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Transciptional regulation of human gonadotropin-releasing hormone (GnRH) and GnRH receptor genes Cheng, Chi Keung 2003

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TRANSCRIPTIONAL REGULATION OF HUMAN GONADOTROPIN-RELEASING HORMONE (GnRH) AND GnRH RECEPTOR GENES by CHI KEUNG CHENG B. Sc. (Hons.), The University of Hong Kong, 1998 M. Phil., The University of Hong Kong, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Obstetrics and Gynecology; Program of Reproductive and Developmental Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 2003 © Chi Keung Cheng, 2003 Abstract In addition to its pivotal role in controlling gonadotropin synthesis and secretion, mammalian gonadotropin-releasing hormone (termed GnRH-I) functions as an autocrine and/or paracrine factor in certain extrapituitary tissues, where expression of its receptor (GnRH-I receptor) has been demonstrated. Although our laboratory has previously addressed a number of issues regarding the transcriptional regulation of the human GnRH-I receptor gene, some aspects including tissue-specific and hormonal regulation of the gene expression remain unclear. In the present study, we have identified by stepwise deletion analysis a new upstream GnRH-I receptor promoter that is primarily used by ovarian granulosa-luteal (GL) cells. Site-directed mutagenesis indicated that the activity of this promoter was governed by a cooperative action among two CCAAT/enhancer-binding protein (C/EBP) and one GATA motifs. On the one hand, these data strengthen the notion that tissue-specific expression of the human GnRH-I receptor gene is mediated by differential promoter usage in various cell types. On the other hand, we have identified an evolutionarily conserved octamer sequence, which constitutively and ubiquitously suppresses the GnRH-I receptor promoter. Electrophoretic mobility shift assays (EMSAs) showed that the repressor binding to this element was the POU homeodomain transcription factor Oct-1. These findings thus uncover a novel role of Oct-1 in silencing GnRH-I receptor gene transcription. Furthermore, we have demonstrated that an activator protein-1 (AP-l)-like motif mediates 17p-estradiol (E2) repression of the GnRH-I receptor promoter via an estrogen receptor (ER)ct-dependent mechanism that may involve competition for the transcriptional coactivator cAMP-i i responsive element (CRE)-binding protein (CREB)-binding protein (CBP) between the steroid receptor and c-Jun/c-Fos heterodimer. The identification of a second form of GnRH from chicken hypothalamus (termed GnRH-II) revealed that it is the most widely expressed form of GnRH and that its structure is conserved over all vertebrate classes from primitive fishes to humans. However, the molecular mechanism governing human GnRH-II gene transcription is poorly understood. Here we have shown that two E-box binding sites (EBSs) and one Ets-like element (ELE) in the untranslated first exon function cooperatively to stimulate the basal transcription of the GnRH-II gene and demonstrated that the basic helix-loop-helix (bHLH) transcription factor AP-4 is an enhancer protein for the GnRH-II promoter. The results of this research provide novel and significant insights into the molecular mechanisms underlying the multi-faceted actions of GnRH in humans. in Table of Contents Abstract - - ii Table of Contents - iv List of Tables ix List of Figures — - — x List of Abbreviations xv Preface - xx Acknowledgements - xxii CHAPTER I Introduction 1.1 GnRH: Overview - 1 1.2 Human GnRH-I Complementary Deoxyribonucleic Acid (cDNA) 3 1.3 Human GnRH-I Gene - 4 1.4 Human GnRH-II cDNA and Gene 6 1.5 Tissue Distribution of Human GnRH-I and GnRH-II Gene Transcripts 1.5.1 Brain - - - 7 1.5.2 Placenta - 8 1.5.3 Uterus 9 1.5.4 Ovary 10 1.5.5 Other Tissues 10 1.6 Regulation of Human GnRH-I and GnRH-II Gene Expression 1.6.1 GnRH-I and GnRH-II 11 1.6.2 Gonadal Steroid Hormones 13 1.6.3 Gonadotropins 14 1.6.4 Other Physiological Regulators - 14 1.7 GnRH-I Receptor cDNAs 1.7.1 Cloning — 15 1.7.2 General Structural Features 18 1.7.3 Unique Structural Features 19 1.8 Tissue Distribution of Human GnRH-I Receptor Gene Transcripts 1.8.1 Pituitary 20 1.8.2 Placenta - 22 iv 1.8.3 Ovary 23 1.8.4 Uterus 23 1.8.5 Prostate Gland 24 1.8.6 Breast 25 1.8.7 Other Extrapituitary Tissues — 25 1.9 Regulation of Human GnRH-I Receptor Gene Expression 1.9.1 GnRH-I and GnRH-II - 26 1.9.2 Gonadal Steroid Hormones - 27 1.9.3 Gonadotropins 28 1.9.4 Melatonin - 28 1.10 Signal Transduction Mechanism of the GnRH-I Receptor 1.10.1 G protein Coupling 29 1.10.2 Mitogen-Activated Protein Kinases (MAPKs) 30 1.10.3 Receptor Desensitization and Internalization 37 1.11 Biological Functions of GnRH-I and GnRH-II in Humans 1.11.1 Gonadotropin Expression and Secretion 39 1.11.2 Ovarian Steroidogenesis 43 1.11.3 Cell Proliferation 44 1.11.4 Apoptosis - 48 1.11.5 Other Extrapituitary Functions 50 1.12 Human GnRH-I Receptor Gene 1.12.1 Structural Organization - 51 1.12.2 Chromosomal Localization 53 1.12.3 5'-and3'-UTRs - 53 1.12.4 5'-Flanking Region 54 1.13 Transcriptional Regulation of the Human GnRH-I Receptor Gene 1.13.1 Identification and Characterization of a Gonadotrope-Specific GnRH-I Receptor Promoter — 57 1.13.2 Identification and Characterization of a Distal and Proximal GnRH-I Receptor Promoter 59 1.13.3 Identification and Characterization of a Placenta-Specific GnRH-I Receptor Promoter 61 1.13.4 Transcriptional Regulation by GnRH-I — 62 1.13.5 Transcriptional Regulation by the cAMP-Dependent Signal Transduction Pathway 63 1.13.6 Transcriptional Regulation by P4 65 v 1.13.7 Transcriptional Repression 66 1.14 GnRH-II Receptor 1.14.1 Nonhuman Primate GnRH-II Receptor 67 1.14.2 Putative GnRH-II Receptor Genes in the Human Genome 71 1.14.3 Tissue Distribution of Human GnRH-II Receptor Transcripts - 75 CHAPTER II Materials and Methods 2.1 Cells and Cell Culture 2.1.1 Primary GL Cell Culture - - 78 2.1.2 Cell Lines - 78 2.2 Plasmid Construction 2.2.1 Human GnRH-I Receptor Promoter-Luciferase Constructs 79 2.2.2 Human GnRH-II Promoter-Luciferase Constructs — 90 2.2.3 Mammalian Expression Plasmids - 91 2.2.4 Plasmid DNA Preparation - 92 2.3 Transient Transfection and Reporter Gene Assay - 92 2.4 RNA Extraction, RT-PCR, and Southern Blot Analysis 94 2.5 Western Blot Analysis - 94 2.6 Primer Extension Analysis — 95 2.7 5'-RACE 96 2.8 EMS A - — 96 2.9 UV Crosslinking 98 2.10 Southwestern Blot Analysis — 98 2.11 Data Analysis 99 CHAPTER III Characterization of a New Upstream GnRH-I Receptor Promoter in Human Ovarian GL Cells 3.1 Introduction - 100 3.2 Results 3.2.1 Expression of GnRH-I Receptor in the Immortalized Human GL Cell Lines SVOG-4o and SVOG-4m - — 102 3.2.2 Mapping of the Human GnRH-I Receptor Promoter in GL Cells 102 vi 3.2.3 Identification of the Transcription Start Sites for the Human GnRH-I Receptor Gene in GL Cells 113 3.2.4 Two Putative C/EBP Motifs and a GATA Motif Function Cooperatively to Regulate the Upstream Human GnRH-I Receptor Promoter in GL cells 113 3.2.5 Analysis of DNA-Protein Interactions of the Putative C/EBP and GATA Motifs - 121 3.3 Discussion 128 CHAPTER IV Oct-1 Is Involved in the Transcriptional Repression of the GnRH-I Receptor Gene 4.1 Introduction - 133 4.2 Results 4.2.1 Silencing Effect of the NRE on a Heterologous TK Promoter — 134 4.2.2 Positional Effect of the NRE Silencing Activity on the Native Promoter 137 4.2.3 Identification of Critical Nucleotide Sequences Mediating the Silencing Effect of the NRE 137 4.2.4 Binding of the POU Domain Transcription Factor Oct-1 to the Putative Octamer Regulatory Sequence - 147 4.3 Discussion 153 CHAPTER V An AP-l-Like Motif Mediates E 2 Repression of the Human GnRH-I Receptor Promoter via an ERa-Dependent Mechanism in Ovarian and Breast Cancer Cells 5.1 Introduction - 161 5.2 Results 5.2.1 E 2 Repression of the Human GnRH-I Receptor Promoter Requires ERa 163 5.2.2 Domains of ERa Required for Repression of the Human GnRH-I Receptor Promoter - 173 5.2.3 An AP-l-Like Motif at -130/-124 Mediates Both Basal Activity and E 2 Repression of the Human GnRH-I Receptor Promoter 177 5.2.4 Multiple Transcription Factors Including c-Jun and V l l c-Fos Form Complexes with the AP-1 -Like Motif — 183 5.2.5 PMA Antagonizes E2-Dependent Repression of the Human GnRH-I Receptor Promoter 187 5.2.6 Overexpression of the Transcriptional Coactivator CBP Attenuates E2-Dependent Repression of the Human GnRH-I Receptor Promoter 194 5.3 Discussion — - 197 CHAPTER VI Functional Cooperation between Multiple Regulatory Elements in the Untranslated Exon 1 Stimulates the Basal Transcription of the Human GnRH-II Gene 6.1 Introduction 204 6.2 Results 6.2.1 Identification of the Transcription Start Sites for the Human GnRH-II Gene - 206 6.2.2 Transcriptional Activity of Various Regions of the Human GnRH-II Gene 209 6.2.3 Two Putative EBSs and One ELE in the Untranslated Exon 1 Function Cooperatively to Stimulate Basal Human GnRH-II Gene Transcription 219 6.2.4 The bHLH Transcription Factor AP-4 Binds to Both EBSs, whereas an Unknown Protein Binds to the ELE 224 6.2.5 Effect of Overexpression of Sense and Antisense AP-4 cDNAs on Human GnRH-II Promoter Activity 235 6.3 Discussion 244 CHAPTER VII Conclusion and Future work - 251 Bibliography 256 V l l l List of Tables 1. Summary of differential regulation of human GnRH-I and GnRH-II gene expression (page 12). 2. Summary of autocrine actions of GnRH-I (I) and GnRH-II (II) in humans (page 40). 3. Primers used in RT-PCR, plasmid construction, primer extension, 5'-RACE, site-directed mutagenesis, EMSA, ultraviolet (UV) crosslinking, and Southwestern blot studies (pages 82-87). ix List of Figures 1. Amino acid sequences of 15 GnRH forms (page 2). 2. Structural organization and chromosomal localization of human GnRH-I and GnRH-II genes (page 5). 3. Amino acid sequence of the human GnRH-I receptor (pages 16-17). 4. The MAPK cascades (pages 32-33). 5. Activation of MAPKs by the GnRH-I receptor in gonadotrope-derived alphaT3-l cells (pages 34-35). 6. Structural organization of the human GnRH-I receptor gene (page 52). 7. Nucleotide sequence of the human GnRH-I receptor 5'-flanking region (pages 55-56). 8. Alignment of the amino acid sequence of the marmoset GnRH-II and human GnRH-I receptors (pages 68-69). 9. Diagram showing the distribution of the putative GnRH-II receptor, PEXlip, and RBM8 genes in the human genome (pages 72-73). 10. Diagrammatic representation of the three-step PCR mutagenesis method (pages 88-89). 11. Expression of GnRH-I receptor mRNA and protein in immortalized and primary-cultured human GL cells (page 103). 12. Progressive 5'-deletion analysis of the human GnRH-I receptor 5'-flanking region in SVOG-40 and SVOG-4m cells (pages 105-106). 13. Progressive 3'-deletion analysis of the human GnRH-I receptor 5'-flanking region in SVOG-4o and SVOG-4m cells (pages 107-108). 14. Fine mapping of the minimal human GnRH-I receptor promoter in SVOG-4o and SV0G-4m cells {pages 109-110). 15. Transcriptional activity of the upstream human GnRH-I receptor promoter (from -1300 to -1018) in different cell lines (pages 111-112). 16. Identification of the transcription start sites for the human GnRH-I receptor gene in SVOG-4o cells by primer extension analysis (pages 114-115). 17. Mutational analysis of the putative C/EBP and GATA motifs on the promoter activity of p(-1300/-1018)-Luc {pages 117-120). 18. EMS As to characterize the dC/EBP motif using nuclear extracts from human GL cells {pages 122-123). 19. EMS As to characterize the GATAa motif using nuclear extracts from human GL cells (pages 124-125). 20. EMSAs to characterize the pC/EBP motif using nuclear extracts from human GL cells {pages 126-127). 21. Diagrammatic representation of differential promoter usage of the human GnRH-I receptor gene in various cell types (page 132). 22. Silencing activity of the NRE on a heterologous TK promoter {pages 135-136). 23. The silencing activity of the NRE is position-independent (pages 138-139). 24. Progressive 3'-deletion analysis of the human GnRH-I receptor 5'-flanking region in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells {pages 140-141). 25. Fine deletion mapping of critical nucleotide sequences that mediate the silencing activity of the NRE in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells {pages 143-144). 26. Mutational analysis of the putative octamer regulatory sequence {pages 145-146). 27. EMS As to characterize the octamer binding sequence using nuclear extracts from different cell lines {pages 148-151). 28. Western and Southwestern blot analyses to confirm the binding of Oct-1 to the octamer sequence {page 152). 29. Overexpression of Oct-1 augments the silencing activity of the octamer sequence in alphaT3-l cells {pages 154-155). 30. ERct but not ER(3 mediates E2 repression of the human GnRH-I receptor promoter {pages 165-169). 31. Dose- and time-dependent effects of E2 on the human GnRH-I receptor promoter activity in ERa-overexpressing OVCAR-3 cells {pages 171-172). 32. ERa domains required for human GnRH-I receptor promoter repression overlap those for classical transactivation at ERE {pages 174-176). 33. Localization of the E2-response region to -266/-117 of the human GnRH-I receptor 5'-flanking region {pages 178-179). 34. An AP-l-like site at -130/-124 mediates both basal activity and E2 responsiveness of the human GnRH-I receptor promoter {pages 181-182). 35. Binding of the AP-1 transcription factors c-Jun and c-Fos but not ERa to the AP-l-like motif {pages 184-186). 36. Functional antagonism between the ER and AP-1 signaling pathways in regulating the human GnRH-I receptor gene transcription {pages 188-192). 37. Overexpression of the transcriptional coactivator CBP attenuates the E2-dependent repression of the human GnRH-I receptor promoter {pages 195-196). 38. Identification of the transcription start sites for the human GnRH-II gene in TE-671 and JEG-3 cells by primer extension analysis {pages 207-208). 39. Transcriptional activity of various regions of the human GnRH-II gene in TE-671 and JEG-3 cells {pages 210-212). 40. Fine mapping of the minimal human GnRH-II promoter in TE-671 and JEG-3 cells {pages 213-215). 41. The enhancer activity of exon 1 of the human GnRH-II gene is position- and orientation-dependent {pages 217-218). 42. Nucleotide sequence of the minimal human GnRH-II promoter {pages 220-221). 43. Mutational analysis of the putative EBS and ELE motifs on the enhancer activity of the untranslated first exon {pages 222-223). 44. EMS As to characterize the two functional EBSs using nuclear extracts from TE-671 and JEG-3 cells {pages 225-228). 45. Identification of a 48 kJDa-nuclear factor binding to the EBSs by TJV crosslinking and Southwestern blot analysis {pages 229-231). 46. In vitro translated human AP-4 proteins bind specifically to both EBSs {pages 232-234). 47. EMS As to characterize the ELE using nuclear extracts from TE-671 and JEG-3 cells {pages 236-237). xii i 48. Endogenous expression level of AP-4 regulates human GnRH-II promoter activity (pages 238-243). 49. Summary of the transcriptional regulation of the human GnRH-I receptor gene (pages 252-253). XIV List of Abbreviations AF, transactivation function AP-1, activator protein-1 ASK, apoptosis signal-regulated kinase bHLH, basic helix-loop-helix BMK, big MAPK bp, base pairs [Ca 2 +]i, intracellular Ca 2 + concentration cAMP, cyclic adenosine 5'-monophosphate CBP, CREB-binding protein CCV, clathrin-coated vesicle cDNA, complementary DNA C/EBP, CCAAT/enhancer-binding protein cGMP, cyclic guanosine 5'-monophosphate CRE, cAMP-responsive element CREB, CRE-binding protein DBD, DNA-binding domain DMEM, Dulbecco's Modified Eagle Medium DMSO, dimethyl sulfoxide DNA, deoxyribonucleic acid E2,17(3-estradiol EBS, E-box binding site XV EGF, epidermal growth factor Egrl, early growth response 1 ELE, Ets-like element EMSA, electrophoretic mobility shift assay ER, estrogen receptor ERE, estrogen-response element ERK, extracellular signal-regulated kinase FBS, fetal bovine serum FSH, follicle-stimulating hormone GAP, GnRH-associated peptide GAPDH, glyceraldehyde-3-phosphate dehydrogenase GL, granulosa-luteal GnRH, gonadotropin-releasing hormone GPCR, G protein-coupled receptor G protein, heterotrimeric guanine nucleotide-binding prot GR, glucocorticoid receptor GRAS, GnRH receptor activating sequence GSE, gonadotrope-specific element GTP, guanosine triphosphate h, hour hCG, human chorionic gonadotropin HDF, human dermal fibroblast HIV-1, human immunodeficiency virus type 1 x v i IEVT, immortalized extravillous trophoblast IGF, insulin-like growth factor IL, interleukin Inr, initiator INK, c-Jun amino-terminal kinase kb, kilobases kDa, kilodaltons LBD, ligand-binding domain LH, luteinizing hormone MAPK, mitogen-activated protein kinase MEF-2, myocyte enhancer factor 2 MEK, MAPK/ERK kinase MEKK, MEK kinase min, minute MKK, MAPK kinase MKKK, MAPK kinase kinase MMP, matrix metalloproteinase MNK, MAPK-interacting kinase mRNA, messenger RNA N F - K B , nuclear factor-KB NF-Y, nuclear factor-Y NRE, negative regulatory element OSE, ovarian surface epithelial xvii P4, progesterone p90RSK, 90-kDa ribosomal protein S6 kinase PACAP, pituitary adenylate cyclase-activating polypeptide PAGE, polyacrylamide gel electrophoresis PAI, plasminogen activator inhibitor PAK, p21-activated kinase PARE, PACAP-response element PEX1 ip, peroxisomal membrane protein 11-P PKA, protein kinase A PKC, protein kinase C PMA, phorbol-12-myristate 13-acetate poly(A), polyadenylated PR, progesterone receptor PRE, progesterone-response element PTP, phosphotyrosine phosphatase PTX, pertussis toxin RACE, Rapid Amplification of cDNA End RNA, ribonucleic acid RSV, Rous sarcoma virus RT-PCR, reverse transcription-polymerase chain reaction s, second SDS, sodium dodecyl sulfate SEM, standard error of the mean x v i i i SERM, selective ER modulator SF-1, steroidogenic factor 1 SMRT, silencing mediator for retinoid and thyroid hormone receptors SV, simian virus TAB, TAK1 -binding protein TAK, transforming growth factor p-activated protein kinase TBP, TATA-binding protein TFII, transcription factor class II TK, thymidine kinase TM, transmembrane tRNA, transfer RNA UAS, upstream activation sequence uPA, urokinase-type plasminogen activator USF, upstream stimulatory factor UTR, untranslated region UV, ultraviolet vs., versus xix Preface Published papers: 1. Cheng CK, Chow BK, Leung PC 2003 An AP-l-like motif mediates 17p-estradiol repression of GnRH receptor promoter via an estrogen receptor cx-dependent mechanism in ovarian and breast cancer cells. Mol Endocrinol. In press. 2. Leung PC, Cheng CK 2003 The role of GnRH as an autocrine regulator in the human ovary. In: THE OVARY, Second Edition. Academic Press; In press. 3. Cheng CK, Hoo RL, Chow BK, Leung PC 2003 Functional cooperation between multiple regulatory elements in the untranslated exon 1 stimulates the basal transcription of the human GnRH-II gene. Mol Endocrinol 17:1175-1191. 4. Leung PC, Cheng CK, Zhu XM 2003 Multi-factorial role of GnRH-I and GnRH-II in the human ovary. Mol Cell Endocrinol 202:145-153. 5. Cheng CK, Yeung CM, Hoo RL, Chow BK, Leung PC 2002 Oct-1 is involved in the transcriptional repression of the gonadotropin-releasing hormone receptor gene. Endocrinology 143:4693-4701. 6. Cheng CK, Yeung CM, Chow BK, Leung PC 2002 Characterization of a new upstream GnRH receptor promoter in human ovarian granulosa-luteal cells. Mol Endocrinol 16:1552-1564. 7. Cheng KW, Cheng CK, Leung PC 2001 Differential role of PR-A and -B isoforms in transcription regulation of human GnRH receptor gene. Mol Endocrinol 15:2078-2092. Published abstracts: 1. Cheng CK, Yeung CM, Hoo RL, Chow BK, Leung PC 2002 A role of Oct-1 in the transcriptional repression of the gonadotropin-releasing hormone receptor gene. The 2nd Pacific Conference on Reproductive Biology and Environmental Sciences. Kyoto, Japan (Poster presentation). 2. Cheng CK, Leung PC 2001 Characterization of human gonadotropin-releasing hormone receptor promoter in human granulosa cells. 