' Control •E2 E2 • Control | • Ga • Ga (E2+Ga)| Pretreatment •"Treatment • Control — ^ Control • Control — • Ga • E2 — • Ga • E2 — • (E2+Ga) Figure 30. Effect of 17p-estradiol treatment on the growth inhibitory effect of GnRH-a in OVCAR-3 (A) and hOSE cells (B). The OVCAR-3 and hOSE cells were plated at 2 x 104 cells/well and cultured for 24h. OVCAR-3 cells were pretreated with 17p-estradiol (E2; 10"7 M) and vehicle for 24 h, and then treated with (D-Ala 6)-GnRH (Ga; 10"7 M), 17p-estradiol (10"7 M) plus (D-Ala )-GnRH (10" M) for 2, 4, and 6 days. The same treatment was applied to hOSE cells for 6 days. Control cultures were treated with vehicle. Prior to the day of collection, the cells were incubated with medium containing the hormone and 1 pCi [3H]thymidine for 24 h and were collected. Proliferative index was measured using the [3H]thymidine incorporation assay. Data are shown as the means of three individual experiments with triplicate samples, and are presented as the mean ± SD. a, PO.05 vs. control; b, PO.05 vs. Ga. 112 IV. Discussion Recent studies have revealed different tissue distributions and expression levels of E R a and ERP in the ovary (Kuiper et al, 1996; Mosselman et al., 1996; Sar and Welsch, 1999; Saunders, 1998; Telleria et al., 1998; Tremblay et al., 1997). It has been demonstrated that E R a is localized primarily in the ovarian stromal and theca cells, whereas ERp is predominantly detected in the granulosa cells of small, developing and preovulatory follicles (Mosselman et al., 1996; Saunders, 1998; Telleria et al., 1998). In the present study, the differential expression levels of E R a and ERp mRNA was demonstrated in normal hOSE cells, and in epithelial cancers of the ovary, i.e. OVCAR-3 cells, and PCO. Expression levels of E R a were higher in PCO and OVCAR-3, whereas only OVCAR-3 cells expressed higher levels of ERp when compared to hOSE cells. Despite the abundance of information on ERs mRNA, there is little characterization of the ER protein in the ovary. The open reading frame predicted from the ERp cDNAs encodes a protein of molecular weight of approximately 54 kDa, which contrasts with the size of E R a (approximately 67 kDa) detected by western blotting (Green et al., 1986; Kuiper et al., 1996). In the present study, the predicted size of E R a and ERp protein was demonstrated in hOSE and OVCAR-3 cells using immunoblot analysis. In parallel with mRNA levels, different expression levels of E R a and ERp protein were observed in hOSE and OVCAR-3 cells. The different expression levels of E R a and ERp suggest that these receptors may have different biological roles in regulating the functions of normal hOSE and OVCAR-3 cells. In agreement with these results, other studies have demonstrated the expression of E R a and ERp in cultured hOSE cells and primary cultures of ovarian cancer cells (Brandenberger et al., 1998; Lau et al., 1999). 113 At the hypothalamus-pituitary levels (Conn, 1994; Leung and Steele, 1992), estrogen is thought to be a key regulator of GnRH and its receptor system (Braden and Conn, 1993; Conn, 1994). Recently, we have proposed that GnRH may be an autocrine/paracrine regulator in normal hOSE and ovarian cancer cells (Kang et al., 2000; PART IV). However, no information is available on the role of 17p-estradiol in regulating GnRH and GnRHR in these cells. In the present study, it was demonstrated for the first time that estrogen significantly decreased the GnRH mRNA level in OVCAR-3 cells and GnRHR mRNA levels in hOSE and OVCAR-3 cells. The exact mechanism by which estrogen regulates GnRH and its receptor mRNAs in the ovary and ovarian cancer remains unclear. After binding to its nuclear receptor, estrogen may modulate GnRH and GnRHR directly or indirectly. Even though no consensus sequence for an estrogen response element (ERE) is found in the promoter region of the human GnRH (Radovick et al., 1991) and GnRHR gene (Fan et al., 1995), estrogen can directly modulate transcription of the GnRH gene (Radovick et al., 1991; 1994) and GnRHR promoter activity (unpublished data). Alternatively, estrogen may act through indirect pathways to regulate GnRH and its receptor. It 94-has been demonstrated that estrogen can mobilize intracellular Ca (Morley et al., 1992), which may lead to activation of the PKC pathway. Phorbol ester-induced activation of PKC has been shown to modulate GnRHR gene expression (Conn et al., 1984). In the present study, the effect of estrogen appears to be a receptor-mediated event, as co-treatment with a competitive estrogen antagonist, tamoxifen, abolished the effect of estradiol. It remains to be determined which type of receptor mediates the effect of estrogen in these cells. Other studies have demonstrated that homodimers ERa/ERcc and ERB/ERp, or heterodimers ERa/ERp, can be formed in vitro and bind to the EREs, and can stimulate the transcription of a reporter gene (Pettersson et al., 1997; Crowley et al., 1997). Therefore, the relative expression of E R a and ERp and the difference in 114 DNA binding activity between heterodimers and homodimers could determine tissue-specific effects of estrogen action. Estrogen has been implicated in the pathogenesis and growth regulation of carcinomas arising from ovary (Chien et al., 1994; Galtier-Dercure et al., 1992; Langon et al., 1994; Siliva et al., 1997; 1998; Young et al., 1985). In this study, 17p-estradiol stimulated the growth of an ovarian cancer cell line, OVCAR-3, in a time- and dose-dependent manner. The growth stimulation was detected as early as 2 days of estradiol treatment and was further evident at 6 days. In contrast, estrogen had no effect on the growth of hOSE cells. The data are in agreement with the previous finding that estrogen has no effect on hOSE cells growth (Karlan et al., 1995). The exact mechanisms of estrogen insensitivity in ER-positive hOSE cells are not known. The responses to estrogen may be dependent on the expression levels of ERs in the target tissues. This notion is supported by findings that only ovarian cancer cell lines with high levels of ER expression were responsive to estrogen treatment (Chien et al., 1994; Langon et al., 1994). The different levels of E R a and ERp expression between hOSE and OVCAR-3 cells observed in the present study corroborate this view. Alternatively, the regulation of epithelial cell growth by estrogen in reproductive organs may occur through common paracrine mechanisms mediated by stromal hormone receptors. This possibility is well demonstrated in uterine and vaginal epithelium, where their growth is dependent on stromal E R that may induce epithelial cell proliferation by producing a variety of growth factors (reviewed in ref. Cooke et al., 1998). In this regard, it is likely that the OVCAR-3 cells have acquired the capacity to respond mitogenically to estrogen, a capacity not expressed by normal hOSE cells. Furthermore, it is possible that normal OSE cells may express splice variant forms of E R a and ERp, which negatively affect normal ER signaling. This suggestion is supported by the finding of a mutation 115 in the exon of ERa, which may explain why an ovarian cancer cell line, SKOV3, was reported to be ER-positive but estrogen-insensitive (Hua et al., 1995; Lau et al., 1999). It has been suggested that a stimulatory GnRH loop could be inhibited by GnRH agonists and antagonists, resulting in a growth inhibition in GnRHR-bearing tumors (Schally, 1999). Our previous studies have demonstrated that long-term treatment of GnRH-a induced a significant growth inhibition in hOSE and OVCAR-3 cells (Kang et al., 2000; PART TV). Since GnRH actions are mediated through a G-protein coupled receptor, and estrogen down-regulates GnRH and GnRHR levels, the possibility that estrogen may block the growth inhibitory effect of GnRH-a was further explored in OVCAR-3 and hOSE cells. The data demonstrate that pre- or co-treatment with 17p-estradiol induced a significant attenuation of the growth inhibitory effect of GnRH-a in OVCAR-3 cells. These results suggest that estrogen may function as a growth modulatory factor as well as a mitogen in OVCAR-3 cells. However, it cannot be ruled out the possibility that mitogenic activity of estrogen may override the growth inhibitory of GnRH-a independent of down-regulation of GnRHR in OVCAR-3 cells. Despite down-regulation of GnRHR mRNA levels, neither pre- nor co-treatment with 17p-estradiol blocked the growth inhibitory effect of GnRH-a in hOSE cells. These results suggest that estrogen may have no growth regulatory effect in hOSE cells but mediate other functions on hOSE cells. Considering that hOSE cells are reported to produce cytokines, cell adhesion molecules and proteolytic enzymes (Auersperg et al., 1995; Kruk and Auersperg, 1992; Ziltener et al., 1993), it is possible that estrogen may play a role in regulating these factors in hOSE cells. 116 PART 6 Gonadotropin-Releasing Hormone Activates Mitogen-Activated Protein Kinase in Human Ovarian and Placental Cells. I. Introduction GnRH binds to a G protein-coupled receptor on gonadotroph cells, culminating in the activation of multiple signaling pathways (Conn, 1994). It has been demonstrated that GnRH is capable of activating the M A P K cascade in the aT3-l gonadotroph cell line (Sundaresan et al., 1996), pituitary organ culture (Michell et al., 1994; Sundaresan et al., 1996), and in the pituitary in vivo (Haisenleder et al., 1998). In addition to its well established function in the pituitary, GnRH is thought to be an autocrine/paracrine regulator in the gonads and placenta in several species, including the human (Bramley et al., 1986; Clayton et al., 1979; Currie et al., 1993; Ertl et al., 1993; Irmer et al., 1995; Kim et al., 1987; Lin et al., 1995; Ny et al., 1987; Olofsson et al., 1995; Peng et al., 1994; Prager et al., 1992; Siler-Khodr et al., 1986; Tan and Rousseau, 1982; Wong and Richards, 1992). However, little is known about the molecular events that mediate actions of GnRH in the extrapituitary tissues. Considering that the action of GnRH may be mediated via different signaling pathways in extrapituitary tissues (Emons et al., 1998), the possible effect of GnRH in the activation of M A P K and its roles were investigated in both normal and carcinoma cells of the human ovary and placenta. 117 II. Materials and Methods Treatment and immunoblot assay Cell cultures were washed once with the medium, and serum starved for 4 h prior to treatment with the GnRH agonist, (D-Ala 6)-GnRH (GnRH-a) at various concentrations for 5 min. Forty pg of total protein was run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech), as described in PART II. The membrane was immunoblotted using a mouse monoclonal antibody specific to phosphorylated p44/p42 M A P K (P-MAPK, Thr 2 0 2 /Tyr 2 0 4 )( 1:500 dilution). Alternatively, the membrane was submerged in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubated at 50 C for 30 min with occasional agitation. After incubation for 2 h in blocking solution, the membrane was reprobed with a rabbit polyclonal antibody for p44/42 M A P K (1:1000 dilution), which detects total M A P K (T-MAPK, phosphorylation-state independent) levels. RIA for progesterone After a culture period of 4 days, hGLCs (2 x 105 cells) were treated with various concentrations of (D-Ala 6)-GnRH. To block GnRH-induced M A P K activation, cells were preheated with vehicle or M A P K / E R K kinase (MEK) inhibitor, PD98059 (10 pM, New England Biolabs Inc.) for 1 h. The PD98059 has been shown to inhibit specifically MEK1/2 and not to cross-react with other MEKs (Haisenleder et al., 1998). The cells were then treated with (D-Ala 6)-GnRH for 24 h. After 24 h incubation, the progesterone 118 concentration in culture medium was measured by an established RIA, as described in the PART n. fH] thymidine incorporation assay After a preincubation period of 48 h, 2 x 104 of OVCAR-3 cells were treated with, 10" 7 M GnRH-a or 10 p M PD98059 for 2, 4, and 6 days. In order to block GnRH-induced M A P K activation, the cells were pretreated with vehicle or PD98059 (10 pM) for 1 h prior to 10"7 M GnRH-a treatment. The proliferative index was measured using a [3H]thymidine assay, as described in PART n. Northern blot analysis for BhCG After a preincubation of 48 h, 1 x 106 of JEG-3 cells were pretreated with vehicle and PD98059 (10 or 50 pM) for 1 h. The cells were then treated with (D-Ala 6)-GnRH for 24 h. Total RNA (40 pg) was isolated and Northern blot analysis was performed using radioactive labeled phCG and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes, as described in PART U. Data analysis M A P K levels are expressed as fold changes compared to basal levels. In RIA for progesterone, the amounts of progesterone are normalized to protein contents, and expressed as the percentage of change to control. Values are represented as the mean ± SD of five individual experiments from five different patients. Expression levels of phCG mRNA expressed as the percent change from the control value and standardized against 119 G3PDH mRNA level. Data are shown as means of three individual experiments, and presented as the mean ± SD. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. P< 0.05 was considered statistically significant. III. Results GnRH-induced MAPK activation in ovarian and pituitary cells The GnRH agonist, (D-Ala 6)-GnRH, stimulated a rapid activation of M A P K in primary cultures of hGLCs cells at concentrations of 10"9 to 10"7 M . Maximal activity (7.3-fold over basal levels) was observed at a concentration of 10"7 M (Fig. 31). The treatment of the ovarian carcinoma cell line, OVCAR-3, with the GnRH-a induced a biphasic pattern of M A P K activation. As shown in Fig. 32, a low concentration of the GnRH-a (10"10 M) significantly decreased M A P K activity (to 50% of basal levels), whereas high concentrations (10"7 and 10"6 M) resulted in a significant stimulation of M A P K activity (2.5-fold and 1.7-fold over basal levels, respectively). In aT3-l cells, which are a mouse-derived pituitary gonadotroph cell line (Windle et al., 1990), a rapid activation of M A P K was observed in all concentrations used. Maximal activity (10.1-fold over basal levels) was observed at the concentration of 10"7 M (Fig. 33). In contrast to the alterations of P-MAPK activity induced by (D-Ala 6)-GnRH, the T-MAPK levels in hGLCs, OVCAR-3 and aT3-l cells were unchanged by the GnRH-a treatment (Figs. 31, 32, and 33). 