47th Annual Meeting of the Canadian Fertility and Andrology Society. Whistler, Canada (Oral presentation). XXI Acknowledgements I would like to express my greatest gratitude to Dr. Peter C. K. Leung for his endless support and experienced training during the course of my Ph.D. study. In addition, I would like to thank my wife, parents, and other family members for their continuous encourage throughout this period. I also wish to thank Dr. Billy K. C. Chow for his valuable scientific advice and all my dearest colleagues especially Dr. K. W. Cheng, Dr. C. M. Yeung, and K. Y. Kim for their technical support. Lastly, I would like to thank the Genesis Fertility Centre (Vancouver, Canada), Dr. A. A. Karande, Dr. P. L. Mellon, Dr. V. Giguere, Dr. H. Singh, Dr. B. S. Katzenellenbogen, Dr. D. P. McDonnell, Dr. P. Chambon, Dr. J. L. Jameson, Dr. R. H. Goodman, Dr. R. Tjian, L. T. O. Lee, and Y. Y. Kwok for providing the human GL cells, antibodies, cell lines, and plasmid constructs used in this study. x x i i C H A P T E R I Introduction 1.1 GnRH: Overview Mammalian GnRH (termed GnRH-I) is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) that plays a key role in the process of reproduction. It is produced by hypothalamic neurosecretory cells and released in a pulsatile manner into the hypothalamo-hypophyseal portal circulation, via which the hormone is transported to the anterior pituitary gland. After binding to its high-affinity receptor on pituitary gonadotropes, the hormone stimulates the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn regulate gonadal steroidogenesis and gametogenesis in both sexes (Fink, 1988). In addition to this well-known endocrine function, GnRH-I has been shown to be an important autocrine and/or paracrine regulator in some extrapituitary tissues (Bussenot et al, 1993; Lin et al, 1995; Chegini et al, 1996; Emons et al, 1998; Kang et al, 2000c; Chen et al, 2002a; Chou et al, 2002). Since its discovery some thirty years ago, about a thousand GnRH-I analogs have been developed and studied extensively (Conn and Crowley, 1994). Clinically, some of these synthetic analogs have been used as an effective treatment for a variety of reproductive endocrinopathies such as prostate cancer (Gommersall et al, 2002), whereas others have been adopted as a component of the regimen for ovulation induction in women undergoing in vitro fertilization (Fauser et al, 1999). Until now, more than a dozen isoforms of GnRH sharing 10-50 % amino acid identity have been found in vertebrates (Sherwood et al, 1993; Dubois et al, 2002) (Figure 1). It is generally believed that most vertebrate species possess at least two, and 1 Figure 1. Amino acid sequences of 15 GnRH forms. Residues that differ from those of the mammalian GnRH are typed in bold. G n R H 1 2 3 4 5 6 7 8 9 10 Mammalian pGlu His Trp Ser Tyr Gly Leu Arg Pro G l y - N H 2 Chicken II pGlu His Trp Ser His Gly Trp Tyr Pro G l y - N H 2 Rana pGlu His Trp Ser Tyr Gly Leu Trp Pro Gly-NHz Catfish pGlu His Trp Ser His Gly Leu Asn Pro G l y - N H 2 Salmon pGlu His Trp Ser Tyr Gly Trp Leu Pro G l y - N H 2 Herring pGlu His Trp Ser His Gly Leu Ser Pro G l y - N H 2 Medaka pGlu His Trp Ser Phe Gly Leu Ser Pro G l y - N H 2 Seabream pGlu His Trp Ser Tyr Gly Leu Ser Pro G l y - N H 2 Dogfish pGlu His Trp Ser His Gly Trp Leu Pro G l y - N H 2 Chicken 1 pGlu His Trp Ser Tyr Gly Leu Gin Pro G l y - N H 2 Guinea pig pGlu Tyr Trp Ser Tyr Gly Val Arg Pro G l y - N H 2 Lamprey 1 pGlu His Tyr Ser Leu Glu Trp Lys Pro G l y - N H 2 Lamprey III pGlu His Trp Ser His Asp Trp Lys Pro G ly -NH 2 Tunicate 1 pGlu His Trp Ser Asp Tyr Phe Lys Pro G ly -NH 2 Tunicate II pGlu His Trp Ser Leu Cys His Ala Pro G l y - N H 2 2 usually three forms of GnRH, which differ in their amino acid sequences, localization, and embryonic origins. In addition to GnRH-I, a second subtype of GnRH (termed GnRH-II), originally identified from chicken hypothalamus (Miyamoto et al, 1984), has been found in humans. This second GnRH form differs from GnRH-I by three amino acid residues at positions 5, 7, and 8 (His5Trp7Tyr8GnRH-I) and is conserved over all vertebrate classes from primitive fishes to humans (Sherwood et al, 1993; White et al, 1998). One of the established biological functions specific to GnRH-II is to serve as a potent inhibitor of K + channels in amphibian sympathetic ganglia (Bosma et al, 1990). Inhibition of these K + channels by GnRH-II facilitates fast excitatory transmission by conventional neurotransmitters (Bosma et al, 1990). This phenomenon may provide a general neuromodulatory mechanism for GnRH-II in the nervous system, and specifically in reproductive behavior, by enhancing neurotransmitter signaling. Recently, Temple and coworkers have shown that GnRH-II, but not GnRH-I, activates mating in energetically challenged musk shrews (Temple et al, 2003), and they suggest a role of the evolutionarily conserved GnRH form in coordinating energy and reproductive behavior. In humans, a growing number of extrapituitary GnRH-II functions such as suppression of tumor proliferation, have been demonstrated although the full-length GnRH-II receptor transcript has not yet been identified. 1.2 Human GnRH-I Complementary Deoxyribonucleic Acid (cDNA) Cloning of GnRH-I cDNAs from human hypothalamus (Adelman et al, 1986) and placenta (Seeburg and Adelman, 1984) revealed that they possess identical coding and 3'-untranslated regions (3'-UTRs). However, the placental cDNA has a much 3 longer 5'-UTR than the hypothalamic one, because of the inclusion of the first intron in the transcript (Adelman et al, 1986; Radovick et al, 1990). The coding region of the GnRH-I cDNA contains an open reading frame of 276 base pairs (bp), which encodes a precursor protein of 92 amino acids. Immediately followed the reading frame are 160 nucleotides of the 3'-UTR, which contain an AATAAA sequence for polyadenylation shortly upstream of a polyadenylated [poly(A)] tail. The first 23 amino acids form the signal sequence features the typical hydrophobic middle section (Perlman and Halvorson, 1983) and ends in two serines followed by the GnRH-I decapeptide. Cleavage of the signal peptide generates an amino-terminal glutamic acid residue, which spontaneously or enzymatically undergoes cyclization to pyroglutamic acid. The decapeptide is followed by a Gly-Lys-Arg sequence and a 56-amino acid peptide termed GnRH-associated peptide (GAP). The Gly-Lys-Arg sequence signals amidation of the carboxyl terminus and enzymatic cleavage of the decapeptide from the precursor. In vitro experiments have demonstrated that the GAP inhibits the release of prolactin but stimulates that of gonadotropins in rat pituitary cell cultures (Nikolics et al, 1985), indicating that the hypothalamic control of gonadotropin and prolactin secretion is coupled through the synthesis of a common precursor protein by neurosecretory cells. 1.3 Human GnRH-I Gene The human GnRH-I gene is composed of four exons separated by three introns and has been assigned as a single gene copy to the short arm of chromosome 8 (region 8pl 1.2-p21) by in situ hybridization and Southern blot analysis (Yang-Feng et al, 1986) (Figure 2). The first exon of the gene is untranslated and consists of 61 bp in ribonucleic 4 Human GnRH-I gene (Chromosome 8p11.2-p21) I 869 bp —II— \ 1.5 kb 2.1 kb —II— 61 bp 142 bp 96 bp 201 bp Human GnRH-II gene (Chromosome 20p13) 741 bp —//— 109 bp 844 bp Z - / / -44 bp Exon 1 161 bp Exon 2 158 bp Exon 3 60 bp Exon 4 • 5'-UTR • Signal sequence BGnRH 0GAP LT3 3'-UTR Figure 2. Structural organization and chromosomal localization of human GnRH-I and GnRH-II genes. Boxes represent exons and thin lines represent introns. The size of each exon and intron is indicated. Both human GnRH-I and GnRH-II genes consist of four exons separated by three introns, and their encoded preprohormones are organized identically such that they all have a signal sequence, followed by a GnRH decapeptide, a conserved Gly-Lys-Arg cleavage site, and a GAP. These genes are present as a single copy in different chromosomal regions in the human genome. The diagram is not drawn to scale. acid (RNA) expressed in the hypothalamus. The second exon encodes the signal sequence, the GnRH-I decapeptide, the Gly-Lys-Arg processing signal, and the first 11 GAP residues. The third exon codes for the next 32 GAP residues, and the fourth exon contains the remaining 13 GAP residues, the translation termination codon, as well as the entire 3'-UTR (Adelman et al, 1986; Radovick et al, 1990). 1.4 Human GnRH-II cDNA and Gene The gene encoding human GnRH-II has been cloned and mapped to chromosome 20pl3 by fluorescence in situ hybridization (White et al, 1998). Nucleotide sequencing revealed that the GnRH-II gene is also comprised of four exons and three introns and that the predicted preprohormone is organized identically to the GnRH-I precursor such that they all have a signal sequence, followed by a GnRH decapeptide, a conserved proteolytic Gly-Lys-Arg site, and a GAP (Figure 2). However, the human GnRH-II gene [2.1 kilobases (kb)] is remarkably shorter than the GnRH-I gene (5 kb) because introns 2 and 3 of the latter are much larger. Moreover, although their corresponding precursor proteins are quite similar in length, the GAP is 50 % longer in human GnRH-II than in human GnRH-I [84 versus (vs.) 56 amino acids]. In fact, a similar disparity in GAP has also been reported in the placental mammal, tree shrew (76 vs. 56 amino acids) (Kasten et al, 1996), indicating that a relatively larger GAP may be a common characteristic among mammalian GnRH-II precursors. 6 1.5 Tissue Distribution of Human GnRH-I and GnRH-II Gene Transcripts 1.5.1 Brain The most prominent difference in the tissue distribution of the GnRH-I and GnRH-II gene transcripts in humans is that the latter is expressed at significantly higher levels outside the brain. For instance, the expression level of GnRH-II in the kidney is 30-fold higher than that in any brain region, whereas a 4-fold higher expression level is detected in the bone marrow and prostate gland (White et al, 1998). Conversely, GnRH-I expression is not observed at a high level outside the brain (White et al, 1998). In humans, immunocytochemcial studies have revealed a considerable amount of GnRH-I neurons in both the external and internal zones of the median eminence (Anthony et al, 1984). In addition, a large number of GnRH-I immunoreactive fibers have been found to traverse the infundibular stalk and enter the neural lobe of the anterior pituitary gland (Anthony et al, 1984). Furthermore, substantial extrahypothalamic projections to the posterior pituitary, habenular complex, and amygdale have been observed (Anthony et al, 1984; King and Anthony, 1984). Interestingly, using in situ reverse transcription-polymerase chain reaction (RT-PCR), GnRH-I messenger RNA (mRNA) has been demonstrated in normal human pituitary as well as in various types of pituitary adenomas (Miller et al, 1996; Sanno et al, 1997). The molecular mechanism underlying neuron-specific expression of the human GnRH-I gene has been studied recently. By means of deletion analysis, a region between -992 and -795 of the GnRH-I gene is found to be essential and sufficient for targeting luciferase expression in the hypothalamus and olfactory tissue in vivo (Wolfe et al, 2002). Two specific DNA-binding sites for the POU homeodomain transcription 7 factors Brn-2 and Oct-1 are identified in this regulatory region, and functional studies by overexpressing Brn-2 reveal that the transcription factor can transactivate both the human and mouse GnRH-I promoters (Wolfe et al, 2002). Thus, these findings strongly support a role of the POU homeodomain proteins in regulating GnRH-I gene transcription in GnRH-I-producing neurons. In human and rodent brains, immunoreactive GnRH-II peptides are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain (Chen et al, 1998). In addition, lower expression levels are detected in the hypothalamus, medulla oblongata, and pons (Chen et al, 1998). A similar distribution pattern of GnRH-II immunoreactivity has also been reported in amphibians, where the neuropeptide is restricted to the midbrain tegmentum (Conlon et al, 1993). RT-PCR and Southern blot analysis reveal that two GnRH-II mRNA variants are expressed in human fetal brain and adult thalamus (White et al, 1998). These transcripts differ in the size of their GAPs, which are predicted to contain 77 and 84 amino acid residues. Interestingly, this alternative splicing process appears to be tissue-specific since the longer GnRH-II transcript is not found in other organs such as the adult kidney (White et al, 1998). 1.5.2 Placenta It has long been known that human placenta in vitro synthesizes and releases GnRH-I that is immunologically indistinguishable from the hypothalamic counterpart (Siler-Khodr and Khodr, 1978; Siler-Khodr and Khodr, 1979; Khodr and Siler-Khodr, 1980). Using immunocytochemistry, GnRH-I has been found to colocalize with its 8 receptor in both cytotrophoblasts and syncytiotrophoblasts (Wolfahrt et al, 1998). Consistently, a steady temporal expression of GnRH-I mRNA has been demonstrated in the trophoblast layers as well as in the stroma (Kelly et al, 1991; Wolfahrt et al, 1998). Recently, Siler-Khodr and Grayson have revealed that GnRH-II is also released from human placenta in vitro and that this second GnRH form is more resistant than GnRH-I to degradation by placental enzymes (Siler-Khodr and Grayson, 2001). 1.5.3 Uterus Expression of GnRH-I mRNA has been demonstrated in virtually all human uterine compartments (Irmer et al, 1994; Chatzaki et al, 1996; Chegini et al, 1996; Kobayashi et al, 1997; Dong et al, 1998; Raga et al, 1998). Interestingly, a dynamic expression pattern is observed in the endometrium as well as in isolated endometrial stromal and epithelial cells during the menstrual cycle such that a significant increase in transcript level occurs in the secretory phase (Dong et al, 1998; Raga et al, 1998). In support of this observation, GnRH-I immunoreactivity has been localized in all endometrial cell types, with the most intense staining being detected in the luteal phase (Raga et al, 1998). The spatiotemporal expression of the GnRH-II gene has also been investigated in human endometrium recently. Throughout the entire reproductive cycle, two splice variants of GnRH-II mRNA are found to be expressed, with the shorter transcript carrying a 21-bp deletion, which reduces the length of GAP from 77 to 70 amino acids (Cheon et al, 2001). Like GnRH-I, GnRH-II immunoreactivity is dynamically expressed in both stromal and glandular epithelial cells such that stronger signals are 9 detected in the early and mid-secretory phases than in the proliferative and late-secretory phases (Cheon et al, 2001). 1.5.4 Ovary Expression of GnRH-I and GnRH-II mRNAs that are identical to their brain counterparts has been demonstrated in various human ovarian compartments including GL cells, ovarian surface epithelial (OSE) cells, as well as in ovarian carcinoma (Ohno et al, 1993; Peng et al, 1994; Irmer et al, 1995; Kang et al, 2000a; Kang et al, 2000c; Choi et al, 2001; Kang et al, 2001c). In addition, a cycle-dependent expression of GnRH-I mRNA and protein has been reported in human fallopian tube, where the hormone is localized in the tubal epithelium (Casan et al, 2000). 1.5.5 Other Tissues A recent study from Chen and coworkers has indicated that two forms of GnRH are expressed in normal human breast tissue and are overexpressed in breast cancer (Chen et al, 2002b). In addition, certain immune cell populations such as T lymphocytes and peripheral blood mononuclear cells have been shown to produce both GnRH-I and GnRH-II (Azad et al, 1997; Chen et al, 1999a; Chen et al, 2002a). Expression of the GnRH-I gene has also been demonstrated in human preimplantation embryo, in which GnRH-I immunoreactivity is present in all blastomeres as well as in the trophectoderm and inner cell mass of the blastocyst (Casan et al, 1999). The findings that GnRH-I is expressed in the preimplantation embryo, endometrium (Dong et al, 1998; Raga et al, 1998), and oviduct (Casan et al, 2000) strongly support an 10 autocrine and/or paracrine role of the hormone in human fertilization, early embryonic development, and implantation. 1.6 Regulation of Human GnRH-I and GnRH-II Gene Expression Expression of human GnRH-I and GnRH-II genes is regulated by a wide variety of endocrine hormones as well as autocrine and paracrine factors, which functionally interact with each other in a highly coordinated fashion. Interestingly, there is growing evidence that the expression of these decapeptides is differentially controlled by their own ligands, gonadal steroids, and gonadotropins (Table 1), and a number of studies have indicated that this occurs at the transcriptional level. 1.6.1 GnRH-I and GnRH-II In human OSE, GL, and ovarian cancer OVCAR-3 cells, treatment with GnRH-I produces a biphasic effect on its mRNA level such that high concentrations decrease whereas low concentrations increase the expression (Peng et al, 1994; Kang et al, 2000a; Kang et al, 2000c; Kang et al, 2001c). In contrast, GnRH-I dose-dependently suppresses its own expression in peripheral blood mononuclear cells (Chen et al, 1999a). Heterologous regulation of GnRH-I gene expression has only been studied in human GL cells, where GnRH-II causes a downregulation of GnRH-I mRNA level at a wide range of concentrations (Kang et al, 2001c). 11 Table 1. Summary of differential regulation of human GnRH-I and GnRH-II gene expression. Treatment Cell type GnRH-I expression GnRH-II expression References G n R H - I G L Biphasic ( m R N A ) Not determined Peng et al, 1994; Kang et al, 2001c O S E Biphasic ( m R N A ) Not determined K a n g etal, 2000c O V C A R - 3 Biphasic ( m R N A ) Not determined K a n g et al, 2000a G n R H - I I G L Decrease ( m R N A ) No t determined K a n g et al, 2001c E 2 G L Decrease ( m R N A ) Increase ( m R N A ) Nathwani et al, 2000; Khosrav i and Leung, 2003 O V C A R - 3 Decrease ( m R N A ) No t determined K a n g etal, 2001a TE-671 Decrease ( m R N A ) Increase ( m R N A ) Chen et al, 2002c R U 4 8 6 G L N o effect Increase ( m R N A ) Khosrav i and Leung, 2003 Gonadotropins G L Decrease ( m R N A ) Increase ( m R N A ) K a n g e / al, 2001c c A M P TE-671 M i l d increase ( m R N A and protein) Drastic increase ( m R N A and protein) Chen et al, 2001a 12 1.6.2 Gonadal Steroid Hormones In addition to their ligands, the expression of the GnRH-I and GnRH-II genes is differentially regulated by gonadal steroids. It has been demonstrated that the steady-state GnRH-I mRNA level is downregulated by E2 in human GL, OVCAR-3, and neuronal TE-671 cells (Nathwani et al, 2000; Kang et al, 2001a; Chen et al, 2002c; Khosravi and Leung, 2003). This E 2 action is believed to be mediated via the nuclear ER since cotreatment with the antiestrogen tamoxifen can abolish the inhibitory effect (Nathwani et al, 2000; Kang et al, 2001a; Khosravi and Leung, 2003). Using the ER-negative CHO-K1 cell line as a model, Chen and coworkers have revealed that E2 can dose-dependently repress the human GnRH-I promoter when ERa is overexpressed (Chen et al, 1999b). Also, the authors have located the estrogen responsiveness to a region between 169 and 548 bp 5' of the upstream transcription start site of the GnRH-I gene (Chen et al, 1999b). In contrast, our laboratory has recently found that in human GL cells, E2 increases the GnRH-II mRNA level in a dose- and time-dependent manner (Khosravi and Leung, 2003). Similarly, a stimulatory effect of the estrogen on GnRH-II gene expression has been reported in TE-671 cells (Chen et al, 2002c). The role of progesterone (P4) in regulating GnRH-I and GnRH-II gene expression has been investigated in human GL cells. While treatment with the progesterone receptor (PR) antagonist RU486 does not affect GnRH-I mRNA level, the content of GnRH-II transcript is dose- and time-dependently stimulated by the antagonist (Khosravi and Leung, 2003), indicating that P4 has an inhibitory effect on GnRH-II gene expression in the ovary. However, it should be noted that the effect of gonadal steroids on GnRH gene expression may be tissue-specific, because it has been 13 demonstrated that cotreatment with E2 and P4 can upregulate GnRH-I secretion in human olfactory cells (Barni et al, 1999), which share a common origin with GnRH-I neurons during organogenesis (Schwanzel-Fukuda and Pfaff, 1989; Wray et al, 1989). 1.6.3 Gonadotropins Further evidence that the expression of the two forms of GnRH is differentially modulated comes from studies of their regulation by gonadotropins, which mediate their actions by stimulating the production of intracellular cyclic adenosine 5'-monophosphate (cAMP) and activating the protein kinase A (PKA) signal transduction pathway. In human GL cells, treatment with FSH or human chorionic gonadotropin (hCG) upregulates the mRNA level of GnRH-II but downregulates that of GnRH-I in a dose-dependent manner (Kang et al, 2001c). Consistently, an increase in GnRH-II mRNA and protein contents in response to cAMP has been observed in TE-671 cells using RT-PCR and immunocytochemical analysis (Chen et al, 2001a). This cAMP-activated GnRH-II gene expression is promoter-mediated since mutation of a putative CRE in the human GnRH-II 5'-flanking region drastically reduces both the cAMP-stimulated and basal promoter activities (Chen et al, 2001a). 1.6.4 Other Physiological Regulators Recently, Huang and coworkers have demonstrated that interleukin (IL)-1B can dose-dependently upregulate GnRH-I mRNA levels in human endometrial stromal cells via a receptor-mediated process (Huang et al, 2003). Since the IL-1 system is known to be a major factor in embryo implantation and decidualization of stromal cells (Kauma et 14 al, 1990; Simon et al, 1993; Simon et al, 1994; De los Santos et al, 1996; De los Santos et al, 1998), this finding further indicates a local action of GnRH-I in the preparation of pregnancy. Also, an increase in GnRH-I gene transcription has been observed in neuronal NLT cells following insulin-like growth factor (IGF)-I treatment, and a consensus AP-1 motif in the proximal promoter region is shown to mediate this effect (Zhen et al, 1997). Moreover, peptides of the endothelin family have been demonstrated to stimulate GnRH-I release from human olfactory cells via activation of the endothelin A receptor (Maggi et al, 2000). 