120 hGLCs tUi- -fifc"~i iiTi'iTiilil •—"• ilUhiililliflfcii-' • -rmnir- ^  p44 T - M A P K ^ » P42 ^^^^^^^^^^^^^ i:^:::::^ ^I^PPIPPPpr •..U-JJ-MSBIP. -™!M!P!'W~ • K 6 1 W - ^jj||§§R|gi}|•••••• • • 0 -10 -9 -8 -7 ODncentrationd([>Ala6)-GnRH (LogM) Figure 31. The effect of GnRH on M A P K activation in hGLCs. Human GLCs were cultured and treated with (D-Ala 6)-GnRH for 5 min as described in the Materials and Methods. The total (T-MAPK) and activated M A P K (P-MAPK) levels were analyzed by immunoblot assay and the intensities of the signals were quantitated. M A P K levels are expressed as relative fold change to basal levels. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs untreated control. 121 O V C A R - 3 T-MAPK P-MAPK <D Ui c CTj O < Q_ < 2.5 1.5 1 0.5 0 ^ll^gg I^^ HMrip ^ MttMP ^KHMJ^ 4^tHBfl^1 ^ H p K i ^ ^^SPPI^^^ ^^^^^SP^ ^PPIIP^^^ ^SHHB^^ _p44 p42 .p44 .p42 HHHH 0 -10 -9 -8 -7 -6 Concentration of (D-Ala6)-GnRH (Log M) Figure 32. The effect of GnRH on M A P K activation in OVCAR-3 cells. A human ovarian epithelial carcinoma cell line, OVCAR-3, was cultured and treated with (D-Ala6)-GnRH for 5 min as described in the Materials and Methods. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs untreated control. 122 aT3-l Concentration of (D-Ala6)-GnRH (Log M) Figure 33. The effect of GnRH on M A P K activation in aT3-l cells. A mouse-derived pituitary gonadotroph cell line, ctT3-l, was cultured and treated with (D-Ala 6)-GnRH for 5 min as described in the Materials and Methods. Values are represented as the mean + SD of three individual experiments, a, P < 0.05 vs untreated control. 123 Activation of MAPK by GnRH in placental cells Treatment of an immortalized trophoblast cell line, IEVT, with GnRH-a induced a significant activation of M A P K at the concentration of 10" and 10" M . Maximal activity (3.8-fold over basal levels) was observed at the concentration of 10"7 M (Fig. 34). Interestingly, GnRH-a induced a biphasic regulatory pattern of M A P K activation in the chorionic carcinoma cell line (JEG-3 cells). As shown in Fig. 35, a low concentration of the GnRH-a (10"10 M) decreased M A P K activity (to 40% of basal levels), whereas high concentrations (10" to 10" M) resulted in a significant increase of M A P K activity (3- to 6.7-fold over basal levels). In contrast to the changes in P-MAPK activity, the T-MAPK levels in IEVT and JEG-3 cells were unaffected by the GnRH-a treatment (Figs. 34 and 35). 124 IEVT T - M A P K P - M A P K WIKKmmKKmw : ^ml^lmmKW ^MHMRHHP ! ^ % ^ « < 4 ^ m j p ij^ f^t _p44 -p42 p44 ~p42 D) C CO o o o < Q_ < a - , n n r m 0 10 •8 -7 Concentration of (D-Ala >GnRH (Log M) Figure 34. The effect of GnRH on M A P K activation in IEVT cells. Immortalized extravillous trophoblast cells were cultured and treated with (D-Ala 6)-GnRH for 5 min as described in the Materials and Methods. Values are represented as the mean + SD of three individual experiments, a, P < 0.05 vs untreated control. 125 JEG-3 T-MAPK P-MAPK wtfl i M i ^ . H p -l H fe^^ ... « ^ ..... l^>| jjfc ,. :,. _p44 _p42 _p44 -p42 Concentration of (OAJa6)-GnRH (LogM) Figure 35. The effect of GnRH on M A P K activation in JEG-3 cells. A placental chorionic carcinoma cell line, JEG-3, were cultured and treated with (D-Ala 6)-GnRH for 5 min as described in the Materials and Methods. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs untreated control. 126 Effect of GnRH and PD98059 on progesterone secretion A significant decrease in progesterone secretion from hGLCs was observed in response to 10"9 to 10"6 M GnRH-a (Fig. 36A). Maximal inhibition (a 45% decrease over basal level) was observed after the treatment with 10"7 M GnRH-a. To investigate whether GnRH-induced activation of M A P K play a role in the regulation of the ovarian steroidogenesis, cells were pretreated with the compound, PD98089, which has been shown to be a specific inhibitor of the M A P K activator, M E K (Haisenleder et al., 1998). Pretreatment with 10 pM PD98059 completely reversed the inhibitory effect of the GnRH-a (Fig. 36B). Treatment with PD98059 and vehicle had no effect on progesterone secretion. Effect ofGnRHandPD98059 on OVCAR-3 cell growth As shown in Fig. 37, (D-Ala 6)-GnRH inhibited the growth of OVCAR-3 cells in a time-dependent manner. A significant inhibition of proliferation was detected as early as the second day of treatment at concentrations of 10"7 M . Inhibition of growth continued through to the fourth day and further evident in the sixth day of treatment. On day 6, 10"7 M (D-Ala 6)-GnRH reduced growth down to 59% of control. Pretreatment with 10 pM PD98059 on a daily basis blocked the inhibitory effect of GnRH-a (Fig. 37). Treatment with PD98059 had no effect on OVCAR-3 cell growth. 127 A 120 100 20 0 J - I I I T i i o •10 -9 -8 Concentration of (D-Ala )-GnRH (Log M) B 140 120 1 2 CO -g 5 ° d) o w ^? a) O 100 Control GnRH-a 10 ' 7 M DMSO PD98059 PD98059 10 u M +GnRH-a Figure 36. The effects of GnRH and PD98059 on progesterone secretion. Human GLCs were cultured for 4 days and treated with 10"10 to 10"6 M GnRH-a for 24 h (A). The cell cultures were pretreated with vehicle and 10 pM PD98059 for 1 h, followed by the treatment of 10"7 M GnRH-a for 24 (B). The progesterone concentration in the culture medium was measured by an established RIA. The amounts of progesterone are normalized to protein contents, and expressed as the percentage of change to control. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. Values are represented as the mean + SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. PD98059 + GnRH-a. 128 0 0 2 4 Time of treatments (Days) Figure 37. The effects of GnRH and PD98059 on OVCAR-3 cell growth. After a preincubation period of 48 h, 2 x 104 of OVCAR-3 cells were treated with 10"7 M GnRH-a (Ga) for 2, 4, and 6 days. In order to block GnRH-induced M A P K activation, the cells were pretreated with vehicle or PD98059 (PD, 10 pM) for 1 h prior to 10"7 M GnRH-a treatment. The cells were collected and proliferative index was measured using a [ H]thymidine assay. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. PD98059 + GnRH-a. 129 Effect ofGnRHandPD98059 on BhCG mRNA levels Treatment of JEG-3 cells with GnRH-a induced a significant increase in (3hCG mRNA expression. As shown in Fig. 38, phCG mRNA levels were increased to 160% of the control value after a 24 h treatment with 10"7 M GnRH-a. Pretreatment with 10 pM or 50 pM PD98059 failed to block the stimulatory effect of GnRH-a. Treatment with PD98059 and vehicle had no effect on phCG mRNA level. 130 Control GnRH-a PD98059 PD98059 PD98059 PD98059 10"7M 10 pM 50 uM 10 pM 50 pM + GnRH-a + GnRH-a Treatment Figure 38. The effect of GnRH and PD98059 treatment on phCG mRNA in JEG-3 cells. JEG-3 cells were plated onto 60 mm culture dishes and cultured for 48 h prior to the treatments. Cells were preheated with vehicles and PD98059 (10 or 50 pM) for 1 h. The cells were then treated with (D-Ala 6)-GnRH for 24 h. After 24 h incubation, total RNA was prepared and subjected to Northern blot analysis as described in the Materials and Methods. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs untreated control. 131 IV. Discussion Activation of M A P K occurs by phosphorylation of specific tyrosine and threonine residues. In this study, the phospho-specific M A P K antibody was used to measure M A P K activation by Western blot analysis (Haisenleder et al, 1998). This assay provides a sensitive, specific, and simple method to determine alterations in M A P K activation, when compared to other M A P K activation assays such as immunoprecipitation/substrate assay (Reuter et al., 1995) and in-gel kinase renaturation assay (Wang and Erikson, 1992). The phosphorylation state of M A P K in response to GnRH was first investigated in human ovarian and placental cells, and compared the response to that of aT3-lcells. In aT3-l cells, GnRH-a stimulated a substantial increase in M A P K activation, with maximal activity at 10"7 M (10.1-fold over basal levels). In addition to confirming GnRH-induced activation of M A P K in the pituitary, a novel finding of the present study is the demonstration that GnRH-a stimulated a rapid and dose-dependent activation of M A P K in normal and carcinoma cells of the human ovary and placenta. In hGLCs, maximal activity (7.3-fold over basal levels) was observed following 5 min of GnRH-a (10"' M) treatment. Likewise, a stimulation of P-MAPK (3.8-fold over basal levels) was observed in IEVT cells at the same dose of GnRH-a. Interestingly, treatment of the human ovarian and placental carcinoma cell lines, OVCAR-3 and JEG-3 cells, with the GnRH-a induced a biphasic regulatory pattern of P-MAPK phosphorylation. A low concentration of the GnRH-a decreased P-MAPK level, whereas high concentrations resulted in a significant increase of P-MAPK levels in both OVCAR-3 and JEG-3 cells. In contrast to the alterations in P-MAPK levels by GnRH-a, no change of T-MAPK levels was observed in 132 the ovarian, placental and pituitary cells studied. This result is consistent with a recent report using a lactotrope-derived cell line stably expressing the GnRHR (GGH3), where a GnRH-a, buserelin, induced a rapid activation of P-MAPK but no change of T-MAPK levels (Han and Conn, 1999). The differential GnRH effects on M A P K activation observed in the present study may reflect differences in the intrinsic activation states of the GnRH signaling pathways in normal vs carcinoma tissues. Activation of signal transduction systems by G protein-coupled receptors are dependent on factors such as the cell type, receptor density, receptor/G-protein affinity, compartmentalization of signal transduction machinery, and accessibility of G proteins to the receptors (Ashkenazi et al., 1987; Zhu et al., 1994). Altered combinations of these factors may result in cell-specific activation of markedly different intracellular signaling pathways. In this regard, the GnRHR in extrapituitary tissues may be different from that of the pituitary in terms of its binding affinity and basal expression levels. In the human ovary, ovarian tumors and placenta, low affinity binding sites for GnRH have been reported using radioreceptor assays (Bramley et al., 1986; Emons et al., 1989; Siler-Khodr et al., 1986). A relatively lower level of GnRHR gene expression has also been demonstrated in the rat ovary and testis when compared to the pituitary gland (Kakar et al., 1994). Furthermore, the GnRHRs expressed on different cell types have been shown to couple with different subtypes of G proteins. For instance, in tumors of the human reproductive tract, GnRHRs are thought to couple to Gj a proteins (Imai et al., 1996), whereas in ocT3-l and GGH3 cells, it has been demonstrated that the GnRHR is coupled to G q / n a (Hsieh and Martin, 1992; Janovick and Conn, 1994). Studies with cholera toxin (an activator of G s a ) have indicated that G s a may also be coupled to 133 the GnRHR (Janovick and Conn, 1993). The evidence aforementioned suggests that multiple factors are involved in activation of the GnRHR. The same receptor may act via differential intracellular signaling pathways in distinct cells or tissues, further determining the cell- or tissue-specific function exerted by GnRH on these cells. Thus, the differential response in M A P K activity to GnRH observed in this study may be a novel component in the signal transduction pathway of GnRH in the human ovary and placenta, which may mediate differential functions of GnRH in the normal versus carcinoma states. To seek physiological relevance to our findings, in terms of altered cellular functions, the possible involvement of GnRH-induced M A P K activation in the ovarian and placental functions was investigated. Functionally, in the ovary, GnRH has been shown to modulate basal and gonadotropin-stimulated steroidogenesis (Clayton et al., 1979; Olofsson et al., 1995; Peng et al., 1994). In this study, a significant decrease in progesterone secretion from hGLCs was observed in response to 10"9 to 10"6 M GnRH-a (Fig. 36A). Maximal inhibition (a 45% decrease over basal level) was observed after the treatment with 10"7 M GnRH-a. This inhibitory effect was completely reversed by pretreatment with PD98059, suggesting the involvement of the M A P K pathway in hGLCs. The mechanism by which GnRH-induced M A P K activation inhibits progesterone synthesis is not known. GnRH has been shown to act directly on the corpus luteum, leading to inhibition of the production and release of progesterone. Sridaran et al. have demonstrated that GnRH leads to reduced steroidogenesis by coordinated suppression of peripheral-type benzodiazepine receptors and steroidogenic acute regulatory protein, which results in impaired cholesterol transport into the mitochondria 134 (Sridaran et al., 1999b). In addition, GnRH has been shown to decrease P450 side-chain cleavage enzyme activity and mRNA content, and 3p-hydroxy-steroid dehydrogenase content in the corpus luteum (Sridaran et al., 1999a). Thus, it is likely that GnRH-induced M A P K activation inhibits progesterone secretion by regulating cholesterol transport and/or the enzymes involved in the steroidogenesis in hGLCs. In the ovarian cancer cells, GnRH is thought to be an autocrine growth regulator. In this study, the treatment of OVCAR-3 cells with GnRH-a induced a time-dependent growth inhibition. Pretreatment with PD98059 completely reversed the growth inhibitory effect of GnRH-a, suggesting that the involvement of the M A P K pathway in OVCAR-3 cells. This result is in agreement with a recent finding that GnRH-a induced a rapid and sustained activation of ERK and resulted in the growth inhibition of an ovarian cancer cell line, CaOV-3 (Kimura et al., 1999). The mechanism by which GnRH-induced M A P K activation inhibits the growth of OVCAR-3 cells growth is not known. Considering that MAPKs is reported to be involved in Gi-specific cell cycle arrest of CaOV-3 (Kimura et al., 1999), human breast cancer cells (Alblas et al.,1998), NTH 3T3 murine fibroblast (Sewing et al., 1997), and human myeloblastic leukemia cells (Yen et al., 1998), it is likely that GnRH exerts its growth inhibitory effect in OVCAR-3 cells through ERKl/2-induced cell cycle arrest in Gi-S transition. In the placenta, GnRH is thought to be a primary regulator of the synthesis and secretion of hCG (Currie et al., 1993; Ertl et al , 1993; Kim et al., 1987; Prager et al., 1992; Siler-Khodr et al., 1986). In the previous (Cheng et al., 2000) and present study, the GnRH-a stimulated the expression of phCG mRNA in placental JEG-3 cells with maximum effect at 10" M . In contrast to the ovarian cells, pretreatment with PD98059 failed to block the GnRH-induced effect on phCG mRNA levels, indicating the 135 involvement of other signal transduction pathways. Considering that multiple-intracellular signaling pathways are activated by GnRH, it is possible that other intracellular pathways may be involved in the regulation of BhCG mRNA by GnRH in JEG-3 cells. This possibility is supported by the recent findings that pretreatment of PD98059 failed to block the GnRH-induced LHB gene transcription in cultured rat pituitary cells (Haisenleder et al., 1998). Furthermore, a M A P K phosphatase or kinase-defective MAPKs vectors partially blocked the effect of GnRH induced-MAPK activation on the transcriptional regulation of glycoprotein a-subunit gene (Roberson et al., 1995). At present, it is not possible to distinguish exact intracellular signaling pathways that mediate GnRH-induced stimulation of B-hCG mRNA expression in JEG-3 cells. Considering multiple cellular functions of GnRH in the human ovary and placenta (Hsueh et al., 1984; Leung and Steel, 1992), M A P K activation in hGLCs and JEG-3 cells may be associated with other cellular function such as cell growth, apart from the involvement in steroidogenesis. 136 PART 7 Stimulation of Mitogen-Activated Protein Kinase by Gonadotropin-Releasing Hormone in Human Granulosa-Luteal Cells: Its Intracellular Signaling Pathways. I. Introduction The GnRHR is a G-protein coupled receptor and is hypothesized to couple to multiple G-proteins (Alarid and Mellon, 1995; Conn, 1994; Ishizaka et al., 1993; Limonta et al., 1999; Stojilkovic and Catt, 1995; Strarzec et al., 1989). GnRHR couples to G q a on gonadotroph cells, culminating in the activation of multiple signaling pathways, including phosphoinositol turnover, release of intracellular calcium, influx of extracellular calcium, and activation of PKC (Conn, 1994; Stojilkovic and Catt, 1995). In addition, coupling of the GnRHR to the G s a subunit has been suggested to increase the biosynthesis of L H (Strarzec et al., 1989), regulating the gonadotropin subunit mRNA (Ishizaka et al., 1993) or GnRHR mRNA in aT3-l cells (Alarid and Mellon, 1995). On the other hand, there is evidence that the GnRHR is coupled to G;a in reproductive tract tumors (Limonta et al., 1999). Since the same receptor couples to multiple G-proteins, it is suggested that the GnRH activates different intracellular signaling pathways, and exerts a cell- or tissue-specific function. Previously, it was demonstrated that GnRH was capable of activating M A P K in hGLCs (PART VI). In this study, GnRH-induced activation of M A P K and its intracellular signaling pathway was further investigated in hGLCs. 137 II. Materials and Methods Treatment and immunoblot assay Cell cultures were washed once with medium, and serum starved for 4 h prior to treatment with a GnRH-a, (D-Ala 6)-GnRH, PMA, 8-Bromo-cAMP, PTX in a time and/or dose dependent manner. Al l agents were purchased from Sigma-Aldrich Corp., Oakville, Canada. Prior to the GnRH-a treatment, the appropriate cultures were pretreated with the GnRH antagonist, antide (10~7 M) and a M E K inhibitor, PD98059 (10 pM) for 10 and 60 min, respectively. To block PKC activation, cells were pretreated with a specific PKC inhibitor, GF109203X (2 pM, Calbiochem, San Diego, CA) for 15 min, followed by treatment with 10"7 M GnRH-a and PMA for 20 and 30 min, respectively. To investigate the involvement of the Gsoc and G;a in GnRH-induced M A P K activation, cells were pretreated with 100 ng/ml PTX and C T X (Sigma-Aldrich Corp.) for 15 min prior to the GnRH-a treatment for 20 min. Total protein was isolated and immunoblot assay was performed using a mouse monoclonal antibody specific to phosphorylated p44/p42 M A P K and a rabbit polyclonal antibody for total p44/42 MAPK, as described in PART II and VI. RIA for intracellular cAMP Human GLCs (2 x 105 cells) were preincubated in serum-free medium containing 0.1% BSA and 0.5 mM 3-isobutyl-l-methylxanthine (IBMX, Sigma-Aldrich Corp.) for 30 min, and treated with 50 pM forskolin (Sigma-Aldrich Corp.), 10"7 M GnRH-a for 0, 5, 20, 60, 120 and 240 min. To determine if GnRH modulates forskolin- or hCG-induced 138 cAMP accumulation, cells were co-treated with GmRH-a (10~7 M) and forskolin (50 pM) or hCG (1 IU, Sigma-Aldrich Corp.) for 20 min. Cells were also treated with PTX (100 ng/ml) for 20 min. Control cells were treated with vehicle. After hormone treatment, intracellular cAMP levels were measured using a [ 3H]-cAMP assay system, as described in PART n. Samples were assayed in duplicate. In vitro MAPK assay Human GLCs were serum starved for 4 h and pretreated with vehicles and PD98059 for 1 h. The cells were then treated with 10"7 M GnRH-a for 20 min, and washed twice with ice-cold PBS and lysed in lx Lysis Buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM B-glycerolphosphate, 1 mM Na3V04, 1 pg/ml leupeptin, 1 mM PMSF). The cells were scraped off the plates and transferred to microcentrifuge tube. The extracts were placed on ice for 15 min and centrifuged to remove cellular debris, and protein content of the supematants was determined. Cellular protein (300 pg) was immunoprecipitated with 15 pi of resuspended immobilized phospho-p44/42 M A P K (Thr 2 0 2 /Tyr 2 0 4 ) monoclonal antibody for 16 h at 4 C with gentle rocking. Active M A P K (phosphorylated p42 MAPK; 20 ng) was also immunoprecipitated as a postive control. The tubes were centrifuged and pellet was washed once with 500 pi of lx Lysis Buffer and twice with 500 pi of lx Kinase Buffer (25 mM Tris (pH 7.5), 5 mM B-glycerolphosphate, 2 mM DTT, 0.1 mM Na3V04, 10 mM MgCL.). In vitro M A P K assays were performed using the Elk-1 fusion protein as a substrate for the MAPKs, according to the manufacturer's suggested procedure (New England Biolabs Inc.). The pellet was resuspended in 50 pi of lx Kinase 139 Buffer supplemented with 200 pM ATP and 2 pg of Elk-1 fusion protein and incubated 30 min at 30 C. The reaction was terminated by adding 25 ul of 3x SDS sample buffer. The reaction mixture was boiled for 5 min and subjected to immunoblot assay. Forty ul of reaction mixture was run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane, as described in the PART U. The membrane was immunoblotted using a rabbit polyclonal antibody specific to the phosphorylated Elk-1 (Ser383). After washing, the signals were detected with horseradish peroxidase-conjugated secondary antibody, and visualized using the E C L chemiluminescent system, followed by autoradiography. Northern blot analysis for c-fos Human GLCs (1.0 x 106 cells) were plated onto 60 mm culture dishes and cultured for 4 days. The cells were then serum starved for 6 h and treated with 10"7 M GnRH-a for 0, 10, 20, 30 and 60 min. Total RNA (25 pg) was isolated and Northern blot analysis was performed using radioactive labeled c-fos and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes, as described in the PART n. Data analysis The data were analyzed by A N O V A followed by Tukey's multiple comparison test. P< 0.05 was considered statistically significant. 140 III. Results GnRH-induced MAPK activation The GnRH-a, (D-Ala 6)-GnRH, stimulated a rapid and sustained activation of M A P K in primary cultures of hGLCs cells at a concentration of 10"7 M (Fig. 39A). Stimulation of M A P K activity was observed within 5 min and was sustained for 60 min after treatment. Maximal activity (6.1-fold over basal levels) was observed within 20 min. Dose response studies indicated that GnRH-a stimulated M A P K activation in a dose-dependent manner, with maximum stimulation (6.7-fold over basal levels) at 10"7 M (Fig. 39B). Effects of antide and PD98059 on GnRH-induced MAPK activation As shown in Fig. 40A, the GnRH-a induced a significant increase in M A P K activation. This stimulatory effect was completely reversed by pretreatment with antide, demonstrating that the activation of the M A P K signaling pathway is a receptor-mediated event. Treatment with antide alone had no effect on M A P K activation. To investigate whether GnRH-induced M A P K activation is mediated through activation of M E K , cells were pretreated with 10 pM PD98059 prior to GnRH-a treatment. As shown in Fig. 40B, a 68% decrease in GnRH-induced M A P K activation was observed in hGLCs pretreated with PD98059, demonstrating that GnRH-induced M A P K activation is mediated through the activation of M E K . Treatment with PD98059 alone had no effect on M A P K activation. 141 B T-MAPK P-MAPK 8 o ro CD a co 6 CL -D < o £ 2 0 T-MAPK P - M A P K 8 ±i 'aT 6 > D) Q_ ^ < O o 5 10 20 30 Time of GnRH-a treatment (min) 60 _p44 -p42 -10 -9 -8 -7 Concentration of GnRH-a (Log M) Figure 39. A time- and dose-dependent effect of GnRH on M A P K activation in hGLCs. Human GLCs were cultured and treated with (D-Ala 6)-GnRH (GnRH-a) in a time (A)-and a dose-dependent manner (B). Control cultures were treated with vehicle. The total (T-MAPK) and activated M A P K (P-MAPK) levels were analyzed by immunoblot assay. P-MAPK levels were quantitated by densitometry (NIH Image beta 3) and standardized against the levels of T-MAPK per sample. M A P K levels are expressed as a relative fold change compared to basal levels. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control. 142 + GnRH-a Control DMSO PD98059 GnRH-a PD98059 + GnRH-a Figure 40. The effects of antide and PD98059 on GnRH-induced M A P K activation in hGLCs. Human GLCs were cultured and pretreated with a GnRH antagonist, antide (10"7 M) for 10 min (A) and a specific inhibitor for MEK, PD98059 (10 uM) for 60 min (B). The cells were then treated with 10"7 M (d-Ala 6)-GnRH (GnRH-a) for 20 min. Control cultures were treated with vehicle. The total (T-MAPK) and activated M A P K (P-MAPK) levels were analyzed by immunoblot assay. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. antide + GnRH-a; c, P < 0.05 V5.PD98059 + GnRH-a. 143 Effects of PMA, GnRH, and PKC inhibitor GF109203Xon MAPK activation To assess whether the PKC signal transduction pathway is involved in M A P K activation in hGLCs, the cells were treated with a PKC activator, PMA (10"7 M) in a time dependent manner. The results showed that PMA stimulated a rapid M A P K activation, with maximum stimulation (9.8-fold over basal levels) within 30 min (Fig. 41 A). To determine whether PKC is necessary for M A P K activation by GnRH and PMA, hGLCs were pretreated with the PKC inhibitor GF109203X (2 pM) for 10 min, followed by stimulation with 10" M GnRH-a and PMA for 20 and 30 min, respectively. As shown in Fig. 4IB, GF109203X completely blocked the GnRH-a-induced M A P K activation, and decreased PMA-induced M A P K activity to 33% of the control value. GF109203X alone had no effect on the M A P K activity. To further determine the role of the PKC pathway in GnRH-induced M A P K activation, the cells were pretreated with P M A (10"7 M) for 5 min, followed by stimulation with GnRH-a (10~7 M) for 20 min. As shown in Fig. 41C, the GnRH-a or PMA treatment induced a substantial increase in M A P K activation. Pretreatment of the cells with PMA significantly potentiated the M A P K activity in response to GnRH-a, suggesting GnRHR couples to G q a and stimulates M A P K activation in a PKC-dependent manner. Effects of8-Br-cAMP and PTX on the MAPK activity Human GLCs were cultured and treated with 8-Br-cAMP (1 mM) and with various doses of PTX. As shown in Fig. 42A, stimulation of M A P K activity was observed within 5 min and reached maximum level within 20 min after treatment. No activation of M A P K was observed in response to various doses of PTX in hGLCs (Fig. 42B). 144 Time of P M A treatment (min) + PMA + GnRH-a Figure 41A and B. The effects of PMA, GnRH, and PKC inhibitor GF109203X on M A P K activation. Human GLCs were cultured and treated with a PKC activator, PMA (100 ng/ml) in a time dependent manner (A). The cells were pretreated with the PKC inhibitor, GF109203X (GF, 2 pM) for lOmin, followed by stimulation with 10"7 M (D-Ala 6)-GnRH (GnRH-a) and PMA (100 ng/ml) for 20 or 30 min, respectively (B). Control cultures were treated with vehicle. The total (T-MAPK) and activated M A P K (P-MAPK) levels were analyzed by immunoblot assay. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. GF + PMA; c, P < 0.05 vs. GF + GnRH-a; d, P < 0.05 vs. GnRH-a; e, P < 0.05 vs. PMA. 145 Control DMSO GnRH-a PMA PMA +CmRH-a Figure 41C. The effects of PMA, GnRH, and PKC inhibitor GF109203X on M A P K activation. Human GLCs were cultured and pretreated with PMA (100 ng/ml) for 5 min, followed by stimulation with 10"7 M GnRH-a for 20 min (C). Control cultures were treated with vehicle. The total (T-MAPK) and activated M A P K (P-MAPK) levels were analyzed by immunoblot assay. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. GF + PMA; c, P < 0.05 vs. GF + GnRH-a; d, P < 0.05 vs. GnRH-a; e, P < 0.05 vs. PMA. 146 T-MAPK P-MAPK 0 10 30 100 500 PTX concentration (ng/ml) Figure 42. The effects of 8-Br-cAMP and PTX on the M A P K activation. Human GLCs were cultured and treated with 8-Br-cAMP (1 mM) in a time-dependent manner (A), and with PTX in a dose-dependent manner (B). Control cultures were treated with vehicle. The total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control. -p44 -p42 147 The Effects of CTX and PTX on GnRH-induced MAPK activation To further determine the role of the Gsoc and Gjcc in M A P K activation, hGLCs were pretreated with C T X (100 ng/ml) and PTX (100 ng/ml) for 15 min, followed by stimulation with 10"7 M GnRH-a for 20 min. As shown in Fig. 43A, GnRH-a and C T X treatment stimulated M A P K activation. An additive rather than a potentiative action of C T X and GnRH-a was observed in hGLCs cells, suggesting that activation of M A P K by C T X and GnRH-a is independent. In contrast to CTX, PTX alone did not affect either basal or GnRH-induced M A P K levels (Fig. 43B). Effects of forskolin, hCG, and GnRH-a on intracellular cAMP accumulation To investigate whether GnRH modulates intracellular cAMP levels, hGLCs were treated with forskolin (50 pM) or GnRH-a (10" M), and intracellular cAMP levels were measured. Forskolin substantially stimulated a rapid cAMP accumulation within 5 min and a maximum increase was observed within 20 min (Fig. 44A). In contrast, GnRH-a did not affect basal intracellular cAMP levels (Fig. 44B), suggesting that GnRHR may not be coupled to the G s a in hGLCs. Activation of G;a subunit protein is negatively correlated with cAMP production. To explore the possibility that GnRHR may be coupled to G;a and affect cAMP production in hGLCs, cells were co-treated with GnRH-a (10"7 M) and forskolin (50 pM) or hCG (1 KJ) for 20 min. As shown in Fig. 44C, co-treatment of cells with GnRH-a (10"7 M) did not attenuate forskolin or hCG-stimulated cAMP production. PTX alone did not affect basal intracellular cAMP levels. 148 T - M A P K §> 12 c m o o ' > '-+-* o ro CL < 10 8 6 4 2 0 T - M A P K H • • • • • |"""""7B| CT) ro 6 o CT O M — > o ro £ 2 < 0 Control GnRH-a CTX CTX + GnRH-a a ad _ H H 1 • H i Control GnRH-a PTX PTX + GnRH-a Figure 43 . The effects of CTX and PTX pretreatment on GnRH-induced MAPK activation. Human GLCs were pretreated with 100 ng/ml CTX (A), 100 ng/ml PTX (B) for 15 min, followed by stimulation with 10"7 M (D-Ala6)-GnRH (GnRH-a) for 20 min. Control cultures were treated with vehicle. The total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. GnRH-a; c, P < 0.05 vs. CTX; d, P < 0.05 vs. PTX. 149 U s « ° 4 1 3 " o ro c -i= *= 9 1 0 5 20 60 120 240 Time of GnRH-a treatment (min) Figure 44A and B. The effects of forskolin, hCG, and GnRH-a on intracellular cAMP accumulation. Human GLCs were preincubated with serum-free medium containing 0.1% BSA and 0.5 mM 3-isobutyl-l-methylxanthine (IBMX) for 30min, and then treated with 50 pM forskolin (A) or 10"7 M GnRH-a (B). Intracellular cAMP levels were measured by an established RIA. Control cultures were treated with vehicle. The amount of cAMP was calculated from standard curve. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. Values are represented as the mean + SD of five individual experiments from five different patients, a, P < 0.05 vs. control. 150 c 6 0 0 Control DMSO GnRH-a PTX Forskolin GnRH-a hCG GnRH-a + Forskolin + hCG Figure 44C. The effects of forskolin, hCG, and GnRH-a on intracellular cAMP accumulation. Human GLCs were preincubated with serum-free medium containing 0.1% BSA and 0.5 m M 3-isobutyl-l-methylxanthine (IBMX) for 30min, and then treated with 50 p M forskolin or 1 IU hCG plus IO"7 M (D-Ala 6)-GnRH (GnRH-a) and intracellular cAMP levels were measured (C). Control cultures were treated with vehicle. The amount of cAMP was calculated from standard curve. The data were analyzed by A N O V A followed by Tukey's multiple comparison test. Values are represented as the mean ± SD of five individual experiments from five different patients, a, P < 0.05 vs. control. 151 Effects of GnRH and PD98059 on Elk-1 phosphorylation The Ets family transcription factor, Elk-1 is a physiological substrate for p42 M A P K and p44 M A P K (Janknecht et al., 1993; Treisman, 1994; Cille et al., 1995). To investigate whether the GnRH-induced activation of M A P K leads to phosphorylation of Elk-1 in vitro, hGLCs were pretreated with 10 pM PD98059 for 1 h prior to treatment with GnRH-a (10 7 M). As shown in Fig. 45, GnRH-a (10 7 M) stimulated a significant increase in Elk-1 phosphorylation. Pretreatment with 10 pM PD98089 for 1 h completely blocked the GnRH-induced activation of Elk-1. As expected, active p42 M A P K (ERK-2) substantially stimulated Elk-1 phosphorylation. Effect of GnRH on c-fos mRNA levels Several studies have shown that Elk-1 phosphorylation stimulates the transcription of the immediate early response gene, c-fos by facilitating the formation of a ternary complex with serum response element and the serum response factor (Janknecht et al., 1993; Treisman, 1994; Cille et al., 1995). To investigate whether GnRH stimulates the transcription of the c-fos gene, hGLCs were treated with 10"7 M GnRH-a in a time dependent manner. As shown in Fig. 46, GnRH-a stimulated a significant increase in c-fos mRNA expression. Maximal stimulation (4.1-fold over basal levels) was observed within 30 min. 152 8 CO co 6 sz o O 1 5 >> 4 > o CO LU 2 1 ^ Phospho Elk-1 Control GnRH-a DMSO PD98059 PD98059 ERK-2 +GnRH-a Figure 45. The Effects of GnRH and PD98059 on Elk-1 phosphorylation. Human GLCs were cultured and pretreated 10 pM with PD98059 for 1 h, followed by stimulation with 10"7 M (D-Ala 6)-GnRH (GnRH-a) for 20 min. Control cultures were treated with vehicle. Activated M A P K in the cell lysate was immunoprecipitated with immobilized phospho-p44/42 M A P K antibody at 4 C overnight. In vitro M A P K assays were performed using the Elk-1 fusion protein as a substrate for the MAPKs. Active p42 M A P K (ERK-2) was included as a positive control. The phosphorylation state of Elk-1 was analyzed by immunoblot assay using a specific antibody for phospho-Elk-1. Values are represented as the mean ± SD of three individual experiments, a, P < 0.05 vs. control; b, P < 0.05 vs. PD98059 + GnRH-a. 153 3Di 0 10 20 30 60 Time of GnRH-a treatment (min) Figure 46. The effect of GnRH on c-fos mRNA levels. Human GLCs were cultured and treated 10"7 M (D-Ala 6)-GnRH (GnRH-a) in a time dependent manner. Control cultures were treated with vehicle. Total RNA (25 pg) was prepared and resolved by formaldehyde denaturing agarose gel electrophoresis. Northern blot analysis was performed using a radioactive labeled c-fos probe. Expression levels of c-fos mRNA expressed as the percent change from the control value and standardized against G3PDH mRNA level. Data are shown as means of three individual experiments, and presented as the mean ± SD. The data were analyzed by A N O V A followed by Tukey's multiple comparison test, a, P < 0.05 vs. control. 154 IV. Discussion Its mechanism of action of GnRH in the human ovary is unclear. Previously, it was demonstrated that GnRH was capable of activating M A P K in hGLCs (PART VI). In this study, the CrnRH-induced activation of M A P K and its intracellular signaling pathway was further investigated in hGLCs. It was demonstrated that GnRH stimulates a rapid and sustained M A P K activation via a PKC-dependent pathway. The activated M A P K phosphorylates Elk-1 transcription factor, and may result in the transcription of the c-fos gene. Interestingly, M A P K activation in the present study was rapid and sustained. It is hypothesized that cellular responses to M A P K may be influenced by the duration of its activation. Sustained activation of M A P K is associated with cell differentiation by nerve growth factor (NGF) in PC 12 cells, whereas transient activation of M A P K by epidermal growth factor (EGF) leads to cell proliferation (Heasley and Johnson, 1992; Nguyen et al., 1993). GnRH-a was found capable of inducing a sustained M A P K signal in the gonadotroph ocT3-l cell line, functionally leading to trigger differentiated cellular functions such as gonadotropin secretion and synthesis (Mitchell et al., 1994; Roberson et al., 1995). Thus, sustained activation of the M A P K by GnRH-a in hGLCs may be associated with differentiated cellular functions such as steroidogenesis. GnRHR is thought to couple to multiple G-protein subunits (G q a, G s a , G;a), and activate multiple signaling pathways (Conn, 1994; Stanislaus et al., 1998; Stojilkovic and Catt, 1995). Increasing evidence that multiple G-proteins mediate the effects of GnRHR, raised the possibility that the same kind of G-protein coupled receptor exerts regulation 155 via differential signal transduction pathways in distinct tissues or cells, thereby determining the specific function mediated by the receptor in these cells. The PKC pathway has been well studied in response to GnRH stimulation (Andrews and Conn, 1986; Zheng et al., 1994) including in hGLCs (Hori et al., 1998). GnRH induces translocation of PKC activity from the cytosol to the plasma membrane and stimulates enzyme activity. The data indicate that the activation of M A P K by GnRH is mediated via PKC, as a specific PKC inhibitor GF109203X completely blocked the M A P K stimulation. Furthermore, pretreatment of the cells with PMA significantly potentiated GnRH-induced M A P K activity. These results indicate that the GnRHR couples to G q a to stimulate M A P K activation. There is evidence that the GnRHR is coupled to GjO, in reproductive tract tumors (Imai et al., 1996; Limonta et al., 1999). On the other hand, in insect cells expressing GnRHR and in the stable cell line GGH3I cells, GnRHR coupled to Gsoc, which activates adenylate cyclase and results in the production of cAMP (Delahaye et al., 1997; Kuphal et al., 1994). The roles of G ; a and G s a i n M A P K activation in response to GnRH are still controversial in aT3-l cells (Reiss et al., 1997; Sim et al., 1995). In this study, the possible involvement of the PKA pathway in GnRH-induced M A P K activation was investigated. The results suggest that the PKA pathway is not involved in the GnRH-induced activation of MAPK. This conclusion is based on the following observations. First, in contrast to forskolin, GnRH-a did not affect basal intracellular cAMP levels. Although 8-Br-cAMP and C T X activate M A P K in hGLCs, it is unlikely that the effect of GnRH-a in stimulation of M A P K involves GnRHR/G s a coupling. Second, activation of the GnRHR by its ligand did not attenuate forskolin or hCG induced cAMP accumulation. Taken with the lack of effect with PTX on basal or 156 GnRH-a-induced M A P K activation, it seems unlikely that the effect of GnRH-a in hGLCs involves G;a coupling to GnRHR. The ability of GnRH to activate a downstream effector of the M A P K pathway was investigated. Several studies have shown that M A P K phosphorylates ternary complex factor (TCF) proteins such as Elk-1 and SAP-1 (Cille et al., 1995; Janknecht et al., 1993; Treisman, 1994). The activated TCF protein regulates the expression of c-fos and other co-regulated genes through their actions on the serum response element. It was demonstrated that treatment of hGLCs with GnRH-a resulted in substantial activation of Elk-1 fusion protein in vitro (Fig. 45). This effect appears to be mediated by the activation of MAPK, as treatment of hGLCs with PD98059 completely reversed the effect of the GnRH-a on Elk-1 phosphorylation. Furthermore, GnRH-a stimulates the expression of c-fos mRNA in hGLCs (Fig. 46). These results are consistent with the finding that GnRH stimulates transcriptional activation of the Elk-1 (Roberson et al., 1995), and increased immediate early response gene mRNA such as c-fos and c-jun in gonadotroph-derived ocT3-l cells (Cesnjaj et al., 1994). Taken together, these results suggest that activated Ets family transcription factors may regulate expression of immediate early genes or other co-regulated gene, which possibly mediate GnRH-induced inhibition of progesterone secretion in hGLCs (see PART VI). 157 PART 8 Differential Expression of Human Gonadotropin-Releasing Hormone Receptor Gene in Pituitary and Ovarian Cells I. Introduction There is some evidence to suggest that the GnRHR in the extrapituitary tissues may be different from that of the pituitary in terms of basal expression levels. A lower level of receptor expression has been demonstrated in the rat ovary and testis compared to that in the pituitary (Kakar et al., 1994). Demonstration of GnRHR mRNA by RT-PCR provides further evidence for the low abundance of receptor mRNA in extrapituitary tissues such as ovarian and endometrial carcinomas (Imai et al., 1994a; 1994b; 1994c; Irmer et al., 1995). The expression level of GnRHR gene is different among tissues in the hypothalamo-pituitary-gonadal axis, suggesting that different factors may be involved in the regulation of its gene expression. The mechanism by which the expression of one gene can be specifically directed in a range of functionally and developmentally diverse tissues may be based on the presence of tissue- or cell-specific regulators (Bodner et al., 1988; Mangalam et al., 1989; Orkin, 1990). These regulators are able to control the expression of the gene by means of recognizing specific DNA sequences to enhance or repress transcription. To elucidate its transcriptional regulation, the 5'-flanking region of the human GnRHR (hGnRHR) gene has been cloned and characterized (Fan et al., 1994; 1995). Analysis of the 5'-flanking region of the hGnRHR gene suggests multiple transcription initiation sites and binding sites for several putative trans-acting factors, thereby reflecting the possible 158 involvement of specific regulatory factors on the hGnRHR gene expression in diverse tissues. Recently, we have also shown that steroidogenic factor-1 (SF-1), by interacting with a gonadotroph specific element (GSE) motif within the first exon of the hGnRHR gene, is largely responsible for its gonadotroph-specific expression (Ngan et al., 1999). However, little is known about the molecular mechanisms governing the differential expression of the hGnRHR gene in the pituitary and extrapituitary tissues. The present study investigates the molecular basis that may account for the different expression levels of hGnRHR gene in various cells. The use of cell-specific promoters and differential trans-acting factors in the transcriptional regulation of hGnRHR gene expression was investigated in the pituitary and ovarian cell lines. The elucidation of the mechanisms controlling different regulation of the receptor in the extrapituitary tissues may provide insight into the understanding of its roles in the control of reproductive functions. II. Materials and Methods Quantification of GnRHR mRNA To investigate the relative expression levels of GnRHR mRNA, semi-quantitative PCRs were performed. For comparison of different expression