1.7 GnRH-I Receptor cDNAs 1.7.1 Cloning The cDNA for the GnRH-I receptor was first cloned and characterized from the mouse gonadotrope-derived cell line alphaT3-l using a PCR-based homology cloning strategy (Tsutsumi et al, 1992). Elucidation of the mouse sequence has led to the identification of the homologous pituitary GnRH-I receptor cDNAs in five additional mammalian species including human (Kakar et al, 1992; Chi et al, 1993), rat (Eidne et al, 1992; Kaiser et al, 1992; Perrin et al, 1993), sheep (Brooks et al, 1993; Illing et al, 1993), bovine (Kakar et al, 1993), and pig (Weesner and Matteri, 1994). The predicted amino acid sequences for the GnRH-I receptors are more than 85 % conserved among the six species and are nearly identical within their putative transmembrane (TM) domains. The human receptor cDNA contains an open reading frame of 987 bp, which encodes a single polypeptide chain of 328 amino acid residues (Figure 3), whereas the mouse and rat receptors are composed of 327 amino acids owing to the absence of a 15 Figure 3. Amino acid sequence of the human GnRH-I receptor. The receptor is composed of a single polypeptide chain, with seven hydrophobic TM domains connected by a series of extracellular and intracellular loops. Unlike other GPCRs, the GnRH-I receptor lacks a carboxyl-terminal tail. The known glycosylation site at residue 18 in the extracellular ammo-terminal tail is marked, and certain key functional residues are numbered according to a consensus numbering scheme described elsewhere (Ballesteros and Weinstein, 1995; Sealfon et al, 1997). The unusual modification of the highly conserved Asp-Arg-Tyr (DRY) to Asp-Arg-Ser (DRS) in the 2nd intracellular loop as well as the unusual exchange of conserved aspartate (D) and asparagine (N) residues in the 2nd and 7th TM domains are indicated with white arrows. The shaded area represents the plasma membrane. 16 17 residue in the second extracellular loop. Analysis of the GnRH-I receptor's primary sequence reveals that the receptor is a member of the rhodopsin-like heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) family. Three distinct families of GPCRs have been identified by molecular cloning, and they include the metabotropic glutamate receptors (Tanabe et al, 1992; Pin and Duvoisin, 1995), the secretin-calcitonin-parathyroid hormone class (Ishihara et al, 1991; Lin et al, 1991; Abou-Samra et al, 1992; Chen et al, 1993; Juppner, 1994), and the large rhodopsin-like GPCR superfamily (Probst et al, 1992; Strader et al, 1995). When sequences of the members of the three classes of GPCRs are analyzed for hydrophobicity, all are found to contain seven putative TM domains though they do not share any noticeable sequence homology. Included in the rhodopsin-like GPCR subfamily are opsins, G protein-coupled neurotransmitter receptors, glycoprotein hormone receptors, and a variety of peptide receptors (Probst et al, 1992; Strader et al, 1995). 1.7.2 General Structural Features Biochemical studies of the GnRH-I receptor have indicated that it is a sialic acid residue-containing glycoprotein (Hazum, 1982; Schvartz and Hazum, 1985). Two putative N-linked glycosylation sites (Asn-X-Ser/Thr) (X can be any amino acid except Pro), one in the amino terminus and the other in the first extracellular domain, are present in the human, bovine, and ovine receptors, whereas the rodent receptors possess an additional glycosylation site at the amino terminus (Stojilkovic et al, 1994; Sealfon et al, 1997). Site-directed mutagenesis and photoaffmity labeling have indicated that only the amino-terminal sites undergo glycosylation, which plays a role in receptor expression 18 level but not in receptor binding affinity (Keinan and Hazum, 1985; Schvartz and Hazum, 1985). Also, it has been shown that glycosylation is not required for proper transport of the receptor to the cell surface (Davidson et al, 1995). Several cytoplasmic serine and threonine residues are present in the intracellular domains of the GnRH-I receptor and may serve as regulatory phosphorylation sites for various protein kinases. Like most GPCRs that contain conserved cysteines in the first and second extracellular loops, a conserved cystine bridge between Cys 1 1 4 and Cys 1 9 6 has been demonstrated in the mouse receptor. In addition, evidence for a second disulfide bond between Cys1 4 and Cys 2 0 0 has been obtained for the human receptor (Sealfon et al, 1997). These covalent modifications are important for proper receptor folding as mutation of these conserved residues disrupts the function of the rhodopsin, muscarinic, [^ -adrenergic, and thyrotropin-releasing hormone receptors (Dixon et al, 1987; Karnik et al, 1988; Fraser, 1989; Savarese et al, 1992; Perlman et al, 1995). 1.7.3 Unique Structural Features Unlike other members of the GPCRs, there is a modification of the highly conserved Asp-Arg-Tyr triplet, which has been implicated in G protein coupling, to Asp-Arg-Ser in the proximal area of the second intracellular domain of the GnRH-I receptor (Stojilkovic et al, 1994; Sealfon et al, 1997). Another highly unusual feature of the GnRH-I receptor is the exchange of conserved aspartate and asparagine residues that are present in the second and seventh TM domains of most other GPCRs (Stojilkovic et al, 1994; Sealfon et al, 1997). Thus, an aspartate residue in the second TM domain, which is essential for normal agonist binding and G protein coupling in 19 many receptors (Probst et al, 1992), is replaced by an asparagine in the GnRH-I receptor. Also, there is a reverse substitution in the seventh TM domain, in which an aspartate replaces a highly conserved asparagine residue. Nevertheless, the most striking structural feature of the GnRH-I receptor is the absence of the entire intracellular carboxyl-terminal tail, which is important for receptor desensitization and internalization (Heding et al, 1998; Willars et al, 1999). Conversely, recent cloning of the GnRH receptor from catfish (Tensen et al, 1997), goldfish (Idling et al, 1999), frog (Troskie et al, 2000), and chicken (Troskie et al, 1998) has revealed that the nonmammalian receptors possess a carboxyl-terminal domain and can undergo rapid desensitization following agonist stimulation (Heding et al, 1998). Other noteworthy properties of the GnRH-I receptor are its unusually long first intracellular loop with a high content of basic amino acids and its phenylalanine-rich seventh TM domain (Stojilkovic et al, 1994). 1.8 Tissue Distribution of Human GnRH-I Receptor Gene Transcripts 1.8.1 Pituitary The availability of the GnRH-I receptor cDNA has made it possible to study the tissue distribution and the steady-state level of mRNA encoding the receptor. Northern blot analysis of human pituitary poly(A) RNA has revealed a predominant transcript of 4.7-5 kb as well as two fainter bands of 2.5 and 1.5 kb (Chi et al, 1993; Kakar, 1997). Subsequent PCR and nucleotide sequencing indicated that these transcripts contain the full-length coding sequence and are all correctly spliced (Kakar, 1997). The presence of multiple species of GnRH-I receptor mRNA is not unique to human as it has also been 20 reported in other mammalian species such as bovine (Kakar et al, 1993), rat (Kakar et al, 1994a), mouse (Reinhart et al, 1992; Tsutsumi et al, 1992), and sheep (Ming et al, 1993; Turzillo et al, 1994; Wu et al, 1994). In addition to the normal pituitary, expression of GnRH-I receptor mRNA has been demonstrated in certain pituitary adenomas including those derived from gonadotropes as well as growth hormone- and adrenocorticotropic hormone-producing cells (Sanno et al, 1997). Two splice variants of GnRH-I receptor mRNA, termed sb2 and sb3, have been identified in human pituitary (Kottler et al, 1999). The shorter fragment sb3 contains a 220-bp deletion between nucleotides 523 and 742, which causes a frameshift in the coding sequence and generates a translation stop codon immediately downstream of the deletion. This mRNA species encodes a protein of only 177 amino acids, lacking the last four TM domains, the second and third extracellular loops, and the third intracellular loop. The other splice variant sb2 carries a shorter deletion of 128 bp between nucleotides 523 and 650, arising from alternative splicing by accepting a cryptic acceptor site in exon 2. This deletion generates a frameshift in the open reading frame, leading to the production of a truncated protein of 249 amino acids such that the glutamine residue at position 174 is followed by a stretch of 75 new residues. The expression of these splice variants is tissue-specific as they are only found in normal pituitary, pituitary adenoma, and granulosa tumor (Kottler et al, 1999). Interestingly, it has been demonstrated that though sb2 exhibits a membranous expression pattern, the splice variant fails to show any ligand binding and signal transduction capabilities (Grosse et al, 1997). Rather, the splice variant impairs insertion of the wild-type receptor into the plasma membrane and reduces agonist-induced responses when 21 coexpressed with the full-length receptor cDNA. These inhibitory effects are dependent on the amount of the splice variant cDNA cotransfected and are specific for the GnRH-I receptor as signaling via other GPCRs such as thromboxane A2, M5 muscarinic, and VI vasopressin receptors is not affected (Grosse et al, 1997). The distribution of GnRH-I receptor immunoreactivity in normal and tumorous human pituitary has also been determined. In normal adenohypophysis, immunoreactive GnRH-I receptor colocalizes with a-subunit-, FSH(3-, LHP-, thyroid-stimulating hormone P-, and growth hormone-producing cells (La Rosa et al, 2000), indicating that the receptor is expressed in gonadotropes, thyrotropes, as well as in somatotropes. Consistent with the mRNA expression pattern in tumorous pituitary, GnRH-I receptor immunoreactivity is frequently detected in adenomas derived from gonadotropes, somatotropes, and a-subunit/null-cells (Sanno et al, 1997; La Rosa et al, 2000). 1.8.2 Placenta Using in situ hybridization, expression of GnRH-I receptor has been detected in both the cytotrophoblast and syncytiotrophoblast cell layers of human placenta (Lin et al, 1995). The temporal expression of the receptor in the placenta parallels the time course of hCG secretion during pregnancy such that the mRNA signal is abundant at 6 weeks, peaks at 9 weeks, declines at 12 and 20 weeks, and then becomes undetectable at term (Lin et al, 1995). Importantly, these changes in receptor mRNA level correlate with the number of GnRH-I receptors in the placental cells (Bramley et al, 1994). The full-length GnRH-I receptor cDNA has been cloned from various human placental cell types, and nucleotide sequencing reveals that the placental receptor shares 22 complete identity with the pituitary counterpart (Cheng et al, 2000a). Northern blot analysis has shown that 2.5- and 1.2-kb transcripts, instead of the major 4.7-5-kb one detected in the pituitary, are expressed in the placental cells (Cheng et al, 2000a). 1.8.3 Ovary High-affinity binding sites specific for GnRH-I have been detected in human corpus luteum, luteinized granulosa cells, epithelial ovarian carcinoma, and a number of ovarian cancer cell lines (Bramley et al, 1987; Emons et al, 1989; Pahwa et al, 1989; Emons et al, 1993a; Brus et al, 1997). Interestingly, an additional type of GnRH-I binding site, which is of low affinity but high capacity, has also been found in the epithelial ovarian cancer cell lines EFO-21 and EFO-27 (Emons et al, 1993a). Using RT-PCR and Southern blot analysis, expression of GnRH-I receptor mRNA that is indistinguishable from the pituitary counterpart has been demonstrated in various ovarian compartments (Kakar et al, 1994b; Peng et al, 1994; Irmer et al, 1995; Kang et al, 2000c; Choi et al, 2001; Volker et al, 2002) though quantitative measurements indicate that the mRNA level is much lower in the ovary than in the pituitary (Minaretzis et al, 1995; Fraser et al, 1996). 1.8.4 Uterus Similar to EFO-21 and EFO-27 cells, two types of GnRH-I binding sites have been identified in the human endometrial carcinoma cell lines HEC-1A and Ishikawa (Emons et al, 1993b). In contrast, others have reported that only one class of high-affinity receptor is present in normal endometrial tissue, endometrial carcinoma, and 23 certain endometrial cancer cell lines (Srkalovic et al, 1990; Imai et al, 1994). Membrane receptors for GnRH-I have also been demonstrated in myometrial and leiomyomal cells using immunocytochemistry (Kobayashi et al, 1997). Results from RT-PCR indicate that GnRH-I receptor mRNA is expressed in both normal and neoplastic uterine cells including those derived from stromal and ectopic endometrial tissues (Imai et al, 1994; Chatzaki et al, 1996; Chegini et al, 1996; Kobayashi et al, 1997; Borroni et al, 2000; Grundker et al, 2001; Huang et al, 2003). Like the placental and ovarian receptors, sequence analysis of the entire coding region of the GnRH-I receptor cDNAs cloned from HEC-1A and Ishikawa cells has revealed neither mutations nor splice variants of the receptor in the cancer cells (Grundker et al, 2001). 1.8.5 Prostate Gland The presence of specific binding sites for GnRH-I has been demonstrated in human prostate cancer (Fekete et al, 1989) as well as in the prostatic cancer cell lines LNCaP and PC3 (Limonta et al, 1992; Ravenna et al, 2000). However, displacement experiments reveal that the affinity of these binding sites is generally lower than that of the pituitary GnRH-I receptor (Limonta et al, 1992; Ravenna et al, 2000). Using RT-PCR and Southern blot analysis, products of expected size for the GnRH-I receptor have been obtained from both normal and neoplastic prostate tissues (Bahk et al, 1998; Limonta et al, 1999; Halmos et al, 2000; Tieva et al, 2001). Consistently, expression of GnRH-I receptor immunoreactivity has been demonstrated in benign and malignant prostate tissues as well as in intraprostatic lymphocytes (Tieva et al, 2001). Expression of immunoreactive GnRH-I receptor in the prostate is further supported by the detection 24 of a protein band of approximately 64 kilodaltons (kDa), which corresponds to the molecular mass of the pituitary GnRH-I receptor, in LNCaP and DU 145 (a prostatic cancer cell line) cells (Limonta et al, 1999). 1.8.6 Breast Two distinct types of GnRH binding sites have been found in the MCF-7 mammary cancer cell line, one is of high affinity and specificity for GnRH-I, while the other is only recognizable by GnRH-I antagonists (Segal-Abramson et al, 1992). In addition, GnRH-I receptor immunoreactivity and an mRNA with sequence identical to the pituitary counterpart have been demonstrated in both normal and malignant breast tissues (Kottler et al, 1997; Moriya et al, 2001). However, unlike its ligand (Chen et al, 2002b), the expression of the GnRH-I receptor is not upregulated in breast cancer (Kottler et al, 1997). 1.8.7 Other Extrapituitary Tissues Multiple lines of evidence indicate that the expression of extrapituitary GnRH-I receptor is not limited to reproductive tissues. For instance, it has been demonstrated by RT-PCR and Southern blot hybridization that the receptor is expressed in the liver, heart, skeletal muscle, and kidney (Kakar and Jennes, 1995). Moreover, Chen and coworkers have shown that human peripheral blood mononuclear cells also express GnRH-I receptor mRNA (Chen et al, 1999a). Furthermore, expression of the receptor has recently been demonstrated in the BLM melanoma cell line at both the RNA and protein levels (Moretti et al, 2002). 25 1.9 Regulation of Human GnRH-I Receptor Gene Expression 1.9.1 GnRH-I and GnRH-II It has been well documented that pituitary GnRH-I receptor gene expression is highly regulated by its own ligand such that pulsatile administration upregulates receptor expression and is obligate for gonadotropin secretion, whereas sustained stimulation causes downregulation and desensitization of the receptor, leading to the arrest of gonadotropin release and ovarian steroidogenesis (Loumaye and Catt, 1982; McArdle et al, 1987; Uemura et al, 1992; Tsutsumi et al, 1993; Conn and Crowley, 1994; McArdle et al, 1995; Tsutsumi et al, 1995). In fact, a similar biphasic effect of GnRH-I on GnRH-I receptor gene expression has been demonstrated in ovarian carcinoma as well as in GL, OSE, and peripheral blood mononuclear cells (Peng et al, 1994; Chen et al, 1999a; Kang et al, 2000a; Kang et al, 2000c). On the contrary, a significant increase instead of a decrease in receptor mRNA level is observed in the placental JEG-3 and immortalized extravillous trophoblast (IEVT) cells after chronic GnRH-I stimulation (Cheng et al, 2000a), indicating that the mechanism underlying homologous regulation of GnRH-I receptor expression is tissue-specific. The effect of GnRH-II on GnRH-I receptor gene expression has also been investigated in human GL cells. In contrast to the biphasic response induced by GnRH-I, treatment with GnRH-II significantly inhibits the mRNA level of the receptor in the steroidogenic cells irrespective of the concentration used (Kang et al, 2001c). 26 1.9.2 Gonadal Steroid Hormones The role of gonadal steroid hormones in regulating GnRH-I receptor gene expression has been extensively studied. At the pituitary level, the effect of E2 is dynamic and appears to depend on the administration pattern. Thus, an increase in pituitary GnRH-I receptor mRNA level has been observed in castrated female and male rats, and hormone replacement therapy of the castrated animals with either estrogen or testosterone reverses the stimulatory effect accordingly (Kaiser et al, 1993). Likewise, short-term treatment of primary cultures of rat pituitary cells with E2 has been shown to downregulate GnRH-I receptor concentration (Emons et al, 1988). Conversely, chronic exposure of pituitary cells to the estrogen has been demonstrated to stimulate receptor expression both in vitro and in vivo (Menon et al, 1985; Quinones-Jenab et al, 1996). Modulation of GnRH-I receptor gene expression by E 2 has also been studied in extrapituitary tissues. Using semi-quantitative RT-PCR analysis, the steady-state mRNA level of the receptor is found to be dose- and time-dependently suppressed by E2 in human GL and OVCAR-3 cells (Nathwani et al, 2000; Kang et al, 2001a). This inhibitory effect can be abolished by cotreatment with tamoxifen, supporting mediation through the classical ER. However, it remains undetermined which ER isoform is involved in this process as both ER subtypes, ERa and ER(3, are expressed in these ovarian cells (Chiang et al, 2000; Kang et al, 2001a). Several lines of evidence indicate that P 4 directly inhibits pituitary GnRH-I receptor gene expression. In primary-cultured ovine pituitary cells, treatment with P 4 has been demonstrated to reduce both GnRH-I binding (Laws et al, 1990) and GnRH-I receptor mRNA level (Wu et al, 1994; Sakurai et al, 1997; Kirkpatrick et al, 1998). 27 Interestingly, Cheng and colleagues have recently revealed a differential role of P 4 in regulating human GnRH-I receptor gene transcription such that the steroid suppresses the GnRH-I receptor promoter in gonadotropes but stimulates it in placental cells (Cheng et al, 2001a). The mechanism underlying these opposing effects of P 4 will be discussed in a later section (1.13.6 Transcriptional Regulation by P4). 1.9.3 Gonadotropins A considerable number of reports have indicated that the regulation of GnRH-I receptor gene expression by gonadotropins is tissue-specific. In human GL cells, treatment with hCG for 24 hours (h) induces a dose-dependent inhibition of GnRH-I receptor mRNA level (Peng et al, 1994), an observation that has also been demonstrated in rat granulosa cells, rat testis, and GT1-7 neurons (Olofsson et al, 1995; Li et al, 1996; Botte et al, 1999). On the contrary, opposing data are obtained from JEG-3 cells, in which the gonadotropin stimulates the mRNA content of the receptor possibly via a promoter-mediated process (Cheng et al, 2000a; Cheng and Leung, 2002). 1.9.4 Melatonin Melatonin is a pineal hormone that controls dynamic physiological adaptations in response to changes in day length in seasonally breeding mammals. Although its role in reproduction in humans remains obscure, there is increasing evidence that melatonin acts at the level of the ovary to modify local biological functions via a receptor-mediated mechanism (Brzezinski et al, 1987; Ronnberg et al, 1990; Ayre and Pang, 1994). In human GL cells, transcripts encoding the two melatonin receptor subtypes M T i and MT2 28 have been identified, and sequence analysis reveals that the ovarian melatonin receptors are identical to their brain counterparts (Woo et al, 2001). Accordingly, treatment of the steroidogenic cells with melatonin has been shown to significantly decrease the steady-state mRNA level of the GnRH-I receptor but to increase that of the LH receptor in a dose-dependent manner (Woo et al, 2001). Since the GnRH-I/GnRH-I receptor system has been implicated as a luteolytic factor that can trigger apoptosis in granulosa cells (Billig et al, 1994; Zhao et al, 2000a), this melatonin-induced downregulation of GnRH-I receptor gene expression may play a role in interfering with corpus luteum regression during the mid- to late luteal phase of the reproductive cycle. 1.10 Signal Transduction Mechanism of the GnRH-I Receptor 1.10.1 G protein Coupling Members of the GPCR family including the GnRH-I receptor transmit their signals primarily via G proteins, which are heterotrimers composed of an a subunit (Ga) that binds guanine nucleotides and a dimer that consists of a P and y subunit (GpY). Upon stimulation, G a dissociates from the GpY dimer and changes to its active guanosine triphosphate (GTP)-bound form, which thereby influences downstream effector molecules. The Gpy subunit remains attached to the plasma membrane and can by itself initiate certain signaling events. Generally, G proteins can be broadly classified according to the subtype of their a subunits into four categories: G a s , G ai, G a q /n, and Gai2/i3- G a s mainly exerts its effect via activation of adenylyl cyclase, which catalyzes the production of intracellular cAMP. Conversely, G«i has an inhibitory effect on adenylyl cyclase activity. G a q/n principally mediates its action by 29 stimulating the membrane-associated phospholipase Cpi, whereas Gai2/n operates by activation of protein-tyrosine kinases (Birnbaumer, 1992). The nature of G protein-coupled signaling initiated by the GnRH-I receptor depends largely on the cellular context. In gonadotrope-derived alphaT3-l cells, it is generally accepted that the receptor is coupled to G a q/n (Shah and Milligan, 1994), which activates phospholipase Cp upon ligand stimulation. This activation in turn triggers a series of intracellular signaling events including enhanced phosphoinositide turnover, sequential activation of phospholipase D and A2, Ca 2 + mobilization and influx, translocation and activation of various protein kinase C (PKC) subspecies particularly PKC8 and s (Harris et al, 1997), release of arachidonic acid, as well as formation of bioactive lipoxygenase products that culminate in gonadotropin secretion (Stojilkovic and Catt, 1995; Shacham et al, 1999). On the contrary, the GnRH-I receptor has been shown to couple with G a s and stimulate intracellular cAMP production in rat somatolactotrope GH3 cells (Kuphal et al, 1994), Chinese hamster ovary CHO cells (Nelson et al, 1999), and insect SF9 cells (Delahaye et al, 1997). Furthermore, studies on the GnRH-I receptor signal transduction mechanism in some reproductive tract-derived tumors have revealed the coupling of the receptor to Gaj (Imai et al, 1996b; Kimura et al, 1999; Grundker et al, 2001). 1.10.2 Mitogen-Activated Protein Kinases (MAPKs) MAPKs are a family of protein-serine/threonine kinases that are activated in response to a diverse array of extracellular stimuli. Very often, they constitute the major components of signal transduction cascades elicited by growth factors and many 30 hormones. The function and regulation of these kinases have been conserved during evolution from unicellular organisms such as yeast to complex organisms including humans (Widmann et al, 1999). Four distinct MAPK cascades have been identified to date, and they include the extracellular signal-regulated kinases (ERKs), ERK1 and ERK2 (Johnson and Lapadat, 2002); the c-Jun amino-terminal kinases (JNKs), JNK1, JNK2, and JNK3 (Johnson and Lapadat, 2002); the four p38 enzymes, p38a, p38p, p38y, and p385 (Johnson and Lapadat, 2002); and the big MAPK (BMK) 1 (also known as ERK5) (Zhou et al, 1995). The core unit of these signaling cascades consists of three protein kinases that sequentially activate one another by phosphorylation (Widmann et al, 1999) (Figure 4). Additional protein kinases identified biochemically or during the sequencing of the human and mouse genomes may function as MAPKs but are not well characterized. ERK7 is a possible MAPK candidate in this category (Abe et al, 1999). The MAPKs play an integral role in GPCR-mediated intracellular signaling. All four G a subunits (Naor et al, 2000), the GpY subunit (Crespo et al, 1994), as well as receptor-interacting proteins such as c-Src (Luttrell et al, 1996), P-arrestin (Daaka et al, 1998), and dynamin (Ahn et al, 1999) are capable of initiating diverse downstream signaling processes culminating in MAPK activation. In alphaT3-l cells, the GnRH-I receptor has been demonstrated to activate all the four MAPK cascades to various extents by a PKC- and tyrosine kinase-dependent mechanism (Naor et al., 2000) (Figure 5). The highest stimulation (~20 fold) is observed with JNKs, which are activated to a degree similar to that obtained by the strong activator peroxovanadate. Activation of JNKs in the pituitary cells is transient, peaking at 30 minutes (min) and declining thereafter. This process is both PKC- and protein-tyrosine kinase-dependent and is 31 Figure 4. The M A P K cascades. Each MAPK module consists of a MKKK, a MKK, and a MAPK. MKKKs respond to a diverse array of extracellular stimuli such as growth factor, stress, and inflammatory response. The activated MKKKs can stimulate one or several MKKs. In contrast, the MKKs are relatively specific for their target MAPKs. Once activated, MAPKs phosphorylate various substrates including transcription factors (e.g. c-Jun), other kinases (e.g. p90RSK), and upstream regulators (e.g. EGF receptor), which in turn regulate different cellular responses ranging from growth, differentiation, and apoptosis. MKKK, MAPK kinase kinase; MKK, MAPK kinase; MEK, MAPK/ERK kinase; p90RSK, 90-kDa ribosomal protein S6 kinase; EGF, epidermal growth factor; MEKK, MEK kinase; TAK, transforming growth factor (3-activated protein kinase; TAB, TAK1-binding protein; ASK, apoptosis signal-regulated kinase; PAK, p21-activated kinase; MNK, MAPK-interacting kinase; MEF-2, myocyte enhancer factor 2. 32 Stimulus Activator MKKK MKK ERK1/2 JNK p38 MAPK BMK Stress, Oxidative stress, Growth factor growth factor, inflammatory Oxidative stress differentiation cytokine factor RasGTP Rac1 TAB1 Src Raf-1, A-Raf, B-Raf MEKK1, MEKK4 TAK1, ASK1, PAK1 MEKK2 MEK1, MKK4, MKK3, MKK5 MEK2 MKK7 MKK6 MAPK ERK1, JNK1, JNK2, p38a, p38p, BMK1 ERK2 JNK3 p38y, p385 p90RSK, c-Jun, ATF-2, MNK1, ATF-2, Substrate Elk-1, Ets-1, Elk-1, p53 Chop, Max, MEF-2 EGF receptor Elk-1 33 Figure 5. Activation of MAPKs by the GnRH-I receptor in gonadotrope-derived alphaT3-l cells. The GnRH-I receptor is coupled to G a q / 1 x in the gonadotropes and can stimulate all the four MAPK cascades to various degrees primarily via PKC activation. The nonreceptor tyrosine kinase c-Src, which acts downstream of PKC, is also required for both the ERK and INK activation. In addition, a substantial role of Ca 2 + mobilization in the MAPK stimulation has been demonstrated under certain cellular conditions though it is not indicated in the diagram. In contrast, the signaling pathways leading to p38 MAPK and BMK1 activation are less clear. It should be noted that in other cell types, the receptor is coupled to different G a proteins and activates the MAPK cascades via essentially distinct mechanisms. Solid and dashed arrows represent direct and indirect activation of the MAPK cascades, respectively. 34 35 mediated by a unique pathway that involves sequential stimulation of PKC, c-Src, CDC42/Racl, and MEKK1 (Levi et al, 1998). On the contrary, others have reported that JNK activation in alphaT3-l cells is triggered by an elevated intracellular Ca 2 + concentration ([Ca2+]j) and is principally PKC-independent (Mulvaney and Roberson, 2000). Moreover, in another gonadotrope-derived cell line LbetaT2, activation of JNKs has been shown to be independent of either PKC or calcium elevation (Yokoi et al, 2000). Thus, it is apparent that under different cellular conditions, the GnRH-I receptor may utilize distinct intracellular pathways to activate the JNK cascade. Similar to JNKs, ERK1/2 are also substantially stimulated (~12-fold) in alphaT3-1 cells (Naor et al, 2000). Activation of ERKs by the GnRH-I receptor is mainly PKC-dependent and involves two distinct signaling pathways that converge at the level of Raf-1. The main pathway is mediated by direct activation of Raf-1 by PKC, which is supported by a second pathway involving activation of Ras in a c-Src- and dynamin-dependent manner (Benard et al, 2001). Additionally, a substantial role of calcium influx through voltage-gated calcium channels in ERK1/2 activation has been demonstrated in the pituitary cells (Mulvaney and Roberson, 2000). In contrast to the robust stimulation observed with JNKs and ERKs, activation of p38 MAPKs and BMK1 in alphaT3-l cells is much weaker (2 to 3 fold), peaking at about 30-45 min and declining to basal level at about 180 min (Naor et al, 2000; Kraus et al, 2001). Recent findings have shown that the GnRH-I receptor can activate ERKs in COS-7 cells with kinetics similar to those observed in gonadotropes (Kraus et al, 2003). However, the signaling mechanism leading to ERK activation is significantly different in kidney cells as it mainly involves transactivation of the EGF receptor, which either 36 directly activates Ras or induces c-Src-dependent activation of Ras. A minor contribution of this stimulation is also observed with (3-arrestin, which probably operates via an EGF- and PKC-independent cascade (Kraus et al, 2003). Similarly, activation of JNKs by the GnRH-I receptor in COS-7 cells relies on pathways that are distinct from those observed in alphaT3-l cells. This process is fully dependent on a sequential stimulation of c-Src and phosphoinositide 3-kinase, which acts mainly downstream of the EGF receptor but can also be activated by the Gpy subunit (Kraus et al, 2003). 1.10.3 Receptor Desensitization and Internalization It is well known that sustained stimulation of most GPCRs causes their desensitization and internalization from the cell surface (Ferguson and Caron, 1998). For many GPCRs, this process is mediated by G protein-coupled receptor kinases, which phosphorylate the receptor and promote its binding with P-arrestin. This interaction will consequently reduce G protein coupling and lead to desensitization of the receptor (Ferguson and Caron, 1998). Beta-arrestin also serves as an adapter by targeting the desensitized receptor to clathrin-coated vesicles (CCVs) for internalization (Goodman et al, 1996). Once formed, the CCV is pinched off from the plasma membrane by an oligomeric dynamin "collar". This effect of dynamin depends on its intrinsic GTPase activity and can be blocked by the expression of GTPase-inactive mutants (Vieira et al, 1996). The internalized receptors are then either recycled back to the surface membrane or targeted to lysosomes for degradation. Although this general scheme appears applicable to many GPCRs, there are important exceptions. For instance, internalization of the M2 muscarinic acetylcholine and angiotensin receptors is independent of both P-37 arrestin and dynamin (Pals-Rylaarsdam et al, 1997; Vogler et al, 1999), while internalization of the endothelin A receptor is apparently independent on P-arrestin and clathrin (Claing et al, 2000; Okamoto et al, 2000). Thus, although these issues remain controversial (Lee et al, 1998a; Bremnes et al, 2000; Lee et al, 2000; Werbonat et al, 2000) for different GPCRs and perhaps for different cell types, the adapter protein targeting receptors for internalization needs not to be p-arrestin, the fission protein regulating internalization needs not to be dynamin, and the vesicle does not need to be clathrin-coated. As mentioned earlier, the mammalian GnRH-I receptor has undergone a period of accelerated molecular evolution, during which the carboxyl-terminal tail present in all known nonmammalian GnRH receptors is lost. Since the serine and threonine residues that are phosphorylated by G protein-coupled receptor kinases are often located in the cytoplasmic tail, a number of comparative studies have revealed that the GnRH-I receptor does not show rapid homologous desensitization (Davidson et al, 1994; Willars et al, 1999). In contrast, the two nonmammalian (catfish andXenopus) GnRH receptors investigated to date do rapidly desensitize (Heding et al, 1998; Hislop et al, 2000). Similarly, although the GnRH-I receptor undergoes agonist-induced internalization via CCVs, it is internalized at a much slower rate than the nonmammalian GnRH receptors. Moreover, the nonmammalian GnRH receptors exhibit agonist-induced phosphorylation and P-arrestin translocation, as well as P-arrestin-dependent internalization (Blomenrohr et al, 1999), whereas the GnRH-I receptor does not (Vrecl et al, 1998). Furthermore, while internalization of the Xenopus receptor is primarily dynamin-dependent, that of the GnRH-I receptor is not (Hislop et al, 2001). Since such 38 differences in desensitization and internalization are retained at a physiological receptor density in gonadotrope lineage cells (Hislop et al., 2000), it supports the argument that the evolution of the nondesensitizing GnRH-I receptor is related to the development of mammalian reproductive strategies. 1.11 Biological Functions of GnRH-I and GnRH-II in Humans In addition to its pivotal role in stimulating gonadotropin synthesis and secretion, GnRH-I serves as an autocrine and/or paracrine factor in a number of extrapituitary tissues such as the ovary, uterus, and placenta, where the hormone regulates steroidogenesis, cell proliferation, and apoptosis (Table 2). In tumors derived from various reproductive tissues, there is clear evidence that the GnRH-I receptor is coupled to Gaj and mediates its action via pathways that are distinct from the classical cascade operating in gonadotropes. The recent discovery of GnRH-II and its cognate receptor, as well as the demonstration of some extrapituitary functions of the hormone, strongly support the existence of an additional GnRH/GnRH receptor system in humans. 1.11.1 Gonadotropin Expression and Secretion GnRH-I plays a key role in the mammalian reproductive process by stimulating the synthesis and release of FSH and LH, which are heterodimeric glycoprotein hormones composed of a common a-subunit, noncovalently bound to a specific p-subunit (FSHp and LHp) (Pierce and Parsons, 1981; Gharib et al, 1990). Activation of the human a-subunit gene transcription by GnRH-I has been shown to be primarily Ca -dependent. Also, this stimulation can be augmented by PKC and requires ERKs 39 Table 2. Summary of autocrine actions of GnRH-I (I) and GnRH-II (II) in humans. Extrapituitary site Cel l type Function Effect O v a r y Cancer ce l l Prol i ferat ion (I, II) Decrease O S E ce l l Prol i ferat ion (I, II) Decrease G L ce l l Steroidogenesis (I, II) Decrease G L ce l l Apoptos i s (I) Increase Placenta h C G release (I, II) B iphas i c Ext ravi l lous cytotrophoblasts Ext ravi l lous cytotrophoblasts u P A expression (I, II) P A I expression (I, II) Increase Decrease Endomet r ium Cancer ce l l Prol i ferat ion (I, II) Decrease Dec idua l stromal ce l l M M P - 2 and M M P - 9 expression (I) Increase Breast Cancer ce l l Prol i ferat ion (I) Decrease Prostate gland Cancer ce l l Prol i ferat ion (I) Decrease S k i n Cancer ce l l Prol i ferat ion (I) Decrease Immune system T c e l l L a m i n i n receptor expression (I, II) Increase T c e l l T ce l l adhesion, chemotaxis, and Increase homing (I, II) 40 and c-Src (Holdstock et al, 1996; Harris et al, 2003). However, one should note that though c-Src has been shown to be involved in GnRH-I-stimulated ERK1/2 activity (Benard et al, 2001), the ERK1/2 and c-Src-response regions are located at different loci on the a-subunit promoter (Harris et al, 2003), indicating that the c-Src contribution is independent of ERK1/2 activation. The role of PKC, Ca 2 + , and MAPK signaling cascades in mediating GnRH-I stimulation of LHP gene transcription is controversial. While it was shown that the activation was Ca2+-dependent (Week et al, 1998), others reported that PKC was responsible for the effect (Saunders et al, 1998). Similarly, while it was found that both PKC and ERK1/2 were required for the stimulation (Call and Wolfe, 1999), others demonstrated an important role of JNKs (Yokoi et al, 2000). On the one hand, two different regulatory regions, region A (-490/-352) and B (-207/-82), have been shown to mediate the GnRH-I response in the rat LHP promoter (Kaiser et al, 1998a). Region A contains two Spl binding sites (Kaiser et al, 1998b) and a CArG element, which partially overlaps the distal Spl site (Week et al, 2000). On the other hand, region B contains two binding sites for the early growth response 1 (Egrl) transcription factor, which are also present in LHp promoters of other species (Tremblay and Drouin, 1999; Wolfe and Call, 1999). Interestingly, these elements have been suggested to differentially mediate GnRH-I stimulation of LHP gene transcription, such that Egrl works synergistically with other transcription factors including steroidogenic factor 1 (SF-1) and pituitary homeobox 1 to mediate the PKC and MAPK cascades (Call and Wolfe, 1999; Halvorson et al, 1999; Tremblay and Drouin, 1999), while the bipartite 41 Spl/CArG motif, though not investigated yet, may mediate the Ca signaling pathway (Andoef al, 2001). Several studies have indicated that PKC and MAPK pathways are involved in GnRH-I-stimulated FSH(3 gene expression (Haisenleder et al, 1998; Saunders et al, 1998; Strahl et al, 1998). For instance, it has been demonstrated that activation of the ovine FSHP promoter is dependent on PKC activation (Strahl et al, 1998) and requires two proximal AP-1 enhancer elements, which interact with c-Jun and c-Fos (Strahl et al, 1997). Significantly, the upstream AP-1 binding site is completely conserved in five of seven mammals (ovine, cow, pig, rabbit, and human) and only varies at one terminal nucleotide in the rat and mouse FSHP promoters (Strahl et al, 1997; Miller et al, 2002), indicating that it may have important functions in transcriptional regulation of the FSHP gene by GnRH-I. An essential role of [Ca2+]j in mediating GnRH-I-stimulated gonadotropin secretion has been demonstrated. By simultaneous measurements of [Ca2+]j and exocytosis in a single gonadotrope, Tse and coworkers have revealed that individual GnRH-I-induced Ca 2 + spike can trigger a small burst of exocytosis (Tse et al, 1993). Likewise, exocytosis can be triggered by [Ca2+]i elevation caused by photolysis of caged inositol 1, 4, 5-triphosphate (Tse et al, 1993). In contrast, the initial phase of gonadotropin release from pituitary cells is apparently independent of extracellular Ca 2 + (Chang et al, 1988; Naor et al, 1988; Tse et al, 1993). The role of PKC activation in mediating GnRH-I-induced gonadotropin release is less clear (Stojilkovic et al, 1994; Stojilkovic and Catt, 1995). Although phorbol esters have been demonstrated to stimulate LH secretion, GnRH-I-induced LH release is only impaired but not abolished 42 in PKC-depleted gonadotropes (Stojilkovic et al, 1988b; Beggs and Miller, 1989), indicating that PKC activation is not an absolute requirement for exocytosis. It has been suggested that PKC participates in the control of gonadotropin secretion through actions on cytoskeleton elements, Rab proteins, and other elements involved in the exocytotic process (Strulovici et al, 1987; Stojilkovic et al, 1988a; Kiley et al, 1992). 1.11.2 Ovarian Steroidogenesis It is well documented that GnRH-I possesses an antigonadotropic effect in rat ovary by downregulating the expression of FSH and LH receptors (Piquette et al, 1991; Tilly et al, 1992), inhibiting gonadotropin-stimulated cAMP production (Knecht et al, 1985; Richards, 1994), and suppressing steroidogenic enzyme activity (Hsueh and Schaeffer, 1985; Sridaran et al, 1999). In human GL cells, treatment with GnRH-I or its agonists at high doses has been shown to suppress both basal and FSH-stimulated steroidogenesis (Parinaud et al, 1988; Kimura, 1992; Maeda et al, 1996; Kang et al, 2000d). Consistent with these observations, Furger and coworkers have demonstrated that the hormone can inhibit FSH-induced morphological changes in steroidogenic cells (Furger et al, 1996). On the contrary, the role of GnRH-I in LH/hCG-regulated steroidogenesis is controversial (Parinaud et al, 1988; Olsson et al, 1990; Kimura, 1992; Hori et al, 1998; Kang et al, 2001c), and it has been shown that treatment with GnRH-I does not significantly affect hCG- and forskolin-mediated responses (Furger et al, 1996). Therefore, it is speculated that GnRH-I may specifically interact with the FSH-induced cAMP-dependent signaling pathway at a level upstream of cAMP generation in human GL cells. 43 Recently, our laboratory has revealed that treatment of human GL cells with a GnRH-I agonist rapidly stimulates the phosphorylation and activity of ERK1/2 and causes a drastic increase in c-Fos mRNA level (Kang et al, 2001b). This GnRH-I-stimulated ERK activity is mediated via G a q/n and involves PKC activation since the effect can be mimicked by phorbol-12-myristate 13-acetate (PMA) and abolished by the PKC inhibitor GF109203X (Kang et al, 2001b). Interestingly, pretreatment with the MEK inhibitor PD98059 can completely reverse the inhibitory effect of GnRH-I on steroidogenesis in the GL cells (Kang et al, 2000d), indicating that the antisteroidogenic action of GnRH-I is mediated via a PKC- and ERK-dependent signaling cascade. The role of GnRH-II in regulating ovarian steroidogenesis has recently been studied. In human GL cells, treatment with GnRH-II or its agonist suppresses both basal and hCG-stimulated P 4 production (Kang et al, 2001c). Similar to the effects produced by GnRH-I, GnRH-II does not interfere with hCG-stimulated cAMP production in steroidogenic cells. Instead, these hormones downregulate the steady-state mRNA level of both the FSH and LH receptors (Kang et al, 2001c), thus strengthening the notion that GnRH-I and GnRH-II exert their antigonadotropic effects at the receptor level but not at the cAMP level. 