levels, the primers for both mouse and human GnRHR were designed to encompass the same region of the gene, and resulted in the same size of PCR products in both cell types. The oligonucleotides for the mouse GnRHR were sense: 5'-G T A T G C T G G G G A G T T C C T C T G C A - 3 ' (mP44F); and antisense, 5'-G G A T G A T G A A G A G G C A G C C G A A G - 3 ' (mP45R). The primers for the human GnRHR were sense: 5 ' - G T A T G C T G G A G A G T T A C T C T G C A - 3 ' (hP44F); and antisense, 5'-G G A T G A T G A A G A G G C A G C T G A A G - 3 ' (hP45R). Primers for p-actin were derived from the 159 human p-actin cDNA sequence (Ng et al., 1985), and resulted in amplification of the same size of PCR products in all cell types. Using 1 pg to 3 pg total RNA, first strand cDNA was transcribed and subjected to PCR amplification. Initially, to determine the conditions under which PCR amplification for CniRHR mRNA and P-actin mRNA were in the logarithmic phase, different amounts of total RNA were reverse transcribed, and aliquots were amplified using different numbers of cycles. A linear relationship was observed between the amount of RNA and PCR products when 3.0 pg of total RNA was used in the reverse transcription reaction, and when 30 and 18 PCR amplification cycles for GnRHR and P-actin were performed, respectively. PCR amplification for GnRHR was performed as described in the PART in. GnRHR mRNA levels were expressed as the ratio of GnRHR to p-actin. Promoter-Luc vector construction and transient transfection assay A 2.3 kb DNA fragment corresponding to the 5'-fianking region of the human GnRHR gene was prepared by PCR amplification using primer A, 5'-C T G A A G C T T C C C A G G A C A G A G C T T C A A G C C T - 3 ' and primer B, 5'-G G C C T G C T C T G T T T T A G C A C T C T G - 3 ' . A 2.7 kb fragment DNA containing the 5'-flanking region of the hGnRHR gene was used as a template for PCR amplification (Fan et al., 1994). The Hind III restriction site (underlined) was included in primer A for subsequent cloning. The PCR-generated DNA was fused to the promoterless pGL2-Basic luciferase vector (Promega, WI, USA) by digesting with 20 U of: Hind III and ligating with 20 IU of T4 DNA ligase. A series of 3'-deletion clones containing different lengths of the promoter were constructed by indicated restriction enzyme digestion and PCR amplification by specific primers. The positive clones were identified by restriction enzyme mapping, and confirmed by DNA sequence analysis. 160 Plasmid DNAs for transfection studies was isolated using Qiagen Plasmid Kits (Qiagen, Hilden, Germany), and DNA concentration was determined by measuring the absorbance at 260 nm and 280 nm. Transfection assays were carried out using the lipofectin reagent as recommended (Life Technologies, Inc.). In order to normalize for different transfection efficiencies of various luciferase constructs, the p-galactosidase vector RSV-LacZ, was cotransfected into cells with each GnRHR promoter-luciferase construct. Approximately 3 x 105 cells were plated onto 6-well plate 24 h before transfection. Five pg of the GnRHR-Luc vector and 2.5 pg of RSV-LacZ were combined with 16 pi of lipofectin reagent (Life Technologies, Inc.) in 200 pi of serum-free medium. Lipofectin and DNA were incubated for 50 min at RT, diluted to 1 ml with serum-free medium and applied onto cells. Transfections were carried out for 24 h. Subsequently, the medium was removed and 2 ml of fresh medium containing 10% FBS was added. After 24 h incubation, cells were washed with PBS three times and lysed with 150 pi of Cell Culture Lysis lx Reagent (25mM Tris-phosphate (pH 7.8), 2mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100; Promega). Cells were scraped off the plates and transferred to the new tube. The tubes were vortex for 15 sec and centrifuged (10,000 g, 2 min). Cellular lysates were collected and immediately assayed for luciferase activity using Luciferase Assay System (Promega). The cellular lysates (20 pi) was mixed with 100 pi of room temperature Luciferase Assay Reagent. The reaction mixture was placed into the TROPLX OPIOCOMP I Luminometer (Bio/Can Scientific) and luminescence was measured for 20 sec. The background level was also measured and substracted from the luciferase activity. P-galactosidase activity was measured and used to normalize the luciferase activity. The cellular extract (10 pi) was incubated for 30 min at 37 C in the reaction containing 0.75 pi of lOOx Mg (0.1 M MgCl 2 , 4.5 M p-mercaptoethanol), 16.5 pi of CPRG (Chlorophenol-Red-p-D-161 Galactopyranoside; 8 mg/ml in 0.1 M sodium phosphate, pH 7.5) (Sigma-Aldrich Corp.), 50.5 pi of 0.1 M sodium phosphate (164 mM Na 2 HP0 4 , 36 mM NaH 2 P0 4 , pH 7.5). The reaction was stopped by adding 200 pi of 1 M Na 2C03 and (3-galacotosidase activity was measured using spectrophomoter at 570 nm wavelength. A typical standard curve for P-galactosidase assay was represented in Fig. 47. A promoterless pGL2-Basic vector was transfected into the cells and served as a negative control. 162 y = 0.9988X - 0.0085 R 2 = 0.9934 Optical Density Figure 47. A standard curve for p-galactosidase assay. The p-galactosidase standard (0-0.6 unit) was incubated for 30 min at 37 C in the reaction containing 0.75 pi of 100X Mg, 16.5 pi of CPRG, 50.5 pi of 0.1 M sodium phosphate. The reaction was stopped by adding 200 pi of 1 M Na2CG*3 and p-galacotosidase activity was measured using spectrophomoter at 570 nm wavelength. 163 Electrophoretic gel mobility shift assays (EMSAs) For the preparation of nuclear extracts, aT3-l , OVCAR-3 and FJEK-293 cells were grown in 100 mm plates to 60% confluence. Cultured cells were washed three times with TBS (pH 7.6), and then lysed by adding the lysis buffer (20 mM HEPES, pH 7.6, 20% Glycerol, 10 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 1 mM DTT, 1 mM PMSF, 10 ug/ml leupeptin, 100 pg/ml aprotinin). The cells were dislodged by scraping and pelleted by centrifugation for 5 min at 2000 rpm at 4 C. Nuclei pellets were resuspended in 500 ul nuclear extract buffer (lysis buffer + 500 mM NaCl). Nuclei were gently rocked for 1 h at 4 C and centrifuged at 10,000 rpm for 10 min. The supernatant was aliquoted, frozen in liquid nitrogen and stored at -70 C. Protein concentration of the nuclear extract was determined by the Bio-Rad assay kit, as described in the PART II. EMSAs were performed as described previously (Lasser et al., 1991). The DNA fragments were end-labeled in the reaction volume of 15 pi containing 10 U of T4 polynucleotide kinase (New England Biolabs Inc), 1.5 pi of 1 OX T4 polynucleotide kinase buffer, 1 pi of 0.1 M DTT, 1 pi of 1 mM spermidine, 5 pi of [y- 3 2P]-ATP (3000 Ci/mmol, Amersham). The reaction mixture was incubated for 1 h at 37 C and the labeled probe was purified using Sephadex G-50 columm. Ten micrograms of nuclear extracts was incubated for 30 min at RT or 0 C with 2 x 105 cpm of the purified probe in a 30 pi reaction mixture containing 20 mM HEPES (pH 7.6), 5% glycerol, 50 mM NaCl, 1.5 mM MgCl 2 , 2 pg poly(dl-dC). For competitive EMSAs, excess amount of the unlabeled competitor DNA was added with the labeled probe. After incubation, reactions were loaded onto a 4% non-denaturing acrylamide gel containing 50 mM Tris base, 50 mM boric acid, 1 mM E D T A and electrophoresed at 150 V for 3 h at 4 C. The gels were dried and exposed to x-ray films with intensifiers. 164 Data analysis Data are represented as the means of three independent experiments each in duplicates. Statistical analysis was carried out using A N O V A , followed by Tukey's t-test. Values are presented as the mean ± SD and are considered significant when P < 0.05. III. Results Expression levels of GnRHR mRNA To investigate relative expression levels of GnRHR mRNA in the pituitary tissues and the primary culture of ovarian cells (PCO), semi-quantitative RT-PCR was performed. Different amounts of total RNA were reverse transcribed, and aliquots were amplified with primers specific for the human GnRHR. The expected PCR products were obtained as visualized by agarose gel electrophoresis and ethidium bromide staining (Fig. 48A, left panel). The authenticity of PCR products was confirmed by Southern blot analysis using a specific probe for hGnRHR (Fig. 48A, right panel). Furthermore, the PCR products were sequenced and were found to be identical to the receptors found in the human pituitary glands (data not shown). A linear relationship was observed between RNA input and PCR products (Fig. 48B). Quantitative analysis of the present study showed that the expression level of hGnRHR is 9-fold higher in the pituitary tissues than the PCO. Different expression levels of GnRHR mRNA in the pituitary and ovarian cells were also investigated using semi-quantitative RT-PCR. The expected PCR products were obtained from both cells (Fig. 49A). A linear relationship was observed between RNA input and PCR products in both cells (Fig. 49B). Quantitative analysis of the present study showed that the expression level of hGnRHR is a 10-fold higher in aT3-l than OVCAR-3 cells. 165 Analysis of cell-specific expression of the hGnRHR promoter by transient transfection assay To investigate the potential usage of a cell specific promoter in the two cells, a series of constructs containing 3'-deletions of the hGnRHR promoter (Fig. 50) were constructed, and transfected into aT3-l (Fig. 51A), OVCAR-3 (Fig. 51B) and HEK-293 cells (Fig. 51C). The promoterless vector, pGL2-Basic, served as a control to determine the basal level of luciferase expression. A similar pattern of luciferase activity was observed in the ccT3-l and OVCAR-3 cells (Figs. 51A and B). As a result, the same promoter regions were found to be functional in both cell lines. When the region between -771 to -557 was deleted, a significant decrease in luciferase activity was observed in aT3-l and OVCAR-3 cells. Furthermore, deletion of the promoter between -1351 to -1022 resulted in a decrease in luciferase activity in both cell types. Conversely, luciferase activity was significantly increased when the region between -1022 to -771 was deleted in aT3-l and OVCAR-3 cells. No significant increase in luciferase activity was observed in HEK-293 cells (Fig. 51C). 166 A Pituitary PCO Pituitary PCO GnRHR P-actin -347 bp -524 bp mmim m RNA lpg 2pg 3pg lpg 2pg 3pg lpg 2pg 3pg lpg 2pg 3pg Figure 48. Different expression levels of GnRHR mRNA in pituitary tissues and primary culture of ovarian carcinomas (PCO). Various amounts of total RNA were reverse transcribed and PCR-amplified. The expected PCR products were observed by ethidium bromide staining (A, left panel), and confirmed as GnRHR by Southern blot analysis with a digoxigenin-labeled 364 bp hGnRHR cDNA probe (A, right panel). The level of mRNA was quantified using densitometer and plotted. A liner relationship was observed between RNA input and mRNA expression in both cells (B). When compared, a 9-fold higher expression of GnRHR mRNA was observed in the pituitary tissues than the PCO. Data are shown as the means of three individual experiments and are represented as the mean ± S.D. 167 A o t T 3 - l OVCAR-3 < x T 3 - l OVCAR-3 ygFNr\ M RNA Figure 49. Different expression levels of GnRHR mRNA in aT3-l and OVCAR-3 cells. Various amounts of total RNA were reverse transcribed and PCR-amplified. The expected PCR products were observed by ethidium bromide staining (A, left panel), and confirmed as GnRHR by Southern blot analysis with a digoxigenin-labeled 364 bp hGnRHR cDNA probe (A, right panel). The level of mRNA was quantified using densitometer and plotted. A liner relationship was observed between RNA input and mRNA expression in both cells (B). When compared, a 10-fold higher expression of GnRHR mRNA was observed in the ctT3-l than the OVCAR-3 cells. Data are shown as the means of three individual experiments and are represented as the mean ± S.D. 168 Hindm BglH Ndel SstI PstI Spel -557 -227 Hpa I Hind m Luc pGL2-Basic -2297 Luc p2300-LucF -2197 Luc p2200-Luc -2197 -173 Luc p2200/-173 Luc -2197 -557 Luc p2200/-557 Luc -2197 -771 Luc p2200/-771 Luc -2197 -1022 Luc p2200/-1022 Luc -2197 -1351 Luc p2200/-1351 Luc Figure 50. Construction a series of 3'-deletion clones. The 2.3 kb fragment of the 5'-flanking region of the human GnRFfR gene were prepared by PCR amplification and fused to the promoterless pGL2-Basic luciferase vector. A series of 3'-deletion clones containing different lengths of the promoter were constructed by restriction enzyme digestion and PCR amplifications from the 2.3 kb fragment and fused to the pGL2-Basic vector. Restriction enzyme sites used in the 5'-flanking region of the human GnRHR gene were indicated. 169 A pG L2-B asic p2300-LucF p2200-Luc p2200/-173 Luc p2200/-557 Luc P22007-771 Luc p2200/-1022 Luc P2200/-1351 Luc B p G L 2 - B a s i c p 2 3 0 0 - L u c F p 2 2 0 0 - L u c p 2 2 0 0 / - 1 73 L u c p 2 2 0 0 / - 5 5 7 L u c p 2 2 0 0 / - 7 7 1 L u c P 2 2 0 0 / - 1 0 2 2 L u c p 2 2 0 0 / - 1 3 5 1 L u c c p G L 2 -B a s ic p 2 3 0 0 - L u c F p 2 2 0 0 - L u c P 2 2 0 0 / - 1 73 L u c p 2 2 0 0 / - 5 5 7 L u c P 2 2 0 0 / - 7 7 1 L u c P 2 2 0 0 / - 1 0 2 2 L u c p 2 2 0 0 / - 1 3 5 1 L u c 2 3 4 5 6 Relative Promoter Activity (fold) Relative Promoter Activity (fold) 0.5 1 1 .5 Relative Promoter Activity (fold) cxT3-l 7 8 OVCAR-3 12 14 HEK-293 Figure 51. Functional analysis of the human GnRHR promoter by transient transfection assays. Five pg of the GnRHR-LUC vectors and 2.5 ug of RSV-LacZ were cotransfected into the ctT3-l (A), OVCAR-3 (B), and HEK-293 (C) cells as described in the Materials and Methods. Luciferase activity of GnRHR-LUC was expressed as a fold-induction over the luciferase activity of promoterless pGL2-Basic vector. Values are the means ± S.D. of luciferase activity after adjusting for B-galactosidase activity. 170 Involvement of regulatory proteins that confer different expression levels of the hGnRHR gene To investigate the factors binding to the hCmRHR promoter, we performed electrophoretic mobility shift assays. Two putative promoter regions were identified from 3'-deletion transfection study, and were designated as PR1 (-771 to -557) and PR2 (-1351 to -1022). The DNA fragments were end-labeled and incubated with nuclear extracts from ccT3-l, OVCAR-3 and HEK-293 cells. When the end-labeled PR1 was incubated with the nuclear extract from aT3-l cells, four protein-DNA complexes, designated A l - A4, were observed. In contrast, using the same probe, two protein-DNA complexes (Bl - B2) were observed in OVCAR-3 cells (Fig. 52A). The binding of each of these complexes were found to be specific, as formation of the retarded complexes were abolished in the presence of a 250-fold molar excess of unlabelled competitor DNA (Fig. 52B). One of the shifted bands in OVCAR-3 cells appeared to represent nonspecific binding, as no competition was observed. When EMSAs were performed with the end-labeled PR2, one protein-DNA complex was observed in aT3-l (CI) and OVCAR-3 cells (Dl). However, patterns of complex formation were different in various cell types (Fig. 53A). The formation of both complexes was prevented by the addition of an excess amount of unlabeled PR2, indicating that these interactions are sequence specific (Fig. 53B). No sequence specific binding activities were observed from the HEK-293 cell nuclear extract when incubated with end-labeled PR1 and PR2 (Figs. 52A and 53A). 171 A FP cxT3-l OVCAR-3 HEK-293 0°C RT 0°C RT 0°C RT Figure 52A. EMSAs performed with PR1 (-771 to -557). The nuclear extracts from the aT3-l , OVCAR-3 and HEK-293 cells were incubated with end-labeled PR1 as a probe at different binding temperatures as described in the Materials and Methods. When end-labeled PR1 was used as a probe, four protein-DNA complexes were observed in nuclear extracts from ccT3-l cells, whereas two different shift complexes were identified in OVCAR-3 cells (A, see arrows). 172 B aT3-l Probe Competitor A l A2 FP PRl(-771/-557) none 5x 50x250x A3 A4 Free • Probe OVCAR-3 FP PRl(-771/-557) none 5x 50x 250x N S B l B2 Figure 52B. Competitive EMSAs performed with PR1 (-771 to -557). The nuclear extracts from the ccT3-l, OVCAR-3 and HEK-293 cells were incubated with end-labeled PR1 as a probe at different binding temperatures as described in the Materials and Methods. Competitive EMSA with excess amount of unlabeled probe was added into the reaction mixture and incubated at RT. As the concentrations of unlabeled competitor increased, the shifts were lost (B, see arrows). Notice a non-specific band (NS) in the OVCAR-3 cells. 173 A FP aT3-l OVCAR-3 HEK-293 0°C RT 0°C RT 0°C RT C I D l Free Probe Figure 53A. EMSAs performed with the PR2 (-1351 to -1022). The nuclear extracts from aT3-1, OVCAR-3 and HEK-293 cells were incubated with end-labeled PR2 as a probe at different binding temperature as described in the Materials and Methods. A different protein-DNA complex was observed when incubated with end-labeled PR2 as probe in nuclear extracts from aT3-l and OVCAR-3 cells, respectively (A, see arrows). 174 B aT3-l OVCAR -3 Probe FP PR2(-1351/-1022) FP PR2(-1351/-1022) Competitor none 5x 50x 250x none 5x 50x 250x Figure 53B. Competitive EMSAs performed with the PR2 (-1351 to -1022). The nuclear extracts from ocT3-l, OVCAR-3 and HEK-293 cells were incubated with end-labeled PR2 as a probe at different binding temperature as described in the Materials and Methods. Competitive EMSA with excess amount of unlabeled probe was added into reaction mixture and incubated at RT. The formation of DNA-protein complex was prevented by the addition of unlabeled probe (B, see arrows). 175 IV. Discussion As evident by the different expression levels, it has been suggested that GnRHR found in extrapituitary tissues may be different from that of the pituitary. In the present study, using pituitary aT3-l and ovarian OVCAR-3 cell lines, it was demonstrated that different regulatory factors may be the molecular basis of these differences. Since human gonadotroph-derived cell line is unavailable, we utilized the mouse gonadotroph-dervied aT3-l cell line for our study. It has been demonstrated that aT3-l cells express high levels of GnRHR that exhibits binding characteristic similar to those found in the normal mouse and rat pituitary (Perrin et al., 1993; Reinhart et al., 1992; Shah et al., 1994). Conservation of transcription factors among species further substantiates the feasibility of using this gonadotroph-derived cell line for our studies (Farmerie et al., 1997; Nilson et al., 1991). For comparison, the GnRHR expressing ovarian cancer cell line, OVCAR-3 was employed. In the previous studies, we demonstrated that OVCAR-3 cells express functional GnRHR (Yin et al., 1998; PART TV). Different basal expression levels of GnRHR were addressed using semi-quantitative RT-PCR. The present study demonstrated a higher level of receptor expression in the primary pituitary tissues and pituitary aT3-l cells than in PCO and the OVCAR-3 cells. Initially, Northern blot analysis was employed but failed to detect the transcript in the ovarian cells. By Northern blot analysis, a previous study in the rat has shown that GnRHR gene expression is the highest in the pituitary and is followed by the ovary and testis (Kakar et al., 1994). By RNase protection assay, the pituitary expressed 200 times greater hGnRHR mRNA level than the granulosa-luteal cells in the human (Minaretzis et al., 1995). In an attempt to elucidate the underlying mechanisms for different basal transcription levels of the hGnRHR gene, the use of various cell-specific promoters for the 176 regulation of gene transcription was investigated in the pituitary and ovarian cells. This possibility has been documented in the transcriptional regulation of human GnRH and a-glycoprotein subunit genes (Dong et al., 1993; 1997; Hamemik et al., 1992; Schoderbek et al., 1992). To address the potential usage of a cell specific promoter, a series of constructs containing 3'-deletions of the hGnRHR promoter were fused to a luciferase reporter gene and tested for their abilities to drive expression in various cells. Two putative promoters and one repressor were identified in both cell types. The similar patterns of luciferase activity of the 3'-deletion constructs suggest that there is no cell specific promoter usage in pituitary aT3-l versus ovarian OVCAR-3 cell lines. However, it is possible that a unique and currently undefined ex-acting element is required for different expression levels of the hGnRHR gene in the two tissues. This possibility was demonstrated in the tissue specific expression of several genes (Hu et al., 1996; Means et al., 1989; Radovick et al., 1984; Zennaro et al., 1996). For instance, unlike in the ovary, a distal promoter which was located at least 40 kb upstream of the ovarian proximal promoter was employed to drive placenta-specific expression of the human P450arom gene (Means et al., 1989). Therefore, it cannot be ruled out that the possibilities of other regulatory elements for cell/tissue-specific expression of the hGiiRHR gene may reside outside of the 2.3 kb promoter region. An alternative explanation is that different combinations of transcription factors may be used in gonadotroph and ovarian cells, thereby contributing to different levels of gene expression. It has been well documented that a cell type-specific expression of genes can be coordinately controlled by an individual or combinations of tissue-specific regulators (Bodner et al., 1988; Farmerie et al., 1997; Mangalam et al., 1989; Nilson et al., 1991; Steger et al., 1993). To examine this hypothesis, gel-shift assays were performed. The present study demonstrated the 177 binding of different nuclear proteins in both promoter regions (PR1 and PR2). This observation provided strong evidence supporting the involvement of different transcription factors in the regulation of the hGnRHR promoter. Each of these complexes appeared to be specific, as they can be competed with excess amount of unlabelled competitor PR1 and PR2. No sequence specific binding activities were evident from the nuclear extract of HEK-293 cells when incubated with end-labeled PR1 and PR2, thus suggesting that binding activities observed in aT3-l and OVCAR-3 cells are important in mediating cell specific expression of the hGnRHR gene. The demonstration of multiple nuclear protein bindings probably reflects the synergistic functional interactions of protein factors that are often observed in other transcription factors (Drean et al., 1996; Nilson et al., 1991; Piao et al., 1997; Schule et al., 1988). Therefore, it was concluded that different and/or combinations of regulatory factors may contribute to the different expression levels of the hGnRHR gene in gonadotroph and ovarian cells. However, these results may reflect species differences rather than cell type difference. At this moment, it cannot be addressed this point, as no human gonadotroph-derived cell line and mouse ovarian epithelial cancer cell line are available for the comparison study. Inspection of the sequence of the two promoter regions reveals putative binding sites for several transcription factors. A consensus sequence for GATA-1 is identified in both the PR1 and PR2 regions. Potential AP-1 binding site with one base insertion is found in the PR1 region (TGACTGCA, underlined) and with one base difference is present in the PR2 region (TGACTTA, underlined). A putative binding site for GF-1 is also found in the PR1 region with two bases difference ( T T A A T C A G , underlined). A putative binding site for AP4 is found in the PR2 region with two bases difference (TCAGCTTCT). 178 PART 9 Differential Regulation of Two Forms of Gonadotropin-Releasing Hormone Messenger Ribonucleic Acid (mRNA) in Human Granulosa-Luteal Cells I. Introduction Within the brain of a single species, two or three forms of GnRH have been identified. At present, thirteen different forms of GnRH have been identified in lower vertebrates (Carolsfeld et al., 2000). The primate brain was thought to contain only one form of GnRH known as mammalian GnRH (GnRH-I). However, it has been recently demonstrated that a second form of GnRH (GnRH-II) with characteristics of chicken GnRH-II (cGnRH-II), is present in brain extracts from adult stumptail and rhesus monkeys (Lescheid et al., 1997). The unique location and differential expression levels of GnRH-II within the brain and outside brain in a single species including human, suggests that it may have distinct functions from those of GnRH-I (King and Millar, 1995; Sherwood et al., 1993; Urbanski et al., 2000; White et al., 1998). Prompted by this finding, the present study was designed to investigate the physiological role of GnRH-II and its hormonal regulation by homologous ligand (GnRH-II) and gonadotropins (FSH and hCG) in the human ovary. 179 II. Materials and Methods Treatment Human GLCs were cultured for 4 days and treated with 10"11 to 10"7 M of GnRH-II (Peninsula Laboratories, Belmont, CA.), GnRH-II-a (Peninsula Laboratories) or GnRH-I-a (Leuprolide, Sigma-Aldrich Corp.). The appropriate cell cultures were treated with 0.1 to 1000 ng/ml of recombinant FSH (rFSH, a gift from National Hormone and Pituitary Distribution Program, NIDDK, NTH) or hCG (0.001 to 10 IU/ml). After 24h incubation, medium was collected and stored -20 C and subsequently assayed for progesterone content. Cells were lysed and thereafter immediately frozen at -70 C until total RNA was extracted. RT-PCR amplification To clone GnRH-II mRNA, one set of primers was designed based on the published sequences for human GnRH-II (White et al., 1998). Primers for GnRH-II were: sense, 5'-G C C C A C C T T G G A C C C T C A G A G - 3 ' ; and antisense, 5'-C C A G G T G T C G C T T C C T G T G A A - 3 ' . The cDNA (2 pi from 1 pg of total RNA) was PCR-amplified for 33 cycles with denaturing for 1 min at 94 C, annealing for 35 sec at 60 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C. The expected PCR product (225 bp) was isolated, sequenced and used as the template for making digoxigenin-labeled probe for Southern blot analysis. 180 Quantification of GnRH-II, GnRH-I, GnRHR, FSH and LH receptor mRNA To compare different expression levels for GnRH-II mRNA, semi-quantitative PCR was performed. Primers for GnRH-II were: sense, 5'-G C C C A C C T T G G A C C C T C A G A G - 3 ' ; and antisense, 5'-C C A A T A A A G T G T G A G G T T C T C C G - 3 ' . PCRs for GnRH-II were carried out with denaturing for 1 min at 94 C, annealing for 65 sec at 62 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C for 26 cycles. PCRs for GnRH-I, GnRHR, p-actin were performed as described previously (Peng et al., 1994; Nathwani et al., 2000). Semi-quantitative PCRs for FSHR and LHR were carried out with denaturing for 1 min at 94 C, annealing for 35 sec at 55 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C for 25 cycles. The primers for LHR were: sense, 5'-G C C C A C C T T G G A C C C T C A G A G - 3 ' ; and antisense, 5'-C C A A T A A A G T G T G A G G T T C T C C G - 3 ' . Primers for FSHR were described previously (Zheng et al., 1996). The cDNA for human FSHR and LHR were kindly provided by Dr. T. Minegishi (Gunma University, Japan) and used as template for DIG-labeled probes. RIA for progesterone To investigate the role of GnRH-II on basal progesterone secretion, hGLCs was cultured for 4 days and then incubated for 24 h in 2 ml D M E M containing 5% FBS, 10"11 to 10"7 M of GnRH-II and GnRH-II-a. To examine the effect of GnRH-II and GnRH-I on hCG-stimulated progesterone secretion, cell cultures were treated with GnRH-II-a (10 M) or GnRH-I-a (10~7 M) in the presence or absence of hCG (1 IU/ml) for 24 h. The appropriate cell cultures were treated with GnRH-II-a (10" M) plus the GnRH antagonist 181 (antide, 10"7 M) for 24 h to determine if the effect of GnRH-II is mediated through the activation of classical GnRHR. Control cultures were treated with vehicle. The progesterone concentration in the culture medium was measured and standardized against protein content, as described in the PART m. RIA for intracellular cAMP To determine if GnRH-II modulates basal or hCG-induced intracellular cAMP accumulation, hGLCs (2 x 10s cells) was cultured for 4 days and preincubated in serum-free medium containing 0.1% BSA and 0.5 mM EBMX for 30 min. The cells were then treated with GnRH-II (10"7 M) or GnRH-II-a (10"7 M) in the presence or absence of hCG (1 IU/ml) for 20 min. Control cultures were