1.11.3 Cell Proliferation The role of GnRH-I as a negative autocrine growth factor has been well demonstrated in a number of human malignant tumors including those of the ovary (Thompson et al, 1991; Emons et al, 1993a; Yano et al, 1994b; Kang et al, 2000a), 44 endometrium (Emons et al, 1993b; Grundker et al., 2001; Nagai et al., 2002), breast (Blankenstein et al., 1985; Miller et al., 1985), prostate gland (Limonta et al, 1992; Dondi et al, 1994; Loop et al, 1995), and skin (Moretti et al, 2002). On the one hand, since the growth of these cancer cells can be significantly inhibited at nanomolar concentrations of GnRH-I agonists, it is generally believed that the antiproliferative action of the hormone is mediated via the high-affinity GnRH-I receptor. This notion is supported by the fact that the nucleotide sequence of the GnRH-I receptor in these tumors is identical to that in the pituitary (Kakar et al, 1994b; Grundker et al, 2001). On the other hand, a number of studies have indicated that the GnRH-I antagonist cetrorelix can also suppress the growth of certain cancer cell lines in a dose-dependent manner (Eidne et al, 1987; Sharoni et al, 1989; Emons et al, 1993a; Emons et al, 1993b; Kleinman et al, 1994; Yano et al, 1994b; Tang et al, 2002). Interestingly, the antiproliferative effect of cetrorelix in ovarian cancer OV-1063 cells is stronger than that produced by the agonist triptorelin (Yano et al, 1994b). Furthermore, it has even been observed that the proliferation of OV-1063 xenografts in nude mice can only be suppressed by the antagonist but not by the agonist (Yano et al, 1994a). A possible explanation for these observations is that GnRH-I agonists and antagonists elicit their antiproliferative effects via different signal transduction mechanisms from the GnRH-I receptor. Another possibility is that there are high-affinity GnRH-I antagonist binding sites that are not recognized by GnRH-I agonists, as previously reported in MCF-7 cells (Segal-Abramson et al, 1992). The latter hypothesis is supported by an earlier finding, which has shown that the proliferation of some human breast cancer cell lines can only be inhibited by antagonistic analogs of GnRH-I (Eidne et al, 1987). 45 Multiple lines of evidence indicate that GnRH-I (or its agonists) mediates its growth-inhibitory effect via modulation of the ERK1/2 signal transduction cascade. For instance, it has been demonstrated that the antiproliferative effect of the GnRH-I agonist leuprolide can be abolished by PD98059 in ovarian carcinoma Caov-3 cells (Kimura et al, 1999). Detailed characterization of the signaling pathway reveals that the agonist causes a sustained activation of ERK1/2, which is associated with concomitant stimulation of MEK as well as phosphorylation of son of sevenless and She. Activation of ERK1/2 by leuprolide in the cancer cells is independent on both PKC and Ca 2 + , which are important messengers that mediate GnRH-I actions in gonadotropes. Conversely, this GnRH-I-stimulated ERK1/2 phosphotransferase activity can be blocked by pertussis toxin (PTX) or by overexpressing the carboxyl terminus of (3-adrenergic receptor kinase, indicating that the response is mediated via a PTX-sensitive G a protein and involves the GpY subunit. Further analysis of the downstream signaling cascade indicates that prolonged stimulation of the ERK1/2 leads to dephosphorylation of the retinoblastoma protein (Kimura et al, 1999), an event that is known to prevent cell cycle progression from Gi to S phase. In fact, similar observations have also been reported in other gynecological cancer cell lines such that treatment with GnRH-I agonists results in blockage of cell cycle transition and decreased DNA synthesis (Kim et al, 1999; Gunthert et al, 2002). Of particular interest, Gunthert and coworkers have revealed that the growth-inhibitory effect of GnRH-I may be partly attributed to its ability to increase the DNA binding activity of JunD (Gunthert et al, 2002), which has been suggested as a negative regulator of cell proliferation (Pfarr et al, 1994). 46 The involvement of the ERK cascade in mediating the antitumor effect of GnRH-I is further supported by the observation that the hormone is capable of antagonizing growth factor-induced mitogenic signaling via coupling to the PTX-sensitive G a ; protein (Emons et al, 1998). It has been shown that in primary ovarian carcinoma as well as in certain ovarian and endometrial cancer cell lines, treatment with GnRH-I agonists can dose-dependently stimulate phospho tyro sine phosphatase (PTP) activity and cause a remarkable loss of phosphotyrosine residues from a 35-kDa membrane protein (Imai et al, 1996a; Grundker et al, 2001). Subsequent experiments have demonstrated that this GnRH-I-stimulated PTP activity is associated with a significant reduction in EGF-induced tyrosine autophosphorylation of the EGF receptor, EGF-induced ERK 1/2 activation, as well as EGF-induced c-Fos gene expression (Emons et al, 1996; Grundker et al, 2000b; Grundker et al, 2001). Thus, in contrast to the findings from Caov-3 cells (Kimura et al, 1999), GnRH-I can negatively regulate the ERK1/2 signaling cascade to mediate its antiproliferative effect in gynecological cancers. The role of GnRH-II as a negative autocrine growth factor has also been demonstrated. Like GnRH-I, treatment with GnRH-II produces a dose-dependent inhibition of cell proliferation in both nontumorigenic and tumorigenic OSE cells (Choi et al, 2001). In support of the previous identification of an additional type of GnRH binding site (Emons et al, 1993a; Emons et al, 1993b), Grundker and coworkers have recently found an mRNA for a second form of GnRH receptor (GnRH-II receptor) in several endometrial and ovarian cancer cell lines, whose proliferation can be dose- and time-dependently suppressed by GnRH-II (Grundker et al, 2002). Interestingly, this antiproliferative effect is shown to be significantly stronger 47 than that produced by equimolar concentrations of triptorelin (Grundker et al, 2002). It should be noted that in GnRH-II receptor-positive but GnRH-I receptor-negative ovarian cancer SK-OV-3 cells, treatment with GnRH-I agonists has no effect on cell proliferation (Volker et al, 2002). Therefore, it is apparent that the growth-inhibitory effect of GnRH-II is unlikely due to cross-binding to the GnRH-I receptor and that the GnRH-II/GnRH-II receptor system may represent an additional autocrine growth regulatory mechanism in cancer cells. 1.11.4 Apoptosis It is well established that GnRH-I has a stimulatory effect on apoptosis in ovarian granulosa cells. In rat, treatment with a GnRH-I agonist has been demonstrated to produce a dose- and time-dependent increase in DNA fragmentation, a hallmark of apoptotic cell death, in granulosa cells of the preantral and antral stages (Billig et al, 1994). In addition, this treatment can partially abolish the antiapoptotic effect induced by FSH (Billig et al, 1994). Likewise, it has been demonstrated that the GnRH-I agonist buserelin can induce a definitive ladder pattern of oligonucleosomal length DNA fragments in rat granulosa cells isolated from preovulatory follicles via a mechanism without affecting growth factor receptor phosphorylation (Yano et al, 1997). Furthermore, a recent study has also shown that continuous treatment of corpus lutea with GnRH-I can stimulate apoptosis, which is associated with a downregulation of the mitochondrial peripheral-type benzodiazepine receptor and Bcl-X(L) expression and an upregulation of the proapoptotic Bax gene expression (Papadopoulos et al, 1999). These observations thus indicate that stimulation of cytochrome c release from 48 mitochondrial membranes may be involved in GnRH-I-stimulated apoptotic cell death in granulosa cells. Consistent with the findings in rat, treatment with buserelin has been shown to increase the incidence of apoptosis in human GL cells (Zhao et al., 2000a) though the proapoptotic mechanism in the human counterpart is not fully understood. The role of GnRH-I in regulating apoptosis, however, remains controversial in cancer cells. It has been demonstrated that in some ovarian cancer cell lines and in cells isolated from GnRH-I receptor-bearing ovarian tumors, treatment with buserelin can cause a dose-dependent stimulation of Fas ligand mRNA and protein expression. This hormonal activation of Fas ligand expression is believed to be receptor-mediated because it can be blocked by the GnRH-I antagonist antide (Imai et al, 1998). Since Fas ligand is known to be an intrinsic inducer of apoptosis via binding to its cell surface receptor Fas, which is frequently expressed in GnRH-I receptor-positive tumors (Imai et al, 1997), it has been suggested that GnRH-I may function as an autocrine factor to stimulate cell death in Fas-positive tumors and that this proapoptotic action may partly account for its antiproliferative effect in ovarian cancer. In contrast, a recent finding from Grundker and coworkers has shown that treatment with triptorelin does not produce any morphological signs of programmed cell death in the ovarian cancer EFO-21 and EFO-27 cells. Instead, the agonist inhibits cytotoxin-induced apoptosis via activation of the nuclear factor-KB ( N F - K B ) signal transduction cascade (Grundker et al, 2000a). N F - K B is a collective term referring to dimeric transcription factors that belong to the Rel family. The activity of N F - K B is strictly regulated by inhibitor K B , which forms a complex with the transcription factor and sequesters it in the cytoplasm (Ghosh et al, 1998). N F - K B has been implicated as 49 an antiapoptotic transcription factor by activating multiple genes, whose products can block apoptosis triggered by either death receptors or the mitochondrial pathway (Chu et al, 1997; You et al, 1997; Wang et al, 1998; Zong et al, 1999; Kreuz et al, 2001). Although activation of N F - K B by triptorelin is also mediated by Gaj in EFO-21 and EFO-27 cells, the signal transduction mechanism leading to N F - K B stimulation is independent of interference with the growth factor-induced mitogenic signaling as previously discussed (Grundker et al, 2000a). Therefore, in some ovarian cancer cells, GnRH-I appears to possess two counteracting activities (antiproliferative vs. antiapoptotic), which are mediated by two distinct signaling pathways triggered by the same G a protein. Contradictory results also exist regarding the role of GnRH-I in inducing apoptosis in uterine leiomyoma. While it has been demonstrated that GnRH-I agonists inhibit the proliferation of leiomyoma cells partly by stimulating apoptotic cell death (Wang et al, 2002b), opposite data have been obtained such that the analogs decrease the expression of certain proapoptotic factors but increase that of the antiapoptotic Bcl-2 protein, indicating that GnRH-I may suppress apoptosis in uterine leiomyoma (Huang et al, 2002). 1.11.5 Other Extrapituitary Functions Both GnRH-I and GnRH-II have been shown to exert a biphasic effect on hCG release from human placenta (Siler-Khodr and Khodr, 1979; Islami et al, 2001; Siler-Khodr and Grayson, 2001). In addition, both forms of GnRH have been demonstrated to stimulate the mRNA and protein levels of urokinase-type plasminogen activator (uPA) 50 but inhibit those of plasminogen activator inhibitor (PAI) in human extravillous cytotrophoblasts via two distinct GnRH receptors, indicating that these hormones have direct functions in embryo implantation (Chou et al, 2002). This notion is strengthened by the observation that GnRH-I can upregulate the expression of matrix metalloproteinase (MMP)-2 and MMP-9 in primary-cultured decidual stromal cells (Chou et al, 2003). Mediation of GnRH responses via different GnRH receptors has also been reported in normal and cancerous T cells, in which GnRH-I or GnRH-II triggers de novo gene transcription and cell-surface expression of a 67-kDa laminin receptor, leading to stimulation of T cell adhesion, chemotaxis, and homing to specific organs (Chen et al, 2002a). 1.12 Human GnRH-I Receptor Gene 1.12.1 Structural Organization In contrast to the genes of many other GPCRs, which are intronless and believed to have arisen by retroposition (Brosius, 1991), the human GnRH-I receptor gene is composed of three exons separated by two introns and spans over 15 kb along the chromosome (Fan et al, 1995; Kakar, 1997) (Figure 6). Exon 1 contains the 5'-UTR and the first 522 nucleotides of the open reading frame, which encode the first three TM domains and a portion of the fourth TM domain. Exon 2 encodes the next 220 nucleotides of the reading frame, which encompass the remainder of the fourth TM domain, the fifth TM domain, and part of the third intracellular loop. Exon 3 contains the rest of the coding sequence and the 3'-UTR. Comparison of the structural organization of the human receptor gene with that of the mouse, rat, and ovine genes 51 Exon 1 5'-UTP 522 bp Intron 1 -V/— Exon 2 -4.2 kb 220 bp / Intron 2 - / / — ~5 kb Exon 3 245 bp 3-UTR I I I I I I Human GnRH-I receptor cD NA 328 Figure 6. Structural organization of the human GnRH-I receptor gene. The human GnRH-I receptor gene is composed of three exons interrupted by two introns and spans over 15 kb. Boxes represent exons and thin lines represent introns. The corresponding structure of the human GnRH-I receptor cDNA is also shown. Exon 1 contains the 5'-UTR and encodes the first three TM domains and a portion of the fourth TM domain. Exon 2 is 220 bp in length and encodes the remainder of the fourth TM domain, the fifth TM domain, and part of the third intracellular loop. Exon 3 encodes the rest of the open reading frame and contains the 3'-UTR. The diagram is not drawn to scale. 52 indicates that the location of all the exon-intron boundaries is perfectly conserved except the size of the first intron is comparatively much smaller in the human counterpart (Albarracin et al, 1994; Campion et al, 1996; Reinhart et al, 1997). 1.12.2 Chromosomal Localization Genomic Southern blot analysis has revealed the presence of a single gene copy for the GnRH-I receptor in the human genome (Kakar, 1997). In addition, the human gene has been assigned to chromosome 4 by PCR analysis of somatic hybrid cell lines (Fan et al, 1994; Kakar and Neill, 1995) and to the 4q 13.1-21.1 region by cell hybrid mapping panels (Kaiser et al, 1994). Using chromosomal in situ hybridization, three groups have reported the gene location at band 4ql3.2-13.3 (Morrison et al, 1994; Kakar and Neill, 1995; Kottler et al, 1995) and one group at band 4q21.2 (Leung et al, 1995). 1.12.3 5'-and3'-UTRs Five transcription start sites, which are 703, 804, 827, 1372, and 1393 bp upstream of the ATG initiation codon, have been identified for the GnRH-I receptor gene in human brain by primer extension analysis (Fan et al, 1995), while a total of 18 start sites have been found in the pituitary by 5'-Rapid Amplification of cDNA End (5'-RACE) (Kakar, 1997). These start sites are clustered into two regions, which are 579-819 and 1348-1751 bp upstream of the initiation codon. Transcripts containing long 5'-UTRs are not uncommon and have been reported in the 53 human GnRH-I and preproenkephalin B genes (Horikawa et al, 1983; Seeburg and Adelman, 1984). The 3'-end of the human GnRH-I receptor gene has been completely sequenced (Fan et al, 1995). Five typical polyadenylation signals (at +3998, +4124, +4327, +4504, and +4811), which are located entirely within an 800-bp region in a cluster-like format, are present in the 3'-UTR. Also, the UTR contains several ATTTA motifs (at +3885, +4027, +4222, +4306, and +4534), which have been implicated in mRNA instability (Shaw and Kamen, 1986) and are notably present in many RNAs that are rapidly degraded. The size of the human GnRH-I receptor mRNA predicted from the length of the 5'- and 3'-UTRs is about 5.5 kb, which is in close agreement with the reported size of the major transcript detected in the pituitary (4.7-5 kb). 1.12.4 5 '-Flanking Region The 5'-flanking region of the human GnRH-I receptor gene has been isolated and partially characterized (Figure 7). Comparison of the proximal 900 nucleotides of the human flanking sequence with those of the mouse (Albarracin et al, 1994), rat (Reinhart et al, 1997), and sheep (Campion et al, 1996) sequences has revealed a 42 % identity among the four mammalian species. Further phylogenetic analysis of these 5'-flanking sequences identifies a major homology area to the proximal 300-bp region, which contains at least 65 % identical nucleotides. Nonetheless, the human GnRH-I receptor 5'-regulatory region does possess some distinctive features. One significant difference between the human and rodent genes is the location of their transcription start sites. Whereas the start sites for the rodent genes are within 100 nucleotides from the ATG 54 Figure 7. Nucleotide sequence of the human GnRH-I receptor 5'-flanking region. Numbers along side the sequence refer to the positions relative to the ATG initiation codon, of which the nucleotide position is assigned as +1. Transcription start sites used by pituitary (or brain) and placental cells are marked with black and white inverted triangles, respectively. C/s-acting DNA motifs that have been shown to be important for transcriptional regulation of the human GnRH-I receptor gene are indicated. Putative TATA and CAAT boxes in the flanking region are underlined with thick black lines. 55 2 2 97 AGCCTTTCTGAAGCATAAATCTGGCCATACCTACCGTATATTACTCCATTCTTTATATAGGTAAAGCCTA 2227 AACTCCTTTTCTTGGAATATAGGCTCTCCAGATCTGAAGTCTGCCTAATTATTTACACTTTTGCTTTCAC 2157 ATACCCTTTGAACTTTCTCACATTGTCTTCGTGTTTGCATGTGCTGCTCCAGCTTCTAAGCATGCCCTCC 2087 CTGTCCTCATACCCCATTCCCCAGCCACTTATTACGCTATATGCTGTAGTCCCATTCAGCTCGGTTGCAA 2017 CCTCTTCCCTAATGAATCAGTCCATCATTAACAAAGAAAGGGAGGGAGGGAGGGAGGAACAGAGGAATGA 194 7 AAGGAGGAAAAGGGAAGGAAGGGGAAGGGAAGGGGAAGGGAAGGGGAAGGGAAGGGAAGGGGAAGGGAAG 1877 GAATGGGAGGAAAAGGGACAAATAATGAATGATATGCTCTAATCTTTTTCCCCTAGATATAGAAGACAAA T 1807 GAGCAAAATATACTTCACTAAATTGATTTTTACATAAATTTTCTTTCCTTTGTTTTTTGGTTGCTGGTCC T 173 7 ACTTACAAACACTTTTCATATTTGTATGTCTTTCCAATGGTTATCCTGTTTTGTTCATTTCAGGCATATG | Oct-1 | | CAAT | | Inr | V T V 1667 GCCCTGATCAGATTAACTGACATGATGTATATGCAAAGCCTTTTGAGTTCTTCAGAAAAATAAATTATCT | PARE | | CRE | | GATA 1 T 1597 TATTCAAGACTGATTGCTTATAAGGAACTTATTATAGCTAATATAGTAGGCACAATTTTTTTTTGTAATT T 152 7 CTCCTAGATGAGTCAGAACTTAGTTTTGATGTAGGTAAAAATTTTATGGTCACAAATCTCAGGTGTGAGA I AP-1 I V T V 1457 AAATCTCTTTCCTTGATACTCTATATAAATAGAGGATATAAATATTTCAAGTCTGGAAGTAGTGAGAGAA TV T " " _ — _ T 1387 GCTGGTAATTCTGGACATATAGTGACAGTCAAAAAGGAGCTCAGGTACAGGACTGGTCTAAGCTGCTCAA 1317 GATTCAGGAGACAGCCAGTACACAGAGAAGCTGAGGAAATAATACAGATATATCTAAAACACTTATCTAA 124 7 CCTTCTGTGGTAACAAGCTCCTTAAAGGGGCTGGATGATGTTGTGTTCACTTTTTATCACCAGCAAAGGC 1177 TAAGATAATGTATATAGTAAATATTTAGTAACCATTTATTAAATAAATAAATATTTAAGACAGAATAAAC 1107 AAGTATAATAAATGAACCAATAAGAATGCACCATCTAAGTCAAAATAGCCACTTTTATCCTTAACATTGT 1037 ACCTGCTTTGGCTGCTGCAGAAGCAAACTTGTTGGCATTAGACAAATCAAGCTGGTGATTTAATAAATTC AP-1-like 967 CAATGTAAGTCTTACCAGTATTGATGAATAACTATCCAGCACTCACCATGAAAGTTAAAGAAGCAACACA 897 GAAAAAGTTCCTAAGTGGTCCCAATTTGAAATGATCAGATAACCTATAAAAGAACATATTCATATTATAC T T T T 827 TAACATAAACACATATAAATGCACTTACAGCAGTTACACAGTATTCTCTTCAATAACTAGTTTCCTTATG T T T T V 757 CATTAATGTGTAATAACAGCAACTACAATATTTAGATAATTATAAAAACCAAGGCAATAATTTAAAAACT T T T T 687 GATTAACCGTTTTACTCTAACTTAAGCATGGATTGGATCAGTAAGATTGATTAATAAATTTGAATGCAGT T T 617 CAGTTGGATTGATTCTAATTTAAAGTTTTAATTTGTTGTAGAATAATTTTAAGTGAATATATTTGTCCAG \ lnr\\ 1 InrW | | AP/CRE-1 | 547 TGTTCGAGTGCTCAACAGTGTGTTTGAAAAGGAAAACAAAGAAATGTTTTTGAGAAATGTGTTAATTCCT 4 77 TAAGACAATGGATTTTAATTGGATCTAGTTGTTTTCATTTTTCTTCATTATCATTATACATCTGTATGTT 4 07 GGACAGAACACTAACACTAAATAGTTTTTAGAAAAATTTTTTAAAGTTATTTAAATCATAATATCATGAC fAP/CRE-2 I 337 TGACATTTTAAATTCAAAATTAGGCTGTGACTATCCTTCTTCACTTAGGAAGAGTGTTGTGAAAGCCAGA 267 CCATCTGCTGAGGTGCTACAGTTACATGTGGCCCTCAGAATGCGTTTGGCCTGCTCTGTTTTAGCACTCT 197 GTTGGATTACCAATCACAAAACAAGTTAACCTTGATCTTTCACATTAAGTATCTCAGGGACAAAATTTGA GSE 127 CATACGTCTAAACCTGTGAACGTTTCCATCTAAAGAAGGCAGAAATAAAACAGGACTTTAGATTCGGTTA 5 7 CAATAAAATATCAGATGCACCAGAGACACAAGGCTTGAAGCTCTGTCCTGGGAAAATATGGCAAACAGTG +1 56 start codon (Albarracin et al, 1994; Reinhart et al, 1997), those for the human gene are no less than 703 bp from it (Fan et al, 1995). Moreover, the human GnRH-I receptor 5'-flanking region is more complex in that it contains multiple canonical TATA and CAAT boxes residing in close proximity to each other near the transcription start sites (Fan et al, 1995; Kakar, 1997). The presence of consensus TATA boxes is unusual among the GPCRs sequenced to date as many of these genes including the mouse FSH receptor (Huhtaniemi et al, 1992), human thyrotropin receptor (Gross et al, 1991), and human endothelin A receptor (Hosoda et al, 1992) have been shown to contain GC-rich promoter regions. 