treated with vehicle. Intracellular cAMP levels were measured using a [ 3H]-cAMP assay system, as described in the PART U. Data analysis The data were analyzed by one-way A N O V A followed by Tukey's multiple comparison test. P< 0.05 was considered statistically significant. III. Results Validation ofPCRsfor GnRH-II, FSHR andLHR Using RT-PCR, the expected size (225 bp) of DNA fragment was obtained from the hGLCs cells and was validated as GnRH-II by sequence analysis (data not shown). The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed in negative controls (without template and without reverse 182 transcriptase in the RT reaction). Hybridization of the membrane containing GnRH-II PCR product with a probe specific for human GnRH-I revealed no signal, excluding cross-hybridization from GnRH-I (data not shown). To determine the conditions under which PCR amplification for GnRH-II, FSHR and LHR mRNA were in the logarithmic phase, total RNA (1 pg) were reverse transcribed, and aliquots (1 pi) were amplified using different numbers of cycles. A linear relationship between PCR products and amplification cycles was observed in GnRH-II, FSHR and LHR (Fig. 54). Twenty-six cycles for GnRH-II and 25 cycles for FSHR and LHR were employed for quantification. The validation of PCRs for GnRH-I, GnRHR and p-actin was described previously (Peng et al., 1994; Nathwani et al., 2000). Homologous regulation of GnRH-II, GnRH-I and GnRHR mRNA Treatment of hGLCs with GnRH-II induced a significant decrease in GnRH-II mRNA levels at all concentrations used (10"H to 10"7 M), with maximum decrease (55 % of control levels) at 10"7 M (Fig. 55A). Similarly, GnRH-II-a down-regulated GnRH-II mRNA levels at all concentrations used (10"11 to 10"7 M), maximum down-regulation (61 % of control levels) at 10"7 M (Fig. 55B). As shown in Figs. 56A and B, both GnRH-II and GnRH-II-a induced a significant down-regulation of GiiRHR mRNA levels at all concentrations used, with maximum down-regulation at 10" M (44 % and 40 % of control levels, respectively). In contrast, treatment with GnRH-I-a resulted in biphasic effect for GnRH and GnRHR mRNA levels. High concentrations of GnRH-I-a (10"8 and 7 11 10" M) decreased GnRH-I and GnRHR mRNA levels, whereas low concentrations (10" and 10"10M) resulted in an up-regulation of GnRH-I and its receptor (Figs. 55C and 56C). 183 GnRH-II 23 26 29 32 35 hf=0.9911 <«-327bp 25000 20000 1 15000 10000 CJ 5000 0 23 23 29 32 35 Cyderxrrber h L H R C 23 26 29 32 35 3 9 6 b p > « i » ft* B 33 hFSHR 20 23 26 29 343bp> 4000 R2 = 0.993 rf = 0.9856 20 23 26 29 32 35 38 C^le number 17 20 23 26 29 32 35 Cycle number Figure 54. Validation of semi-quantitative RT-PCR for GnRH-II (A), LHR (B) and FSHR (C). Human GLCs was cultured in 35-mm culture dishes at 2 x 105 cells in 2 ml DMEM supplemented with 10 % FBS, 100 U/ml penicillin G and 100 ug/ml streptomycin. Total RNA was isolated and reverse transcribed, and aliquots were amplified using different number of PCR cycles as described in Materials and Methods. A linear relationship was observed between PCR products and amplification cycles when plotted. 184 GnRH-II (Log M) GnRH-II Control -11 -10 B Control GnRH-II-a (Log M) Control GnRH-II -11 -10 -9 -8 -7 GnRH-II treatment (Log M) -11 -10 -9 -8 -7 Control GnRH-I-a (Log M) Control GnRH-I (3-actin - 200 CD > < 1 150 -11 -10 -9 -8 GnRH-II-a treatment (Log M) r 100 Control -11 -10 -9 -8 GnRH-I-a treatment (Log M) Figure 55. Homologous regulation of GnRH-II and GnRH-I mRNA. Human GLCs was cultured for 4 days and treated with various concentrations of GnRH-II (A), GnRH-II-a (B), or GnRH-I-a (C) for 24 h as described in Materials and Methods. Control cultures were treated with vehicle. Total RNA was isolated and reverse transcribed, and semi-quantitative PCR was performed. The PCR products were quantified and normalized against P-actin levels after Southern blot analysis. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, P<0.05 vs. control. 185 GnRH-II (Log M) Control -11 GnRHR P-actin 1 20 j j ^ 1 00 < g 80 | o 6 0 -10 a a a • • • • C o n t r o l -11 •1 0 -8 -7 B GnRH-II-a (Log M) Control GnRHR P-actin 120 GnRH-II treatment (Log M) -11 -10 -9 -I 100 < 3 8 0 S 60 4 0 2 0 c l i i i i H Control -11 -10 -8 GnRH-II-a treatment (Log M) GnRH-I-a (Log M) Control -11 GnRHR p-actin _ 200 -10 -8 -7 < o 150 , ° 100 50 a a a • I I I . . Control -11 -10 -9 -8 -7 GnRH-I-a treatment (Log M) Figure 56. Homologous regulation of GnRHR mRNA. Human GLCs was cultured for 4 days culture and treated with various concentrations of GnRH-II (A), GnRH-II-a (B), or GnRH-I-a (C) for 24 h as described in Materials and Methods. Control cultures were treated with vehicle. Total RNA was isolated and reverse transcribed, and competitive PCR was performed. The PCR products were quantified and the amount of GnRHR transcript was calculated from the ratio of the target to competitive cDNA. Data are shown as the means of four individual experiments and are presented as the mean + SD. a, P<0.05 vs. control. 186 Heterologous regulation of GnRH-II and GnRH-I mRNA As shown in Fig. 5 7A, FSH induced a dose-dependent increase in GnRH-II mRNA levels, with maximum increase (347% of control levels) at 1000 ng/ml. Similarly, hCG induced a significant up-regulation of GnRH-II mRNA levels, maximum increase (337% of control levels) at 10 IU/ml (Fig. 58A). In contrast, treatment with FSH and hCG resulted in a down-regulation of GnRH-I mRNA levels in a dose-dependent manner, with maximum down-regulation at 1000 ng/ml FSH (54% of control levels) and 10 IU/ml hCG (40% of control levels) (Figs. 57B and 58B). Functionally, treatment with FSH and hCG stimulated progesterone secretion from hGLCs (Figs. 57C and 58C). Effect of GnRH-II and GnRH-I on basal and hCG-stimulated progesterone secretion A significant decrease in progesterone secretion from hGLCs was observed in response to 10"10 to 10"7 M GnRH-II (Fig. 59A). Maximum inhibition (a 33%) decrease over basal level) was observed after the treatment with 10" M GnRH-II. As shown in Fig 59B, a further decrease in progesterone secretion was observed in cells treated with 10"10 to 10"7 M GnRH-II-a, with maximum inhibition (a 49%) decrease over basal levels) at 10"7 M GnRH-II-a. Similarly, treatment with 10"7 M GnRH-I-a induced a significant decrease in progesterone secretion (a 52 % decrease over basal levels) (Fig. 59C). As shown in Fig. 59C, hCG alone (1 IU/ml) stimulated progesterone secretion (a 223% increase over basal levels), whereas concomitant treatment with GnRH-II-a or GnRH-I-a attenuated the stimulatory effect of hCG. Co-treatment with antide abolished the inhibitory effect of GnRH-II-a on progesterone secretion (Fig. 59D), while antide alone had no effect on progesterone secretion (Fig. 59D). 187 rFSH (ng/ml) Control 0.1 1 10 100 1000 Control 0.1 1 10 100 1000 rFSH treatment (ng/ml) rFSH (ng/ml) Control 0.1 1 10 100 1000 rFSH treatment (ng/ml) Figure 57. The effect of FSH on GnRH-II (A), GnRH-I mRNA (B) and progesterone secretion (C). Human GLCs was cultured for 4 days and treated with various concentrations of recombinant FSH for 24 h as described in Materials and Methods. Control cultures were treated with vehicle. Total RNA was isolated and reverse transcribed, and semi-quantitative PCR was performed. The PCR products were quantified and normalized against B-actin levels after Southern blot analysis. Progesterone concentration in the medium was determined by established RIA. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, PO.05 vs. control. 188 C o n t r o l 0.001 0.01 0.1 1 10 hCG treatment (IU/ml) hCG treatment (IU/ml) c Control 0.001 0.01 0.1 1 10 hCG treatment (IU/ml) Figure 58. The effect of hCG on GnRH-II (A), GnRH-I mRNA (B) and progesterone secretion (C). Human GLCs was cultured for 4 days and treated with various concentrations of hCG for 24h as described in Materials and Methods. Control cultures were treated with vehicle. Total RNA was isolated and reverse transcribed, and semi-quantitative PCR was performed. The PCR products were quantified and normalized against P-actin levels after Southern blot analysis. Progesterone concentration in the medium was determined by established RIA. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, P<0.05 vs. control. 189 A B Control GnRH-I-a GnRH-II-a hCG GnRH-I-a GnRH-II-a Control GnRH-II-a antide antide + hCG +hCG + GnRH-E-a Figure 59. The effect of GnRH-II and GnRH-I on basal and hCG-stimulated progesterone secretion. Human GLCs was cultured for 4 days and treated with various concentrations of GnRH-II (A) or GnRH-II-a (B). The cells was treated with GnRH-II-a (10-7M) or GnRH-I-a (IO 7 M) in the presence or absence of hCG (1 IU/ml) (C) for 24 h. The appropriate cell cultures were treated with GnRH-II-a (IO"7 M) plus antide (IO"7 M) for 24 h (D). Control cultures were treated with vehicle. The progesterone concentration in the culture medium was measured by an established RIA and was normalized against protein contents. Progesterone secretion is expressed as the percent change from the control value. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, P<0.05 vs. control; b, PO.05 vs. hCG; c, PO.05 vs. antide + GnRH-II-a. 190 Effect of GnRH-II and GnRH-I on FSHR and LHR mRNA levels As shown in Figs. 60A and B, treatment of hGLCs with various concentrations of GnRH-II and GnRH-II-a induced a significant decrease in FSHR mRNA levels, with maximum decrease at 10"7 M of GnRH-II (39% of control levels) and GnRH-II-a (35% of control levels). Similarly, a down-regulation of FSHR mRNA levels was observed in cells treated with GnRH-I-a at all concentrations used, maximum decrease (40% of control levels) at 10"7M (Fig. 60C). Like FSHR mRNA, GnRH-II or GnRH-II-a induced a significant down-regulation of LHR mRNA levels, with a maximum down-regulation at 10"9 M of GnRH-II (65% of control levels) and at 10"7 M of GnRH-II-a (65% of control levels) (Figs. 61A and B). A similar decrease in L H R mRNA levels was observed in cells treated with GnRH-I-a, with a maximum decrease (51%) of control levels) at 10" M (Fig. 61C). Effects of GnRH-II on basal and hCG-stimulated intracellular cAMP accumulation Treatment with hCG (1 IU/ml) stimulated a substantial increase in cAMP accumulation within 20 min. In contrast, GnRH-II and GnRH-II-a did not affect basal intracellular cAMP levels (Fig. 62). Concomitant treatment of cells with 10"7 M GnRH-II or GnRH-IJ-a did not affect hCG-stimulated cAMP production (Fig. 62). 191 GnRH-II (Log M) Control p-actin 1 20 -11 -10 C o n t r o I B -11 -10 -9 -8 GnRH-II treatment (Log M) GnRH-II-a (Log M) Control FSHR -11 -10 -9 -8 -7 -7 P-actin 120 a m o 80 60 < I 4 0 20 a a a Control -11 -10 -9 -8 -7 GnRH-II-a treatment (Log M) GnRH-I-a (Log M) FSHR p-actin 120 S o Control Control -11 -10 -9 -8 GnRH-I-a treatment (Log M) -7 Figure 60. The effect of GnRH-II (A), GnRH-II-a (B) or GnRH-I-a (C) on FSHR mRNA. Human GLCs was cultured for 4 days and treated with various concentrations of GnRH-II, GnRH-II-a or GnRH-I-a for 24h as described in Materials and Methods. Control cultures were treated with vehicle. Total RNA was isolated and reverse transcribed, and semi-quantitative PCR was performed. The PCR products were quantified and normalized against P-actin levels after Southern blot analysis. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, P<0.05 vs. control. 192 GnRH-II (Log M) Control L H R B C ontro l -11 -10 -9 -8 GnRH-II treatment (Log M) GnRH-II-a (Log M) Control -11 -10 ( ( m m m m m -9 -8 • H P €BW* -7 " ^ 1 0 0 u o < § 80 2 £ 60 5? 40 2 0 C ontrol -11 -1 0 -7 GnRH-II-a treatment (Log M) GnRH-I-a (Log M) Control LHR p-actin 120 | ^ 100 ~1 80 3 8 60 -11 -10 40 20 0 I i i Control -11 -10 -9 -8 -7 GnRH-I-a treatment (Log M) Figure 61. The effect of GnRH-II (A), GnRH-II-a (B) or GnRH-I-a (C) on LHR mRNA. Human GLCs was cultured for 4 days and treated with various concentrations of GnRH-II, GnRH-II-a or GnRH-I-a for 24h as described in Materials and Methods. Control cultures were treated with vehicle. Total RNA was isolated and reverse transcribed, and semi-quantitative PCR was performed. The PCR products were quantified and normalized against P-actin levels after Southern blot analysis. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, P<0.05 vs. control. 193 Figure 62. Effects of GnRH-II on basal and hCG-stimulated intracellular cAMP accumulation. Human GLCs (2 x 105 cells) was plated onto 35 mm culture dishes and cultured for 4 days. The cells were then preincubated in serum-free medium containing 0.1% BSA and 0.5 mM 3-isobutyl-l-me%lxanthine (IBMX) for 30 min, and treated with GnRH-II (IO7 M) or GnRH-II-a (IO7 M) in the presence or absence of hCG (1 IU/ml) for 20 min. Control cells were treated with vehicle. Intracellular cAMP levels were measured using a [3H]-cAMP assay system, according to the manufacturer's suggested procedure. Data are shown as the means of four individual experiments and are presented as the mean ± SD. a, PO.05 vs. control. 