1.13 Transcriptional Regulation of the Human GnRH-I Receptor Gene 1.13.1 Identification and Characterization of a Gonadotrope-Specific GnRH-I Receptor Promoter The isolation of the human GnRH-I receptor 5'-flanking region has led to an intensive research on the transcriptional regulation of the gene. Using transient transfection and progressive deletion analysis, it has been demonstrated that the proximal 173 bp of the 5'-flanking region are important for directing gene expression in gonadotropes since deletion of this sequence results in an almost complete loss of promoter activity in alphaT3-l cells (Ngan et al, 1999). Two putative gonadotrope-specific elements (GSEs) with the core sequence TG(A/T)CC (Barnhart and Mellon, 1994) are present at -143/-135 and -13/-5 of the proximal promoter region. Such regulatory elements have been shown to confer cell-specific expression of the glycoprotein hormone a-subunit (Ffeckert et al, 1995) and LHP (Halvorson et al, 1996) 57 genes in pituitary gonadotropes. Site-directed mutagenesis has revealed that the upstream GSE (i.e. at -143/-135, 5TTGTCCCTG3') is essential for gonadotrope-specific transcription of the GnRH-I receptor gene, because mutation of this element drastically decreases the promoter activity in alphaT3-l cells but not in cell lines (SK-OV-3 and COS-7) derived from extrapituitary tissues (Ngan et al, 1999). Results from EMS As indicate that the orphan nuclear receptor SF-1 binds specifically to the upstream GSE and that the second, fifth, sixth, and the ninth nucleotides of the motif are crucial for the DNA-protein interaction (Ngan et al, 1999). The functional significance of SF-1 in regulating human GnRH-I receptor gene transcription is further confirmed by the observation that overexpression of sense and antisense SF-1 mRNAs can stimulate and repress the GnRH-I receptor promoter, respectively (Ngan et al, 1999). It should be noted that SF-1 is also expressed in extrapituitary sites, most prominently in steroidogenic tissues like the gonads and adrenal gland. The nonpituitary expression of SF-1 indicates that GSEs may not be the sole mediator in conferring gonadotrope-specific gene expression. Evidence supporting this view comes from a previous study on the transcriptional regulation of the mouse GnRH-I receptor gene in alphaT3-l cells. It was demonstrated that a proximal tripartite enhancer consisting of a SF-1 binding site, a canonical AP-1 site, and a novel element termed GnRH receptor activating sequence (GRAS) was indispensable for targeting cell-specific gene expression in the gonadotropes (Duval et al, 1997). Each of these DNA motifs was found to contribute equally to the promoter function, and in particular, the GRAS was shown to be capable of functioning as a stand-alone enhancer that could stimulate the activity of a heterologous promoter selectively in alphaT3-l cells (Duval et al, 1997). 58 Despite sharing a high degree of sequence homology with the proximal mouse GnRH-I receptor 5'-flanking region, the human gene is unlikely to contain the same module of regulatory motifs in governing gonadotrope-specific gene expression because neither the AP-1 site nor the GRAS are present in close vicinity of the functional GSE (Ngan et al, 1999). 1.13.2 Identification and Characterization of a Distal and Proximal GnRH-I Receptor Promoter The existence of multiple transcription start sites and TATA boxes in the human GnRH-I receptor 5'-flanking region indicates that multiple promoters participate in controlling the spatiotemporal expression of the gene. Using a stepwise deletion analysis, a distal TATA-less promoter working in an orientation-dependent manner has been identified between -1727 and -1674 of the flanking region (Ngan et al, 2000). This promoter is immediately upstream of a transcription start site (at -1672) determined in the pituitary (Kakar, 1997) and is transcriptionally active in both pituitary (alphaT3-l) and nonpituitary (SK-OV-3, COS-7, and JEG-3) cell lines. Block replacement mutagenesis indicates that the function of this distal promoter is dependent on at least two cw-acting regulating motifs, which include a consensus CAAT box (5'CCAAT3') at -1704/-1700, a pyrimidine-rich initiator (Inr) (5TCATTTC3') at -1683/-1677, and the sequence residing between them (Ngan et al, 2000). The ubiquitous nature of this promoter may be explained by the ability of multiple nuclear factors of 45-50 kDa commonly expressed in the pituitary and nonpituitary cells to interact specifically with the CAAT box and Inr (Ngan et al, 2000). Since it has been suggested that TATA-59 binding protein (TBP)-associated factors are capable of interacting with Inr elements (Kaufmann and Smale, 1994; Purnell et al, 1994; Verrijzer et al, 1995), the functional Inr (at -1683/-1677) may serve as a "TATA box" to accurately direct the positioning of the general transcription apparatus and initiate the transcription of the GnRH-I receptor gene. Recently, an additional TATA-less human GnRH-I receptor promoter that is active in alphaT3-l cells has been found between -677 and -558 of the 5'-flanking region (Hoo et al, 2003). This promoter element works in an orientation-dependent manner and is believed to be responsible for a cluster of proximal transcription start sites identified in the pituitary (Kakar, 1997). Two Inr elements, Inr I (at -603/-598, 5'CTAATT3') and Inr II (at -590/-584, 5'TTAATTT3'), are crucial for the promoter function since mutation of either motif results in a complete loss of promoter activity (Hoo et al, 2003). Using Southwestern blot analysis and competitive EMS As, multiple protein factors of 36-150 kDa and belonging to the transcription factor class II (TFII) regulators have been demonstrated to interact with both Inr motifs as well as the distal Tnr-driven promoter specifically (Hoo et al, 2003). Since the distal and proximal Inr elements discussed are important for the functioning of the GnRH-I receptor promoter in gonadotropes, it is speculated that they cooperate with the GSE (at -143/-135) to mediate full gonadotrope-specific expression of the GnRH-I receptor gene. 60 1.13.3 Identification and Characterization of a Placenta-Specific GnRH-I Receptor Promoter Multiple lines of evidence indicate that tissue-specific gene expression can be mediated via the use of different promoters in various cell types (Flouriot et al., 1998; McCormick et al., 2000; Shields et al., 2001). In accord with this notion, Cheng and coworkers have identified a novel human GnRH-I receptor promoter between -1737 and -1346 of the 5'-flanking region, which is highly active in JEG-3 and IEVT cells (Cheng et ai, 2001b). The usage of this distal promoter is supported by the identification of five transcription start sites at -1629, -1608, -1416, -1391, and -1379 in the placental cells (Cheng et al., 2001b). Four putative transcription factor binding sites, namely hGR-Oct-1 (at -1718/-1711, 5'ATTTGTAT3'), hGR-CRE (at -1650/-1642, 5TGACATGA3'), hGR-GATA (at -1603/-1598, 5TTATCT3'), and hGR-AP-1 (at -1519/-1513, 5'TGAGTCA3'), are present in the distal promoter region. Mutation of the hGR-Oct-1 or hGR-AP-1 motif significantly reduces the promoter activity in various cell lines, indicating that these DNA sequences function ubiquitously. Conversely, the hGR-CRE and hGR-GATA motifs appear to play a specific role in placenta-specific gene transcription since mutation of either motif results in a considerable loss of promoter activity in JEG-3 cells exclusively (Cheng et al, 2001b). Using competitive and antibody supershift EMS As, specific binding of the transcription factors Oct-1, CREB, GATA-2, GATA-3, and c-Jun/c-Fos heterodimer to the corresponding motifs has been demonstrated (Cheng et al, 2001b). Although it is known that CREB, GATA-2, and GATA-3 are important regulators of gene expression in the placenta in vivo (Bokar et al, 1989; Pittman et al, 1994; Heckert et al, 1995; Ma et al, 1997; Scatena and Alder, 61 1998), the mechanism leading to the placenta-specific repression of the human GnRH-I receptor promoter following mutation of the hGR-CRE and hGR-GATA motifs is obscure. It is postulated that a placenta-specific cofactor is required to mediate the CREB and GATA actions as protein-protein interactions have been shown to play an important role in regulating the activity of these transcription factors (Montminy, 1997; Robyr et al, 2000). 1.13.4 Transcriptional Regulation by GnRH-I Homologous regulation of GnRH-I receptor gene expression is an important mechanism for controlling the sensitivity of pituitary gonadotropes to its own ligand. It has been shown that treatment of alphaT3-l cells with a GnRH-I agonist results in a dose- and time-dependent repression of the human GnRH-I receptor promoter (Cheng et al, 2000b). This inhibitory effect can be abolished by pretreatment with the PKC inhibitor GF109203X or depletion of PKC, indicating a role of the PKC signaling cascade in mediating the GnRH-I response. Subsequent experiments have indicated that a 248-bp region between -1018 and -771 is sufficient for mediating the repression and that mutation of an AP-l-like motif (at -1000/-994, 5'TT AG AC A3') can abolish the sensitivity of the promoter to both GnRH-I and phorbol ester (Cheng et al, 2000b). EMSAs reveal that a single DNA-protein complex corresponding to the binding of c-Jun homodimer to the AP-l-like motif is formed when nuclear extracts from untreated alphaT3-l cells are used. However, an additional complex that is recognized by both anti-c-Jun and anti-c-Fos antibodies is produced when extracts from GnRH-I-stimulated cells are used (Cheng et al, 2000b). Therefore, it is apparent that homologous 62 downregulation of the human GnRH-I receptor gene transcription involves induction of c-Fos DNA binding activity at the AP-l-like motif. Interestingly, earlier studies of the homologous activation of the mouse GnRH-I receptor promoter in alphaT3-l cells also revealed an integral role of the PKC signaling pathway and an AP-1 binding site termed Sequence Underlying Responsiveness to GnRH-2 (Norwitz et al, 1999; White et al, 1999). In addition, a substantial contribution of this stimulation is observed with the ERK cascade because addition of PD98059 dramatically reduces the GnRH-I effect (White et al, 1999). However, it should be noted that under the conditions that can produce the maximal stimulatory response of the rodent promoter (Norwitz et al, 1999; White et al, 1999), a significant decrease instead of increase in promoter activity is observed for the human gene (Cheng et al, 2000b). Whether this discrepancy is due to different experimental paradigms or the existence of differential mechanisms in controlling GnRH-I responsiveness of the human and rodent promoters in gonadotropes is not fully understood. 1.13.5 Transcriptional Regulation by the cAMP-Dependent Signal Transduction Pathway It has been well documented that cAMP can enhance the responsiveness of gonadotropes to GnRH-I by upregulating GnRH-I receptor gene expression (Young et al, 1984; Clayton et al, 1985; Turgeon and Waring, 1986; Hawes et al, 1993; Abdilnour and Bourne, 1995; Cassina et al, 1995). Consistently, an increase in GnRH-I receptor mRNA level has been detected in placental cells after forskolin treatment 63 (Cheng et al, 2000a). These stimulatory effects of cAMP are believed to be mediated at the transcriptional level since forskolin can dose- and time-dependently activate the human GnRH-I receptor promoter in both alphaT3-l and JEG-3 cells (Cheng and Leung, 2001; Cheng and Leung, 2002). Similar responses of the human promoter have also been observed with other physiological regulators that activate the cAMP-dependent signaling pathway (Cheng and Leung, 2001; Cheng and Leung, 2002). Using progressive deletion analysis, the forskolin response area has been mapped to a region between -577 and -167 of the 5'-flanking sequence, in which two potential AP-1/CREB binding sites termed hGR-AP/CRE-1 (at -5697-562, 5'TTAAGTGA3') and hGR-AP/CRE-2 (at -341/-334, 5TGACTGAC3') are identified (Cheng and Leung, 2001; Cheng and Leung, 2002). These regulatory motifs contribute partly to the forskolin-stimulated effect since mutation of either motif or both does not completely abrogate the response (Cheng and Leung, 2001; Cheng and Leung, 2002). Results from competitive and supershift EMS As indicate that both hGR-AP/CRE-1 and hGR-AP/CRE-2 motifs interact specifically with CREB in forskolin-stimulated cells. Intriguingly, a differential binding of transcription factors to hGR-AP/CRE-2 is observed in alphaT3-l cells such that the motif binds primarily to AP-1 under nonstimulated condition (Cheng and Leung, 2001; Cheng and Leung, 2002). This observation is not unexpected as CREB and AP-1 have been demonstrated to be capable of exchanging partners and forming heterodimers with altered DNA binding specificity (Hai and Curran, 1991; Chatton et al, 1994, De Casare et al, 1995; Millhouse et al, 1998). 64 1.13.6 Transcriptional Regulation by P4 The dynamic changes in GnRH-I receptor number and its mRNA level in pituitary gonadotropes during the estrous cycle (Bauer-Dantoin et al, 1993; Brooks et al, 1993; Funabashi et al, 1994) or after gonadectomy (Kaiser et al, 1993; Sakurai et al, 1997) strongly suggest a role of gonadal steroids in regulating GnRH-I receptor gene expression. Accordingly, a recent study from Cheng and coworkers has revealed that P 4 can dose- and time-dependently repress the human GnRH-I receptor promoter in alphaT3-l cells (Cheng et al, 2001a). In contrast, P 4 exerts a stimulatory effect on the human promoter in the placenta as treatment with RU486 causes a significant reduction of promoter activity in JEG-3 cells (Cheng et al, 2001a). Consistently, blockage of the endogenous P 4 production in the placental cells with aminoglutethimide represses the GnRH-I receptor promoter, and this suppression can be reversed by P 4 replacement. Progressive 5'-deletion analysis indicates that a region between -577 and -227 of the human gene is responsible for mediating the P 4 responses in both alphaT3-l and JEG-3 cells (Cheng et al, 2001a). Mutation of an imperfect progesterone-response element (PRE) in this region (at -536/-522, 5'TCAACAGTGTGTTTG3') significantly attenuates the repressive effect of P 4 in the gonadotropes and completely abolishes the inhibitory effect of RU486 in the placental cells (Cheng et al, 2001a). Using competitive and supershift EMSAs, a specific binding of PRs to the PRE has been demonstrated, indicating a direct involvement of the steroid hormone receptors in mediating the transcriptional effects (Cheng et al, 2001a). Functional studies of overexpressing the two human PR isoforms (PR-A and PR-B) indicate that PR-B plays a more predominant role than PR-A in mediating the downregulatory effect of P 4 in alphaT3-l cells. 65 Surprisingly, a differential role of PR-A and PR-B in controlling the human GnRH-I receptor gene transcription is observed in JEG-3 cells such that PR-B stimulates while PR-A inhibits the activity of the GnRH-I receptor promoter. In agreement with these findings, PR-B has been shown to be the major PR subtype in the placental cells (Cheng et al, 2001a), thus supporting a positive role of P4 in regulating GnRH-I receptor gene expression in the placenta. 1.13.7 Transcriptional Repression Besides activation by enhancer elements, gene expression may be repressed by negative regulatory genetic elements, of which there are two types, namely classical silencers and negative regulatory elements (NREs). Classical silencers are defined as sequence elements that are capable of repressing promoter activity in an orientation- and position-independent fashion, in the context of a native or a heterologous promoter (Brand et al, 1985). Conversely, NREs are orientation- or position-dependent DNA elements that direct a passive repression mechanism (Ogbourne and Antalis, 1998). Studying the process of transcriptional repression is essential for our complete understanding on the promoter structure and regulation of gene expression. Recently, Ngan and coworkers have identified a putative NRE residing between -1673 and -1351 of the human GnRH-I receptor 5'-flanking region. This NRE can constitutively repress the GnRH-I receptor promoter in many different cell types in an orientation-independent but position-dependent manner (Ngan et al, 2001). Interestingly, the first 97 bp of the NRE (i.e. from -1673 to -1577) is responsive to forskolin such that treatment with the adenylyl cyclase activator relieves most of the NRE silencing activity. Similar effects 66 are also observed with pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 but not with vasoactive intestinal peptide (Ngan et al, 2001), which implicates mediation via the PAC i -receptor. Using block replacement mutagenesis, two czs-acting elements at -1671/-1644 (5'TATGGCCCTGATCAGATTAACTGACATG3') and -1630/-1623 (5'AGCCTTTT3') have been demonstrated to be important for the NRE function. Although both elements can interact specifically with nuclear factors derived from alphaT3-l cells, an increase in complex intensity is only observed for the -1671/-1644 motif following PACAP stimulation (Ngan et al, 2001), indicating that it may serve as a PACAP-response element (PARE). It is postulated that PACAP may upregulate the expression and/or activity of a transcription factor, which is presumably an activator for the human GnRH-I receptor promoter. The increase in binding of the activator to the PARE may attenuate the NRE activity, and thereby retrieve the function of the native promoter. 1.14 GnRH-II Receptor 1.14.1 Nonhuman Primate GnRH-II Receptor The fact that GnRH-II is expressed in humans (White et al, 1998) and that a receptor specific for GnRH-II exists in a number of fish, amphibians, and monkeys (Tensen et al, 1997; tiling et al, 1999; Troskie et al, 2000; Millar et al, 2001; Neill et al, 2001; Okubo et al, 2001; Wang et al, 2001) strongly support the existence of a second GnRH receptor subtype in humans. Molecular cloning of the marmoset GnRH-II receptor cDNA reveals that it codes for a typical seven-TM GPCR that is composed of 380 amino acid residues (Millar et al, 2001) (Figure 8). Strikingly, the monkey GnRH-67 Figure 8. Alignment of the amino acid sequence of the marmoset GnRH-II and human GnRH-I receptors. Numbers along side the sequences represent the amino acid positions. Residues conserved in the two receptors are typed in bold. Seven a-helical regions predicted by homology modeling with the rhodopsin crystal structure are indicated. These helices encompass the membrane spanning regions. Strikingly, the GnRH-II receptor possesses a carboxyl-terminal tail that is absent in the GnRH-I receptor. A putative glycosylation site at the amino terminus and a disulfide bridge in the GnRH-II receptor are marked with white and black circles, respectively. 68 I GnRH-II receptor 1 MSAVNGTPWGSSAREEVWAGSGVEVEGSELPTFSTAAKVR GnRH-I receptor 1 MANSASPEQNQMHCSAINNSIPLMQGN..LPTLTLSGKIR I II GnRH-II receptor 41 VGVTIVLFVSSAGGNLAVLWSVTRPQPSQ...LRPSPVRR GnRH-I receptor 3 9 VTVTif'FLF.Ll'jSATF'NASFLLiKLQKWTQKKEKGKKLS II III GnRH-II receptor 78 LFAHLAAADLLVT FVVMPLDATWNITVQW1.AGDIACRTLM GnRH-I receptor 79 LLKHLTLANLL.ETLIVMPLDGMWNITVQWi'AGELLCKVLS III IV GnRH-II receptor 118 FLK L M A M Y A ? A F L P W I G L D R Q A A V L N P L G S R S . . . GVRK GnRH-I receptor 1 1 9 YLKLFSMYAP A F M M W I S L D R S L A I T R P L A L K S N S K V G Q S IV GnRH-II receptor 155 LLGAAWGLSFLLAl.PQLFLF. . . HTVRRAGPVP . FTQCAT GnRH-I receptor 159 MVGLAWILSSVFAGPQLYIFRMIHLADSSGQTKVFSQCVT GnRH-II receptor 191 KGSFKARWQETTYNLFTFCCLFLLPLTAMAICYSRIVLGV GnRH-I receptor 199 HCSFSQWKHQAFTtNFFTFSCLFI.IPLFIMl.ICNAK.ilFTL VI GnRH-II receptor 231 SSPRTRKGSHAPAGEFALRRSFDNRPRVRLRALRLALLVL GnRH-I receptor 239 TRY LHQDPHKLQLNQSKNNIPRARLKTLKMTVAFA VI VII GnRH-II receptor 271 LTFILCWTPYYLLGLWYWFSPSMLSEVPPSLSHILFLFG:., GnRH-I receptor 2 74 TSFTVCWTPYYVLGIWYWFDPEMLNRLSDPVNHFFFIiFA? VII GnRH-II receptor 311 LNAPLDPLLYGAFTLGCRRGHQELSMDSSREEGSRRMFQQ GnRH-I receptor 314 LNPCFDPLIYGYFSL GnRH-II receptor 351 DIQALRQTEVQKTVTSRRAGETK GIPITSI 6 9 II receptor possesses a carboxyl-terminal tail, which is important for rapid receptor desensitization and internalization (Blomenrohr et al, 1999) and is absent from the GnRH-I receptor (Stojilkovic et al, 1994; Sealfon et al, 1997). The monkey receptor also does not have the unusual Asn/Asp microdomains in TM helices 2 and 7 of the GnRH-I receptor, which play a role in receptor activation (Flanagan et al, 1999). Instead, it contains the Asp/Asp motif as in nonmammalian GnRH receptors recently cloned (Tensen et al, 1997; Illing et al, 1999; Troskie et al, 2000; Sun et al, 2001; Wang et al, 2001). In addition, the Leu-Ser-Asp/Glu-Pro sequence in the third extracellular loop, which is important for ligand selectivity of the GnRH-I receptor (Flanagan et al, 1994; Sealfon et al, 1997), is replaced by Val-Pro-Pro-Ser, which is also found in the reptile and amphibian GnRH-II receptors (Troskie et al, 1998). Pharmacological characterization of the monkey GnRH-II receptor reveals that it is highly selective for GnRH-II in receptor binding assays and in stimulation of inositol phosphate production (40- and 90-fold greater activity relative to GnRH-I). Overall, GnRH-II has an affinity of 24-fold greater for the GnRH-II receptor than for the GnRH-I receptor. Also, the GnRH-II receptor is more selective for salmon GnRH and [D-Arg6]-GnRH-II (Millar et al, 2001). RT-PCR and Northern blot hybridization demonstrate that the GnRH-II receptor is widely expressed in monkey tissues (Millar et al, 2001). Interestingly, results from immunocytochemical studies have revealed that both GnRH-I and GnRH-II receptors are coexpressed in the majority of gonadotropes in sheep anterior pituitary (Millar et al, 2001), indicating that these receptors may differentially regulate FSH and LH biosynthesis and release. In fact, there are substantial lines of evidence invoking the 70 existence of a FSH-releasing hormone to account for the differential secretion of gonadotropins (Lumpkin et al, 1987; Yu et al, 1997; Yu et al, 2000; Padmanabhan and McNeilly, 2001). For instance, it has been demonstrated that chicken GnRH-II has a preferential FSH-releasing activity than chicken GnRH-I in pituitary cells (Millar et al, 1986). Functional studies in COS-7 cells indicate that the monkey GnRH-II receptor, like the GnRH-I receptor, is coupled to G a q/n and can activate ERK1/2. However, the GnRH-II receptor is unique in that it can also stimulate p38 MAPKs (Millar et al, 2001). Furthermore, the GnRH-II receptor is distinct from the GnRH-I receptor in that the former can be activated by GnRH-I antagonists, leading to the production of intracellular inositol phosphate (Millar et al, 2001). This observation may provide a possible explanation for the exhibition of agonistic activity by GnRH-I antagonists in some ovarian cancer cells (Emons et al, 1993a; Yano et al, 1994b; Tang et al, 2002). Thus, although both GnRH receptors are coupled to the same G a protein, they can trigger different signal transduction cascades and may have differential roles in regulating gonadotrope functions such as gonadotropin secretion. 1.14.2 Putative GnRH-II Receptor Genes in the Human Genome Searches of the human genome database have revealed a putative GnRH-II receptor gene on chromosome lql2 (Neill et al, 2001; Morgan et al, 2003) (Figure 9). This gene shares a 40 % sequence identity with the GnRH-I receptor gene and is composed of at least three exons spanning approximately 7.5 kb along the chromosome. The first exon is 509-bp long and encodes the first four TM helices and connecting domains. The second exon is located 4.25 kb downstream and codes for the fifth TM 71 Figure 9. Diagram showing the distribution of the putative GnRH-II receptor, PEXlip, and R B M 8 genes in the human genome. A putative GnRH-II receptor gene containing at least three exons is present on chromosome lql2. Coding exons are represented by open boxes and are numbered. The size of each exon and intron is shown in bp and kb, respectively. The reading frame of this putative receptor gene is disrupted by a -1 frameshift and contains a premature stop codon in exon 2. The PEX1 ip gene lies in the opposite orientation on the same chromosomal region such that its distal promoter region overlaps that of the receptor gene. Putative transcription factor binding sites for these genes are indicated (G, GATA; M, MEF-2; P, Pit-1; S, Spl; U, UAS). On the other hand, the two carboxyl-terminal coding exons of the receptor gene overlap in the antisense orientation with the 3'-UTR of the RBM8A gene. Besides the chromosome 1 locus, a truncated copy of the GnRH-II receptor gene lacking the first coding exon and part of the intron 1 exists on chromosome 14q22. This region contains an intronless RBM8A sequence (termed RBM8B) that lies in the opposite direction along the chromosome. The RBM8B is a pseudogene containing eight nucleotide mutations, including one that generates a premature stop codon. This pseudogene is probably originated from the RBM8A locus by reverse transcription and insertion into the genome. The diagram is not drawn to scale. 72 Chromosome 1q12 GnRH-II receptor gene Frameshift Premature stop codon (TGA) L L L bum ri nidi ie iu  u JU] V im MPSS 5 0 9 I 2 1 l f 4 1 9 (bp) -4.25 0.449 (kb) m m mm mmmmmm S S GM U PEX11P gene RBM8A gene Chromosome 14q22 Truncated GnRH-II receptor gene Premature stop codon (TGA) 5. mm 3. 16I5I*1I3I2I11 Premature stop codon (TAA) RBM8B gene 73 and flanking domains. The third exon is separated from the second one by an intron of 449 bp and encodes the rest of the TM domains and a cytoplasmic tail. Strikingly, the reading frame of this putative GnRH-II receptor gene is apparently disrupted by a -1 frameshift, which is caused by a deletion of a guanine nucleotide located 28 bp downstream of the putative ATG initiation codon in the human sequence. In addition, the gene sequence contains a cytosine to thymine substitution that changes the codon Arg 1 7 9 in the monkey and frog sequences to an in-frame UGA premature stop codon (Morgan ef al, 2003). The putative GnRH-II receptor gene is closely linked to the peroxisomal membrane protein 11-P (PEXlip) gene (Schrader et al, 1998), which lies in an opposite direction along the locus such that the distal promoter region of these genes overlaps with each other (Figure 9). Sequence analysis reveals that putative transcription factor binding sites are well separated on the opposite DNA strands, with the exception of a shared MEF-2 motif. The core promoter of both genes lacks canonical TATA boxes but contains two GC-rich Spl sites. Significantly, the presence of a potential Pit-1 binding site and a motif similar to the yeast upstream activation sequence (UAS) at the 5' end of the receptor and PEXlip genes indicates that their transcription is regulated independently (Gurvitz et al, 2001; Morgan et al, 2003). The two carboxyl-terminal coding exons of the GnRH-II receptor gene overlap in the antisense orientation with the 3'-UTR of the RBM8A gene (Conklin et al, 2000; Salicioni et al, 2000; Faurholm et al, 2001; Neill, 2002) (Figure 9). The RBM8A gene is composed of six exons and five introns, with all exon-intron boundaries containing the consensus splice junctional sequence. Northern blot analysis shows that 74 the gene displays ubiquitous and high levels of expression in human tissues, supporting important cellular functions for its encoded product (Conklin et al, 2000). Two RBM8A transcripts encoding proteins of 173 and 174 amino acids arisen from alternative splicing have been reported (Conklin et al, 2000; Salicioni et al, 2000; Zhao et al, 2000b). These proteins share a substantial homology (55 %) with a C. elegans ribonucleoprotein termed R07E5.3 and contain a recognizable RNA binding domain that is composed of a two-layer a/p sandwich, comprising two amphipathic helices packed against a four-stranded P sheet (Xu et al, 1997). In addition to the putative gene on chromosome 1, a truncated copy of the GnRH-II receptor gene lacking the first exon and part of the intron 1 is present on chromosome 14q22 (Figure 9). Sequence analysis reveals that this locus contains in the opposite direction an intronless RBM8A sequence, which is uniquely flanked by a stretch of 16 adenines and two direct repeats of 7 (TTTAAAT) and 11 (GTTTTTTTTTT) nucleotides (Faurholm et al, 2001). The open reading frame of this intronless gene carries eight nucleotide mutations, including one that generates a premature stop codon at a position equivalent to amino acid 84 of the wild-type protein, indicating that the intronless gene is a pseudogene (termed RBM8B) and is probably originated from the chromosome 1 locus by reverse transcription and insertion into the genome (Faurholm et al, 2001). 1.14.3 Tissue Distribution of Human GnRH-II Receptor Transcripts Expression of GnRH-II receptor mRNA has been demonstrated throughout the human brain as well as in the heart, pancreas, salivary gland, lymph node, peripheral 75 leukocytes, mature sperm, and postmeiotic testicular cells (Millar et al, 2001; van Biljon et al, 2002). In addition, Grundker and colleagues have shown that the receptor is also expressed in certain human endometrial and ovarian cancer cell lines (Grundker et al, 2002). Furthermore, a number of differently spliced receptor transcripts have recently been identified in pituitary adenoma HP-75 and neuroblastoma IMR-32 cells (Morgan et al, 2003), thus strengthening the notion that the human GnRH-II receptor gene is transcriptionally active. Most importantly, GnRH-II receptor immunoreactivity has been demonstrated in human anterior pituitary using an antibody (ECL-3) that is raised against the third extracellular loop of the putative receptor (Millar et al, 2001). Nevertheless, to date, direct proof for the existence of a full-length, properly processed, and functional GnRH-II receptor transcript in humans is lacking. So far, nucleotide sequencing of all the human transcripts has revealed no alteration of the premature stop codon by posttranscriptional RNA editing, though in some instances, alternative splicing of part of the exon 1 to circumvent the frameshift to encode a two-TM domain protein is observed (van Biljon et al, 2002; Morgan et al, 2003). Several potential translation mechanisms have been proposed to explain the immunocytochemical detection of ECL-3 epitope in human pituitary. One possibility is that translation begins at the second ATG start codon (situated at the end of the second TM domain) such that a single base transition occurs in the in-frame termination codon. These events will generate a truncated five-TM domain receptor that lacks the first two TM helices (Morgan et al, 2003). In fact, functional GPCRs having only five TM domains have been reported in mutant chemokine receptors (Ling et al, 1999), supporting the existence of functional five-TM domain GPCRs in nature during the 76 course of evolution. In addition, it is possible that a functional GnRH-II receptor is generated by lateral interaction or domain swapping of the truncated GnRH-II receptor gene products with the GnRH-I receptor or with other GPCRs, as hypothesized for some receptors (Gouldson et al, 1998; Schultz et al, 2000). Evidence from others indicates another unusual mechanism by which a functional GnRH-II receptor may be produced. This process involves the incorporation of a selenocysteine residue at the UGA codon, instead of encoding a translation termination signal. Indeed, a number of mammalian proteins containing selenocysteines encoded by in-frame UGA codons have been identified (Behne and Kyriakopoulos, 2001). Selenocysteine incorporation requires a selenocysteine-insertion sequence of approximately 200 nucleotides that form a stem-loop structure in the 3'-UTR of the mRNA (Berry et al, 1991). The finding that selenocysteine is incorporated more efficiently when the UGA codon is positioned closer to the centre of the coding region, as in the case for the human GnRH-II receptor, than is located close to one of the ends (Wen et al, 1998) supports the selenocysteine incorporation hypothesis. Nonetheless, insertion of a guanine residue to correct the frameshift in the 5'-region of the transcript is still required if a full-length functional receptor is to be generated. A major question remains to be answered is whether a correctly spliced GnRH-II receptor mRNA transcript can be identified in certain human cell types or tissues under appropriate physiological conditions. 77 CHAPTER II Materials and Methods 2.1 Cells and Cell Culture 2.1.1 Primary GL Cell Culture The use of human GL cells was approved by the Clinical Screening Committee for Research and Other Studies involving Human Subjects of the University of British Columbia. Primary GL cell cultures were prepared as previously described using follicular aspirates collected from women undergoing in vitro fertilization (Nathwani et al, 2000). Briefly, follicular contents were centrifuged at 1000 x g for 15 min to remove the fluid. Afterward, cell pellets were resuspended in 6 ml of Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Inc., Burlington, Canada) supplemented with 100 units/ml penicillin and 100 pg/ml streptomycin (Invitrogen, Inc.) and then layered gently onto an equal volume of Ficoll Paque (Amersham Pharmacia Biotech, Morgan, Canada). Following centrifugation to remove the red blood cells, cells in the interface (GL cells) were collected and washed twice with DMEM before resuspending in the same medium supplemented with 10 % fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, UT). The GL cells were cultured at an initial density of 2 x 105 cells in 3 5-mm culture dishes for 4 days at 37 °C in humidified atmosphere of 5 % CO2 in air before RNA and protein extraction. 2.1.2 Cell Lines Human ovarian adenocarcinomas OVCAR-3 and SK-OV-3, human choriocarcinoma JEG-3, human kidney HEK-293, human neuronal medulloblastoma 78 TE-671, human breast adenocarcinoma MCF-7, and African green monkey kidney COS-7 cells were obtained from American Type Culture Collection (Manassas, VA). Mouse gonadotrope-derived alphaT3-l and human neuroblastoma SH-SY5Y cells were kindly provided by Dr. P. L. Mellon (Department of Reproductive Medicine, University of California, San Diego, CA) and L. T. O. Lee (Department of Zoology, University of Hong Kong, HKSAR, China), respectively. Immortalized human GL cell lines (SVOG-4o and SVOG-4m) (Lie et al, 1996), immortalized OSE cell line (IOSE-29EC) (Ong et al, 2000), as well as human dermal fibroblasts (HDFs) were provided by Dr. N. Auersperg (Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada). The SVOG-4o, SVOG-4m, and IOSE-29EC cell lines were maintained in Medium 199/MCDB 105 (1:1) (Sigma-Aldrich, St. Louis, MO), whereas all other cell lines were maintained in DMEM, with both media supplemented with 10 % FBS. Cultures were maintained at 37 °C in humidified atmosphere of 5 % CO2 in air. Culture media were renewed every 3 to 4 days. Cells were passaged when they reached about 80 % confluence using trypsin/EDTA solution (0.05 % trypsin and 0.53 mM EDTA) (Invitrogen, Inc.). 2.2 Plasmid Construction 2.2.1 Human GnRH-I Receptor Promoter-Luciferase Constructs The full-length human GnRH-I receptor promoter-luciferase construct p(-2297/+l)-Luc (all numbering is relative to the ATG start codon, of which the position is designated as +1) and deletion constructs [p(-2197/+l)-Luc, p(-1671/+l)-Luc, p(-1346/+1)-Luc, p(-1018/+l)-Luc, p(-771/+l)-Luc, p(-707/+l)-Luc, p(-2197/-167)-Luc, 79 p(-2197/-771)-Luc, p(-2197/-1018)-Luc, and p(-2197/-1346)-Luc] were prepared as previously described (Cheng et al, 2000b; Cheng et al, 2001b). Constructs for fine deletion mapping [p(-1900/-1018)-Luc, p(-1700/-1018)-Luc, p(-1500/-1018)-Luc, p(-1300/-1018)-Luc, p(-1300/-1009)-Luc, p(-1300/-994)-Luc, p(-1300/-979)-Luc, p(-1300/-964)-Luc, p(-1300/-954)-Luc, p(-1300/-894)-Luc, p(-1300/-834)-Luc, p(-1300/-771)-Luc, p(-487/+l)-Luc, p(-367/+l)-Luc, p(-266/+l)-Luc, p(-214/+l)-Luc, and p(-117/+l)-Luc] were generated by PCR of the corresponding regions of the 5'-flanking region, followed by cloning of the amplified fragments into the promoterless pGL2-Basic vector (Promega Corp., Nepean, Canada). Heterologous thymidine kinase (TK) promoter constructs (sNRE-pTK-Luc and rNRE-pTK-Luc) were generated by PCR of the NRE (from -1017 to -771 of the 5'-flanking region), followed by cloning of the amplified product into the BamHl site of the pTK-Luc vector (kindly provided by Dr. V. Giguere, Molecular Oncology Group, McGill University Health Center, Montreal, Quebec) in both orientations. The same PCR product was also cloned in both directions into the BamRl site of p(-1300/-1018)-Luc to generate sNRE-p(-1300/-1018)-Luc and rNRE-p(-1300/-1018)-Luc. PCR reactions were carried out for 30 cycles with denaturation for 30 seconds (s) at 94 °C, annealing for 1 min at 60 °C, extension for 1 min at 72 °C, and a final extension for 15 min at 72 °C. Mutant human GnRH-I receptor promoter-luciferase constructs [dC/EBP-mut, GATAa-mut, pC/EBP-mut, (dC/EBP + pC/EBP)-mut, (GATAa + pC/EBP)-mut, (dC/EBP + GATAa + pC/EBP)-mut, nuclear factor-Y (NF-Y)-mut, estrogen-response element (ERE)-like-mut, GATAb-mut, AP-l-like-mut (a), and AP-l-like-mut (b)] were prepared by a three-step PCR mutagenesis method described by Chow and coworkers 80 (Chow et al, 1991) using site-specific mutagenic primers MP-dC/EBP, MP-GATAa, MP-pC/EBP, MP-NF-Y, MP-ERE-like, MP-GATAb, MP-AP-l-like (a), and MP-AP-1-like (b) as well as universal primers MP-B, MP-D, pGL2-BasicR, and p+1 (Table 3). Each mutagenic primer contains a restriction enzyme recognition site or a point mutation in the core binding sequence. As shown in Figure 10, the wild-type construct p(-1300/-1018)-Luc or p(-266/+l)-Luc was used as a template in the first and second steps of the PCR reaction. Following the first PCR, products defined by MP-B and mutagenic primers were amplified and then purified by the High Pure PCR Product Purification Kit (Roche Diagnostic Corp., Quebec, Canada). Afterward, PCR reactions of 10 cycles were performed using the purified products from the first amplification as primers. In the third step, primers pGL2-BasicR (or p+1) and MP-D were used with products from the previous reaction as templates, and the final amplified products were cloned into the pGL2-Basic vector. MP-D is designed in such a way that it spans the unique region at the 5'-end of MP-B to avoid amplification of the wild-type sequence. Mutant human GnRH-I receptor promoter-luciferase constructs (mut-a, mut-b, mut-cOct-1, and mut-rOct-1) were prepared by PCR using the forward primer p-1300, reverse primers carrying the desired mutations (Table 3), and the plasmid p(-1300/-1009)-Luc as a template. E-box-l-mut and E-box-2-mut were produced by PCR using forward primers carrying the mutations (MP-E-box-1 and MP-E-box-2), the reverse primer p+1, and p(-266/+l)-Luc as a template. Mutations in all constructs were confirmed by restriction mapping and DNA sequence analysis. 8 1 Table 3. Primers used in RT-PCR, plasmid construction, primer extension, 5'-RACE, site-directed mutagenesis, EMSA, ultraviolet (UV) crosslinking, and Southwestern blot studies. Only the sense oligonucleotides used in EMSAs are shown. Restriction enzyme recognition sites introduced into some oligonucleotides are shown in italic, whereas mutated sequences are underlined. Primers are listed according to the order of their appearance in the thesis. Primer Sequence (5' to 3') Purpose G n R H - I R (F) G G G A T G T G G A A C A T T A C A G T C C R T - P C R G n R H - I R (R) G G A T G A T G A A G A G G C A G C T G A A R T - P C R p-1900 A T C T C G A G A G G G A A G G G A A G G G G A A G G G A A G G Plasmid construction p-1700 ATCTCGA G T G G T T A T C C T G T T T T G T T C A T T T C Plasmid construction p-1500 ATCTCGAGGATGTAGGTAAAAATTTTATGGTC Plasmid construction p-1300 ATCTCGA G G T A C A C A G A G A A G C T G A G G A A A T A Plasmid construction and r site-directed mutagenesis p-1018 A T 4 / 1 G C 7 T C T G C A G C A G C C A A A G C A G G T A C A A Plasmid construction PE-1 T T T A T T A A T C A A T C T T A C T G A T Primer extension PE-2 T C A G C T T C T C T G T G T A C T G G C T Primer extension A T T G C C T G A G T T C A C T G C A C A A A T A A A G C A A T A _ ,. M P - B G C A T C A C A A A T T T C A C A Site-directed mutagenesis M P - D A T T G C C T G A G T T C A C T G C Site-directed mutagenesis 82 p G L 2 - B a s i c R T C C A G C G G T T C C A T C C T C T A G A G G Site-directed mutagenesis M P - d C / E B P ^ G C C ^ ^ ^ ^ ^ A A Q Q S.e-directed mutagenests M P - G A T A a T A C T A A A T A T T T A C T A T A T A G ^ C C ^ T A G C C T s i t e . d i r e c t e d m u t a g e n e s i s Mf u / \ iAa T T G C T G G T G A T A A A 5 M P - p C / E B P T T T A T T T A T T T A A T A A A T G G G ^ C C G C T A T T T A S i t e . d i r e c t e d m u t a g e n e s i s w p u C B r C T A T A T A C A T T A T C 5 G S - d C / E B P C T A A C C T T C T G T G G T A A C A A G C T C C T T E M S A G S - G A T A a A A A G G C T A A G A T A A T G T A T A T A G T E M S A G S - p C / E B P A T A G T A A A T A T T T A G T A A C C A T T T A T T A E M S A G S - d C / E B P - m u t C T A A C C T T C G C G G C C G C C A A G C T C C T T E M S A G S - G A T A a - m u t A A A G G C T A G C G G C C G C T A T A T A G T E M S A G S - p C / E B P - m u t A T A G T A A A T A G C G G C C G C C C A T T T A T T A E M S A N R E (s) A7GGA r C C A A G C A A A C T T G T T G G C A T T A G A C A Plasmid construction N R E (r) ATGGA rCCACTAGTTATTGAAGAGAATACTGT P lasmid construction p-771 ATAA GCrrGTTATTGAAGAGAATACTGTGTA P lasmid construction p-834 ATAA GCr7TATGAATATGTTCTTTTATAGGTT P lasmid construction p-894 ATAA GCr7TTTCTGTGTTGCTTCTTTAACTTT Plasmid construction p-954 ATAA G C r 7 T A A G A C T T A C A T T G G A A T T T A T T A Plasmid construction p-964 ATAA GCrrATTGGAATTTATTAAATCACCAGC P lasmid construction 83 p-979 p-994 p-1009 ATAA G C 7 T A T C A C C A G C T T G A T T T G T C T A A T G Plasmid construction ATAA GCr7TGTCTAATGCCAACAAGTTTGCTT P lasmid construction ATAA GCrrAGTTTGCTTCTGCAGCAGCCAAAG Plasmid construction mut-a ATAA GCrrAGTTTCACACTGCAGC AGCC AAAG Site-directed mutagenesis mut-b ATAA G C 7 T A C A C A G C T T C T G C A G C A G C C A A A G Site-directed mutagenesis mut-cOct-1 ATAA GCrrAATTTGCATCTGCAGCAGCCAAAG Site-directed mutagenesis mut-rOct-1 AT^GCJrACTTTGCTTCTGCAGCAGCCAAAG Site-directed mutagenesis GS-Oct-1 G C T G C A G A A G C A A A C T T G T T G G E M S A and Southwestern blot E R a - 1 ( F ) A T G / M 7 T C A T G A C C A T G A C C C T C C A C A C C A A A Plasmid construction E R a 261 f R i ATGG^ rCCTCATCTCCCTCCTCTTCGGTCTTTTCG E K a / 0 3 ( K ) T A T P lasmid construction E R a 180 m ATGAA 7 T C A T G A A G G A G A C T C G C T A C T G T G C A G T GTGC P lasmid construction E R a - 5 9 5 (R) ATGG^rCCTCAGACTGTGGCAGGGAAACCCTC P lasmid construction ™ ^ /T->\ ATG^TTCATGAGAATGTTGAAACACAAGCG'CCA . , _ E R a - 2 6 3 (F) GAGA Plasmid construction E R a - 1 8 0 (R) C T T G G C A G A T T C C A T A G C C A T A C T Plasmid construction A G T A T G G C T A T G G A A T C T G C C A A G A G A A T G T T G E R a - 1 8 0 + 260 (F) A A A C A C A A G C G C C A G A G A G A T G A T G G G G A G G G Plasmid construction C A G p-777 ATGGTA C C C A A T A A C T A G T T T C C T T A T G C A T T Plasmid construction p-487 AT GGTA C C G T T A A T T C C T T A A G A C A A T G G A T T Plasmid construction 84 p-367 ATGGTACCTTAAAGTTATTTAAATCATAATAT Plasmid construction p-266 A T G G Z 4 C C C A T C T G C T G A G G T G C T A C A G T T A C Plasmid construction p-214 ATGGTA C C C T C T G T T T T A G C A C T C T G T T G G A T Plasmid construction p-117 ATGGTA C C A A C C T G T G A A C G T T T C C A T C T A A A Plasmid