194 IV. Discussion The present study demonstrates for the first time that the two forms of GnRH expressed in hGLCs are differentially regulated by its own ligands (GnRH-I and GnRH-II) and gonadotropins. Nevertheless, GnRH-II inhibits basal and hCG-stimulated progesterone secretion. In addition, GnRH-II exerts its antigonadotropic effect through a down-regulation of receptors for FSH and L H without affecting hCG-stimulated intracellular cAMP accumulation. One of the interesting findings of the present study is the demonstration of differential regulation of GnRH-II gene expression by GnRH-II itself and gonadotropins as compared to that of GnRH-I. It has been demonstrated that GnRH-I regulates its own ligand and receptor in a biphasic manner depending on the concentration and duration of treatment in the hypothalamus, pituitary and ovary (Braden and Conn, 1993; Conn, 1994; Kang et al., 2000; Krsamanovic et al., 1993; 1994; Peng et al., 1994). In the present study, a biphasic response of GnRH-I and GnRHR mRNA was observed in response to GnRH-I-a treatment, confirming previous studies. Low doses of GnRH-I-a (10 and 100 pM) increased GnRH-I and GnRHR mRNA levels, whereas higher doses (10 and 100 nM) of the GnRH-I-a induced a statistically significant decrease in GnRH-I and GnRHR mRNA levels. In contrast to GnRH-I, no biphasic response was observed in response to GnRH-II. GnRH-II at all concentrations used (10 p M to 100 nM) induced a significant down-regulation of both GnRH-II and GnRHR mRNA levels. The exact mechanism for this differential regulation is not clear. It is possible that GnRH-I and GnRH-II may have different binding characteristics for GnRHR, which may induce distinct receptor conformations. These ligand-specific conformations of the GnRHR in hGLCs could 195 induce differential coupling to G-proteins and/or generate different intracellular signal tranduction mechanisms, eventually leading to differential regulation of GnRH and its receptor gene expression. In this regard, GnRHR has been shown to couple with different subtypes of G proteins, leading to activation of differential intracellular signaling pathways in the same or distinct cells (Hsieh and Martin, 1992; Janovick and Conn, 1994; Imai et al., 1996; Stanislaus et al., 1998). Furthermore, two native GnRH forms in goldfish (sGnRH and cGnRH-II) have been shown to activate differential signal transduction pathways that differ in their relative dependence on intracellular and extracellular Ca availability, protein kinase C activation, inositol phosphate production, and arachidonic acid mobilization (Chang et al., 1995; 1996; Jobin and Chang, 1992; Johnson et al., 1999). In addition, gonadotropins have been shown to regulate GnRH-I mRNA expression. It has been demonstrated that hCG induced a dose- and time-dependent decrease in GnRH-I mRNA in GT1-7 neuron (Lei and Rao, 1994), supporting the presence of short feedback mechanism (Conway and McCann, 1990; Ziecik et al., 1988). In the present study, treatment of hGLCs cells with FSH and hCG induced a marked increase in GnRH-II mRNA levels, whereas resulted in an apparent decrease in GnRH-I mRNA. Several studies have demonstrated the differential regulation of two forms of GnRH during various physiological conditions. In the brain of the European female silver eel, steroids regulates differential regulation of two forms of GnRH, with a positive estrogen-dependent feedback on mGnRH and a negative androgen-dependent feedback on cGnRH-II (Montero et al., 1995). As well, in the chicken, only the cGnRH-I levels change with castration in the hypothalamus (Sharp et al., 1990). In the goldfish, the ratio 196 between sGnRH and cGnRH-II changed with sexual maturation, such that a stronger increase in sGnRH than in cGnRH-II has been observed in the pituitary (Rosenblum et al., 1994). Taken together, the differential regulation of two forms of GnRH by gonadotropins in the present study suggest that gonadotropins may regulate the ratio between GnRH-I and GnRH-II, leading to distinct spatial expressions of these peptides. However, the physiological relevance of this differential regulation remains to be determined. Increasing evidence has suggested that two or three identified forms of GnRH in a single species may have similar physiological roles (Gazourian et al., 1997; Lescheid et al., 1997; Miyamoto et al., 1984). Administration of synthetic GnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma L H concentration, suggesting that GnRH-II may also have a physiological role in regulating the release of L H (Lescheid et al., 1997). In the goldfish, the two endogenous forms of GnRH stimulate the release of both gonadotropins and growth hormones from the pituitary, even though there are functional differences in terms of potency (Gazourian et al., 1997; King and Millar, 1995; Sharp et al., 1990). In adult female sea lampreys, two endogenous forms of GnRH (lamprey GnRH-I and -III) have been shown to stimulate ovarian steroidogenesis (Gazourian et al., 1997). Like GnRH-I, in the present study, GnRH-II inhibited both basal and hCG-stimulated progesterone secretion in hGLCs. This result suggests that GnRH-II has similar a biological role with respect to ovarian steroidogenesis. However, it cannot be ruled out the possibility that GnRH-II may have other unique reproductive functions as compared to GnRH-I in the ovary. The inhibitory effect of GnRH-II on progesterone secretion in the present study appears to be mediated via activation of the classical 197 GnRHR, because co-treatment with antide abolished the effect of GnRH-II. However, it is possible that GnRH-II may bind and activate unknown second type of GnRHR whose activation may also be blocked by antide. Even though a second form of GnRHR has not been demonstrated in the human, recent cloning of two GnRHR subtypes with distinct ligand selectivity in the goldfish supports this notion (Illing et al., 1999). Furthermore, binding studies have indicated the presence of two different types of GnRHR in the ovary, as high-affinity-low-capacity and low-affinity-high-capacity GnRH binding sites (Bramely et al., 1986; Latouche et al., 1989). The antigonadotropic action of GnRH-I has been shown to be mediated through a down-regulation of receptors for FSH and L H (Piquette et al., 1991; Tilly et al., 1992), inhibition of gonadotropin-stimulated cAMP production (Knecht et al., 1985; Richard, 1994) and steroidogenic enzymes (Hsueh and Schaffer, 1985; Sridaran et al., 1999a; 1999b). Like GnRH-I, the treatment of hGLCs with GnRH-II induced a significant down-regulation of FSHR and LHR. In contrast, GnRH-II did not affect basal and hCG-stimulated cAMP production. Our previous results showed that GnRH-I also had no effect on either basal or hCG-stimulated cAMP production (PART VII). These results suggest that GnRH-II, like GnRH-I, exerts its anti-gonadotropic effect at the receptor levels in hGLCs, independent of cAMP levels. 198 PART 10 Summary and Future Studies 10.1. SUMMARY The aforementioned studies have investigated the autocrine role of GnRH and its regulation and mechanism of action in the human ovary. Furthermore, the study has examined the expression of GnRH-II and its regulation and functional role in the human ovary. Taken with interaction with estrogen/ER system, these findings strongly suggest that GnRli/GnRHR is an integral part of the intraovarian regulatory complexes that modulate the function of normal and neoplastic human ovarian cells. Autocrine role of GnRH in the growth of normal and neoplastic OSE cells In this study, the presence of an autocrine GnRH/GnRHR loop and its functional role was investigated in normal and neoplastic OSE cells. It was found that: 1. Human OSE and OVCAR-3 cells express GnRH and GnRHR that have sequences identical to those found in the hypothalamus and pituitary, respectively. 2. GnRH regulates its own and receptor mRNA in a biphasic manner in both cell types. 3. The biphasic effects of the GnRH were receptor-mediated in both cell types. 4. GnRH-a had a direct inhibitory effect on the growth of hOSE and OVCAR-3 cells in a time- and dose-dependent manner. 5. The growth inhibitory effect of GnRH-a in OVCAR-3 was associated with altered levels of GnRHR mRNA and programmed cell death. 199 This study strongly suggests that GnRH can act as an autocrine regulator in the growth of normal and neoplastic OSE cells Interaction between GnRH/GnRHR and the estrogen/ER system In this study, for the first time, a potential interaction between the GnRH/GnRHR and estradiol/ER and systems was investigated. The results provided the following evidence: 1. Differential expression levels of E R a and ERB mRNA were observed in hOSE cells when compared to OVCAR-3 cells. Expression levels of E R a and ERB proteins also changed in parallel with mRNA levels in hOSE and OVCAR-3 cells. 2. Treatment with 17 B-estradiol induced a significant down-regulation of GnRH mRNA in OVCAR-3, but not in hOSE cells and of GnRHR mRNA in both hOSE and OVCAR-3 cells. 3. The effect of estrogen on GnRH and GnRHR mRNA levels was specific as tamoxifen, an estrogen antagonist, prevented the effects of 17P-estradiol. 4. 17p-estradiol stimulated the growth of OVCAR-3, but not hOSE cells, in a dose-and time-dependent manner. 5. Pre- or co-treatment with 17p-estradiol significantly attenuated the growth inhibitory effect of the GnRH-a in OVCAR-3, but not in hOSE cells. This study demonstrates for the first time interaction between GriRH/GnRHR and estrogen/ER system which may be important in the growth regulation of normal and neoplastic hOSE cells. 200 GnRH-induced MAPK activation: its role and intracellular signaling pathways In this study, the activation of M A P K and its intracellular signaling pathway was investigated. Furthermore, the physiological relevance of GnRH-induced M A P K activation was examined in the human ovary and placenta. It was demonstrated that: 1. GnRH-a induced a time- and dose-dependent activation of M A P K in hGLCs 2. The M A P K activation appears to be mediated via PKC-dependent pathway (coupled to GqCt) in hGLCs. It seems unlikely that the effect of GnRH-a in hGLCs involves Gioc and G s a coupling to GnRHR in hGLCs 3. The GnRH-induced M A P K activation induced a significant increase in Elk-1 phosphorylation and c-fos mRNA expression in hGLCs. 4. The GnRH-a also stimulated a rapid activation of M A P K in normal placenta-derived cells (IEVT). Interestingly, GnRH-a induced a biphasic regulatory pattern in M A P K activity in ovarian carcinoma (OVCAR-3) and placental carcinoma (JEG-3) cells. 5. Functionally, GnRH-induced M A P K activation in hGLCs resulted in the inhibitory effect of GnRH-a in progesterone secretion. 6. GnRH-induced M A P K activation mediated the growth inhibitory effect of GnRH-a in OVCAR-3 cells. 7. In contrast to hGLCs and OVCAR-3 cells, other signaling pathway(s) may play a more dominant role in GnRH-induced phCG mRNA expression in JEG-3 cells This study demonstrates for the first time GnRH induced activation of MAPK, which plays an important role in regulating the functions of the ovary and placenta. 201 Differential transcriptional regulation of hGnRHR gene in the pituitary and ovary In terms of regulation of gene expression, GnRHR found in extrapituitary tissues has been suggested to be different.from that in the pituitary. In this study, the molecular basis of this difference was examined using the pituitary aT3-l and ovarian carcinoma OVCAR-3 cells. It was found that: 1 . The different expression levels of GnRHR mRNA in the pituitary and ovarian cells were observed. 2. There was no cell-specific promoter usage for the human GnRHR gene, as the same promoters (PR1 and PR2) appeared to be utilized for driving the basal promoter activities in both aT3-l and OVCAR-3 cells. 3. Alternatively, different regulatory protein factors appear to be involved, as differential shifted complexes were observed when the same promoters were used as probes in aT3-l and OVCAR-3 cells. This study clearly demonstrates that one mechanism by which cell-specific expression of the human GnRHR, is achieved is through the binding of distinct and/or combinations of cell-specific regulatory factors to various promoter elements in the 5'-flanking region of the gene. Differential hormonal regulation and functional role of GnRH-II in the human ovary In this study, the expression, hormonal regulation and functional role of GnRH-II in the human ovary were investigated. It was found that: 202 1. GnRH-JJ was expressed in hGLCs 2. Homologous GnRHs and gonadotropins regulated differentially GnRH-I and GnRH-II mRNA expression. 3. Functionally, both GnRH-II and GnRH-II-a inhibited basal and hCG-stimulated progesterone secretion. 4. GnRH-II and GnRH-I exerted their anti-gonadotropic effect by down-regulating gonadotropin receptors. 5. Anti-gonadotropic effect of GnRH-II and GnRH-I was independent of modulation of cAMP levels, as GnRH-II and GnRH-I did not affect both basal and hCG-stimulated intracellular cAMP accumulation These results demonstrate for the first time that GnRH-II may have biological effects similar to those of GnRH-I, but is under differential hormonal regulation in the human ovary. 203 10.2. FUTURE STUDIES 1. The autocrine role of GnRH in normal hOSE Future experiments need to be done to elucidate the exact mechanism of the growth inhibitory effects of GnRH-a in hOSE cells. At the receptor levels, using a blocking antibody for GnRH, it needs to be clarify whether endogenous GnRH has a growth stimulatory effect, which can be blocked by continuous treatment of GnRH-a as shown in this study. At the cellular levels, experiments should clarify whether GnRH-a inhibit OSE cell growth by increasing the portion of cells in the resting phase, Go-Gi or induce cell death. 2. The autocrine role of GnRH in neoplastic OSE cells As in normal OSE, future experiments should clarify whether endogenous GnRH has a stimulatory effect on the growth of ovarian cancer cells. The involvement of the Fas ligand-Fas system should be evaluated to explain molecular mechanism for GnRH-induced apoptosis shown in this study. 3. The interaction with the GnRH/GnRHR and estrogen/ER in normal and neoplastic OSE cells Future experiments need be done to explain the exact mechanisms of estrogen insensitivity and functional role of estrogen in ER-positive hOSE. 4. The activation of M A P K by GnRH in human ovarian and placental cells 204 Future experiments should clarify whether GnRH can induce the other family of M A P K (JNK/SAPK or p38), which are known to be activated by GnRH in the pituitary. In addition, future experiments need be done to determine the mechanism by which GnRH-induced M A P K activation inhibits progesterone secretion in hGLCs. More physiological relevance of GnRH-induced M A P K activation in the ovary and placenta should be investigated. 5. The intracellular pathways in activating M A P K by GnRH in hGLCs The sequential M A P K cascade, which follows GnRH-induced PKC activation as shown in this study, should be dissected to characterize potential regulators for GnRH-induced M A P K activation in hGLCs. 6. The differential transcriptional regulation of the GnRHR gene expression in the ovary Future experiments need to be done to address the potential regulatory factors and their precise D N A binding sequences that contribute to the different expression levels of the hGnRHR gene in gonadotroph and ovarian cells. 7. 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