construction p+1 ATAA G C 7 / 7 T A T T T T C C C A G G A C A G A G C T T C A A G Plasmid construction and site-directed mutagenesis A/rn L . , A T G G 7 M C C T C T A G A C T G A G G T G C T A C A G T T A C A T ,. t , t MP-E-box-1 r-rrr-r^ Site-directed mutagenesis G1GG ^ T , c u A T G G T A C C C A T C T G C T G A G G T G C T A C A G T T A T C T c . t „ , „ M P - E - b o x - 2 . . . „ Site-directed mutagenesis T V ^ X T T , ^ G T T A A C T T G T r T T G T G G A T C C T A A T C C A A C A G A G „ . ,. ^ , ^ M P - N F - Y ~~TGC Site-directed mutagenesis A ™ ™ c ,., T G A G A T A C T T A A T G T G A A A C G C G T A G G G A G C T C c . t ,. t , t M P - E R E - l i k e T G T T T T G T G A T T G G T A A Site-directed mutagenesis A ^ O A ^ A U T C A A A T T T T G T C C C T G C T C G A G T T A A T G T G A A A G c v ,. t , t M P - G A T A b Site-directed mutagenesis A A n A T , 1 i - i ^ T T C A C A G G T T T A G A C G A G G C C T A A A T T T T G T C C C c v ,. t , t M P - A P - l - h k e (a) T G A G Site-directed mutagenesis M P - A P - l - l i k e ( b ) C A G G T T T A G A C G T G A G T C A A A T T T T G T C Site-directed mutagenesis G S - A P - l - l i k e C A A A A T T T G A C A T A C G T C T A A E M S A G S - A P - l - l i k e - m u t C A A A A T T T A G G C C T C G T C T A A E M S A P E - G I I C A A G G T G G G C A G T C A G C A G C A G C A Primer extension ATGGTA C C A G C T T G G C T C T G G T T T A G A T T T T C C A n , . , _ t . p-2103-GII . . P lasmid construction UOAA p-1789-GII ATGGTA C C G C C C A T C C T G C C T T G G C T G C T G A A Plasmid construction 85 p-1324-GII ATGGTA C C C C A G G T G T G T G G A T G T G A A T T G T C Plasmid construction p-1124-GII ATGGTA C C A A T T C T C T T T T G G G A T C A G G G A A G Plasmid construction and site-directed mutagenesis p-924-GII A T G G Z 4 C C A A T G T T C A T G G A C T G G A T G C T C T G Plasmid construction p-864-GII A T G G 7 M C C C T G A A G A C G T C A C T G G A G T C T G G G Plasmid construction p-793-GII p-749-GII p-794-GII p-750-GII p+l -GII A T G G 7 X C C C T G C A G C T G C C T G A A G G A G C C A T C T C A T C C A T G G 7 M C C G T G A G T G G G G A G C C T T C C C T A A G G G C T A G G A T / 4 y 4 G C r 7 " G A C C C C A G C C T G A T G G C C C C A G G A T T T A T A ATAA G C 7 T C A A G G A A G A G C T G T G G A T G A G A T G G C T C C T A T ^ G C r 7 T G G C T G C T C T A A T G G A C A G G G T A C A G C A T T Plasmid construction Plasmid construction Plasmid construction Plasmid construction Plasmid construction R A C E - G I I (n) G C A G T G T C C G T G C C A G G T G T C G C T 5 ' - R A C E R A C E - G I I (o) C C T C A C A C T T T A T T G G A G G A T G G C G 5 ' - R A C E dE-box-mut A T ^ G C T T C A A G G A A G A G C T G T G G A T G A G A T G G C T C C T T C A G G G T T A A C C A G G A C C C Site-directed mutagenesis ATAA GCrrCAAGGAAGXLAACTGGATGAGATGGC c v , . t , t pE-box-mut — ~ Site-directed mutagenesis E L E - m u t A T ^ G C 7 T C A A G G A A G A G C T G T G G A T G A G A T G G C A A A C T C A G G C A G Site-directed mutagenesis (dE-box + p E - ATAA G C 7 T C A A G G A A G T T A A C T G G A T G A G A T G G C box)-mut T C C T T C A G G G T T A A C C A G G A C C C Site-directed mutagenesis (dE-box + E L E ) - ATAA G C 7 7 C A A G G A A G T T A A C T G G A T G A G A T G G C mut A A A C T C A G G C A G Site-directed mutagenesis ( E L E + pE-box)- A T ^ ^ G C 7 7 C A A G G A A G A G C T G T G G A T G A G A T G G ,. ^» » »^rr,^ ,. „ „ „ „ „ , . , . „ „ „ Site-directed muta mut C A A A C T C A G G G T T A A C C A G G A C C C genesis 86 (dE-box + E L E + A T A 4 G C 7 T C A A G G A A G T T A A C T G G A T G A G A T G G C c . t ,. t , t pE-box)-mut A A A C T C A G G C T T A A C C A G G A C C C Sxte-directed mutagenes is GS-dE-box GS-pE-box G T C C T G C A G C T G C C T G A A C A T C C A C A G C T C T T C C T T E M S A , U V crosslinking, and Southwestern blot E M S A , U V crosslinking, and Southwestern blot GS-dE-box-mut G T C C T G G T T A A C C C T G A A E M S A GS-pE-box-mut C A T C C A G T T A A C T T C C T T E M S A AP -4 -c C A C C C G G T C A G C T G G C C T A C A C C E M S A G S - E L E G C C T G A A G G A G C C A T C T C E M S A G S - E L E - m u t G C C T G A G T T T G C C A T C T C E M S A AP -4 (F) AP -4 (R) G A P D H (F) G A P D H (R) A A C A G G T G A G G C T G C T G C A C C A G G T C A T C G T G A A G C C T G T C C G C A G A A T C C A C C C A T G G C A A A T T C G G C A G A G A T G A T G A C C C T R T - P C R R T - P C R R T - P C R R T - P C R 87 Figure 10. Diagrammatic representation of the three-step PCR mutagenesis method. In the 1st PCR, amplifications were performed with MP-B and site-specific mutagenic primers. MP-B consists of a sequence-specific region (black) and an unique region (gray) that does not hybridize with the template. On the other hand, each mutagenic primer carries a desired mutation in the central region flanked by wild-type sequences. The mutation can be a base substitution or a restriction enzyme recognition site. After the 1st PCR reaction, products were separated by electrophoresis and then purified. Afterward, the purified products were used as primers in the 2nd amplification, and intermediate products lacking definite ends were generated. In the 3rd PCR, products from the previous reaction were used as templates, and amplifications were performed with MP-D and pGL2-BasicR (or p+1) such that the ends of the final products were flanked by MP-D and the reverse primer with the desired mutations created at the target site. 88 Template 1 s t PCR i Y/A M//jmmm Product from 1 s t PCR 2 n d PCR V/A Product from 2 n d PCR 3 r d PCR V/A Product from 3 r d PCR c::mmm MP-B m MP-D ^^nyxmmm s ' t e _ s P e c i f i c mutagenic primer • • 1 pGL2-BasicR or p+1 89 1 S T PCR Y/A • M/sjmm Product from 1 s t PCR 2 n d PCR • 3rd PCR Template Product from 2 n d PCR t//mmmmmmmi^mm^m Product from 3rd PCR ' ^ • i MP-B MP-D site-specific mutagenic primer ^ • i pGL2-BasicR or p+1 8 9 2.2.2 Human GnRH-II Promoter-Luciferase Constructs The human GriRH-II promoter-luciferase construct p(-2103/+l)-Luc-GII (all numbering is relative to the ATG start codon, of which the position is designated as +1) was prepared by PCR of the corresponding region of the GnRH-II gene using human genomic DNA (CLONTECH Laboratories, Inc., Palo Alto, CA) as a template, followed by cloning of the amplified fragment into the pGL2-Basic vector. The authenticity of the DNA fragment was confirmed by nucleotide sequencing. Deletion constructs except p(-1524/-750)-Luc-GH and p(-1962/-750)-Luc-GII were generated by amplification of the corresponding regions using p(-2103/+l)-Luc-GII as a template. The constructs p(-1524/-750)-Luc-GII and p(-1962/-750)-Luc-GII were prepared by digesting p(-2103/-750)-Luc-GII with a combination of Nhel/Kpnl and Spel/Kpnl, respectively, followed by self-ligation. Site-directed mutants [dE-box-mut, ELE-mut, pE-box-mut, (dE-box + pE-box)-mut, (dE-box + ELE)-mut, (ELE + pE-box)-mut, (dE-box + ELE + pE-box)-mut] were generated by PCR using the forward primer p-1124-GII, reverse primers containing the desired mutations (Table 3), as well as p(-1124/-750)-Luc-GII as a template. A Hpal restriction site (5'GTTAAC3') was introduced into each of the EBSs, whereas the ELE (5'AGGA3') was mutated to 5'GTTT3'. PCR reactions for wild-type and mutant GnRH-II promoter-luciferase constructs were carried out for 30 cycles with denaturation for 30 s at 94 °C, annealing for 1 min at 50 °C, extension for 1 min at 72 °C, and a final extension for 15 min at 72 °C. Heterologous simian virus (SV) 40 promoter constructs (UsExon l-pGL2-Promoter, DsExon l-pGL2-Promoter, UrExon l-pGL2-Promoter, and DrExon l-pGL2-Promoter) were prepared by amplifying exon 1 of the 90 human GnRH-II gene and subsequent cloning of the amplified product into the Smal (upstream) or HindUI (downstream) site of the pGL2-Promoter vector (Promega Corp.) in both orientations. 2.2.3 Mammalian Expression Plasmids The Oct-1 expression plasmid pcDNA3-HA-Oct-l and the control plasmid pcDNA3-HA were provided by Dr. H. Singh (Howard Hughes Medical Institute, University of Chicago). Full-length human ERa (pCMV5-ERa) and ERp (pRST7-ER(3) expression plasmids were kindly provided by Dr. B. S. Katzenellenbogen (Department of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign) and Dr. D. P. McDonnell (Department of Pharmacology and Cancer Biology, Duke University Medical Center). The ERP coding region was released by restriction digestion and then subcloned into an empty pCMV5 vector to generate pCMV5-ERp. ERa deletion mutants were prepared by PCR of the corresponding coding regions and subsequent cloning of the amplified products into the pCMV5 vector. Initiation codons (ATG) or termination codons (TGA) were introduced into the primers for proper translation (Table 3). PCR reactions were carried out in the presence of 10 % dimethyl sulfoxide (DMSO) for 30 cycles with denaturation for 30 s at 94 °C, annealing for 1 min at 59 °C, extension for 1.5 min at 72 °C, and a final extension for 15 min at 72 °C. The authenticity of the deletion mutants was confirmed by nucleotide sequencing. The human PR-B expression plasmid pSG5-PR-B was provided by Dr. P. Chambon (TNSERM, Universite Louis Pasteur, Paris, France) and has been described previously (Cheng et al, 2001a). The ERE reporter plasmid ERE2-tkl09-Luc and the CBP 91 expression plasmid pRc/RSV-CBP-HA were provided by Dr. J. L. Jameson (Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School) and Dr. R. H. Goodman (Oregon Health Sciences University). Expression plasmid for human AP-4 (pCMV-AP-4) was a generous gift from Dr. R. Tjian (Howard Hughes Medical Institute, University of California, Berkeley). The full-length AP-4 cDNA was released by restriction digestion and then blunt-ended, followed by cloning into the pcDNA3.1 expression vector (Invitrogen, Inc.) in both orientations to generate pcDNA3.1-AP-4 (sense) and pcDNA3.1-AP-4 (antisense). The human secretin receptor promoter-luciferase construct Sp(-223/-158)-Luc was kindly provided by Y. Y. Kwok (Department of Zoology, University of Hong Kong, HKSAR, China). 2.2.4 Plasmid DNA Preparation Plasmid DNA for transient transfection was prepared using the QIAGEN Plasmid Midi Kit (QIAGEN, Chatsworth, CA) following the manufacturer's suggested procedures. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively. 2.3 Transient Transfection and Reporter Gene Assay Transient transfection of SVOG-4o, SVOG-4m, and IOSE-29EC cells was carried out using LIPOFECTAMiNE PLUS Reagent (Invitrogen, Inc.) following the manufacturer's suggested procedures, whereas LIPOFECTAMINE Reagent (Invitrogen, Inc.) was used for the rest of the cell lines. The Rous sarcoma virus (RSV)-/acZ vector was cotransfected into the cells to correct for differences in transfection efficiency. Cells 92 were harvested for reporter gene assays 48 h after transfection unless otherwise stated. Briefly, 4 x 105 of cells were grown on six-well tissue culture plates for 24 h before the day of transfection. For E 2 studies, 2.5 x 105 of cells were grown in phenol red-free DMEM (Invitrogen, Inc.) supplemented with 10 % charcoal-dextran-treated FBS (Hyclone Laboratories, Inc.) for 48 h prior to transfection. One microgram of GnRH-I receptor promoter- or GnRH-II promoter-luciferase constructs, 1 or 0.5 pg of RSV-lacZ plasmid, and an indicated amount of expression plasmids (for overexpression studies) were cotransfected into the cells under serum-free condition. After 3 h (for LJPOFECTAMINE PLUS Reagent) or 5 h (for LIPOFECTAMLNE Reagent) of transfection, 1 ml of medium containing 20 % FBS was added and the cells were allowed to recover for 18 h. Following recovery, the culture medium was removed and the cells were cultured for another 24 h with fresh medium containing 10 % FBS. For E 2 studies, the cells were washed twice with phenol red-free DMEM and then treated with E 2 (Sigma-Aldrich), tamoxifen (Sigma-Aldrich), PMA (Sigma-Aldrich), forskolin (Sigma-Aldrich), or a combination of them for different time periods as indicated before harvest. Ethanol or DMSO was added to the control media in the same final solvent concentration (typically 0.1 %). Cellular lysates were collected with 150 pi of the Reporter Lysis Buffer (Promega Corp.) and assayed for luciferase activity with the Luciferase Assay System (Promega Corp.). Luminescence was measured using the Lumat LB 9507 luminometer (E.G&G, Berthold, Germany). Beta-galactosidase activity was measured using the (3-Galactosidase Enzyme Assay System (Promega Corp.) and used to normalize the transfection efficiency. Promoter activity was calculated as luciferase activity/p-galactosidase activity. 9 3 2.4 RNA Extraction, RT-PCR, and Southern Blot Analysis Total RNA was extracted by the TRIZOL Reagent (Invitrogen, Inc.) and reverse transcribed by the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) following the manufacturer's suggested protocols. Primers specific for the human GnRH-I receptor (GenBank accession no.: XM030222.1), human AP-4 (GenBank accession no.: BC010576), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank accession no.: M33197) were designed based on the published sequences (Table 3). PCR reactions were carried out for 35, 32, and 22 cycles for the GnRH-I receptor, AP-4, and GAPDH cDNAs, respectively, with denaturation for 30 s at 94 °C, annealing for 1 min at 60 °C, extension for 1 min at 72 °C, and a final extension for 15 min at 72 °C. The authenticity of the PCR products was confirmed by Southern blot analysis. For the Southern blot analysis, PCR products were separated by agarose gel electrophoresis and then transferred onto Hybond-N nylon membranes (Amersham Pharmacia Biotech), followed by hybridization with the corresponding [a-32P]-labeled cDNA probes at 65 °C overnight. Hybridized products were detected by autoradiography at -70 °C with Kodak X-OMAT AR films (Eastman Kodak Co., Rochester, NY). 2.5 Western Blot Analysis Briefly, 4 x 105 of cells were lysed in a lysis buffer containing 1 x phosphate buffered saline (pH 7.4), 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 10 pg/ml phenylmethylsulfonyl fluoride, 30 pg/ml aprotinin, and 10 pg/ml leupeptin for 15 min on ice. After that, cell lysates were collected and debris was 94 cleared by centrifugation. Protein concentration was measured by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Thirty five to fifty micrograms of proteins were resolved by 10 % SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech) by electroblotting. Membranes were blocked with 5 % (w/v) nonfat dried milk in Tris buffered saline containing 20 mM Tris hydrochloride (pH 8.0), 140 mM sodium chloride, and 0.05 % (v/v) Tween 20 for 2 h at room temperature before incubating with primary antibodies (All were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA except the F1G4 antibody, which was provided by Dr. A. A. Karande (Department of Biochemistry, Indian Institute of Science, Bangalore, India)) at 4 °C overnight. Irnmunostained proteins were detected using the ECL Western Blot Analysis System (Amersham Pharmacia Biotech). 2.6 Primer Extension Analysis Transcription start sites for the human GnRH-I receptor and GnRH-II genes were identified by primer extension analysis as previously described (Ip et al, 2000; Cheng et al, 2001b). Briefly, gene-specific primers were end-labeled with [y- PJ-ATP by T4 polynucleotide kinase (Invitrogen, Inc.) and then hybridized with 60 pg of total RNA (for the GnRH-I receptor gene) or 5 pg of poly(A) RNA (for the GnRH-II gene) at 42 °C Q overnight. Poly(A) RNA was isolated from 1 x 10 of cells using the PolyATtract System 1000 (Promega Corp.) according to the manufacturer's suggested procedures. Following hybridization, RNAs were reverse transcribed at 42 °C for 2 h with 20 units of Superscript RNaseH-reverse transcriptase (invitrogen, Inc.), and the reactions were 95 stopped by adding 20 pg/ml RNaseA. Extended products were then purified by phenol-chloroform extraction and analyzed on 6 % polyacrylamide/7.0 M urea gels. Sequencing ladders (A, C, G, T) were generated from the M l 3mp 18 DNA using the universal primer provided in the T7 Sequencing Kit (Amersham Pharmacia Biotech) and were used as size standards. 2.7 5'-RACE 5'-RACE was performed using the SMART RACE cDNA Amplification Kit (CLONTECH Laboratories, Inc.) following the manufacturer's suggested procedures. Briefly, first-strand cDNA was constructed from 2 pg of poly(A) RNA. An antisense human GnRH-II exon 4-specific and an exon 3-specific sequence (Table 3) were used as the outer and nested primers, respectively. PCR products were cloned into pUC18 (Invitrogen, Inc.) and sequenced in order to determine the 5'-ends of the transcripts. 2.8 EMSA Oligonucleotides containing the putative C/EBP (GS-dC/EBP and GS-pC/EBP), GATAa (GS-GATAa), octamer sequence (GS-Oct-1), AP-l-like motif (GS-AP-1 -like), EBS (GS-dE-box and GS-pE-box), ELE (GS-ELE), the corresponding mutant sequences (GS-dC/EBP-mut, GS-pC/EBP-mut, GS-GATAa-mut, GS-AP-l-like-mut, GS-dE-box-mut, GS-pE-box-mut, and GS-ELE-mut), as well as the consensus AP-4 motif (AP-4-c) (Table 3) were annealed in the presence of 20 mM Tris hydrochloride (pH 7.5), 10 mM magnesium chloride, and 50 mM sodium chloride. Double-strand probes were end-labeled with [y- P]-ATP by T4 polynucleotide kinase and then purified by the 96 Microspin G-25 columns (Amersham Pharmacia Biotech). Double-strand oligonucleotides containing the consensus sequences for C/EBP (C/EBP-c), Oct-1 (Oct-1-c), GATA (GATA-c), AP-1 (AP-l-c), CREB (CREB-c), N F - K B (NF-KB-C), TFIID (TFIID-c), glucocorticoid receptor (GR) (GR-c), AP-2 (AP-2-c), and Ets transcription factor (Ets-c) were purchased from Santa Cruz Biotechnology, Inc. Nuclear extracts were prepared from primary GL cells, SVOG-4o, OVCAR-3, JEG-3, alphaT3-l, and TE-671 cells as described before (Lassar et al, 1991; Cheng et al, 2001b). For E 2 studies, OVCAR-3 cells were grown on 100-mm tissue culture dishes to about 70 % confluence in phenol red-free DMEM supplemented with 10 % charcoal-dextran-treated FBS. After that, the cells were transiently transfected with 12 pg of pCMV5-ERoc by LIPOFECTAMINE Reagent according to the manufacturer's suggested protocol. Following transfection, the cells were treated with 100 nM E 2 , 100 nM PMA, or 100 nM E 2 and 100 nM PMA for 24 h before harvest. Ethanol or DMSO was added to the control media in the same final solvent concentration (0.1 %). Protein concentration was measured by the Bio-Rad protein assay. In vitro translated human ERa and AP-4 proteins were generated by the TNT Coupled Reticulocyte Lysate System (Promega Corp.). EMSAs were carried out in 20-pl reactions containing 20 mM HEPES (pH 7.5), 50 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM dithiothreitol, 1 mM EDTA, 10 % glycerol, 1 pg of poly (dI:dC) (Amersham Pharmacia Biotech), 5 pg of nuclear proteins (or 2 pi of in vitro translated products), and 50 frnol of radiolabeled probes (30,000 cpm). For competitive assays, competitor oligonucleotides were added simultaneously with the radiolabeled probes. For antibody supershift assays, nuclear extracts were preincubated with anti-Oct-1, anti-GATA-4, anti-c-Jun, anti-c-Fos, anti-97 ERa, anti-CREB, anti-dHAND, anti-eHAND, anti-E2A, anti-upstream stimulatory factor (USF)-l, or anti-USF-2 antibody (Santa Cruz Biotechnology, Inc.) for 30 min at room temperature prior to the addition of the radiolabeled probes. Binding reactions were incubated at room temperature for 15 min and then separated by 6 % native gels containing 0.5 x Tris-Borate-EDTA buffer (0.09 M Tris-Borate and 2 mM EDTA, pH 8.0) at constant 200 V and at 4 °C. After electrophoresis, the gels were dried and then exposed to Kodak X-OMAT AR films at -70 °C. 2.9 UV Crosslinking UV crosslinking was performed essentially the same as previously described (Yoo et al, 2000). Briefly, end-radiolabeled oligonucleotides used in EMS As were incubated with 20 pg of nuclear extracts for 15 min at room temperature in a volume of 40 pi. Next, the binding reactions were exposed to UV (254 nm) for 30 min at a distance of 2 cm from the light source before terminated by adding 8 pi of 6 x Laemmli gel loading buffer. After that, the crosslinked reactions were boiled for 10 min and then separated by 10 % SDS-PAGE. Following electrophoresis, the gels were dried and the products were detected by autoradiography. 2.10 Southwestern Blot Analysis Southwestern blot analysis was performed as described before (Wieczorek et al, 2000). Briefly, one hundred micrograms of nuclear extracts were resolved by 10 % SDS-PAGE and then transferred onto Hybond-C nitrocellulose membranes by electroblotting. Next, transferred proteins were allowed to renature overnight at room 98 temperature in a buffer (TNED) containing 10 mM Tris hydrochloride (pH 7.5), 50 mM sodium chloride, 0.1 mM EDTA (pH 7.5), and 1 mM dithiothreitol supplemented with 5 % milk. Afterward, membranes were rinsed three times with the TNED buffer and then incubated in 11 ml of the same buffer containing 75 pmol of radiolabeled probes used in EMSAs at room temperature for 24 h. Following hybridization, membranes were washed three times (10 min each) and dried before subjected to autoradiography. 2.11 Data Analysis For transient transfection studies, data were shown as mean ± standard error of the mean (SEM) of triplicate assays in three independent experiments. Data were analyzed by one-way analysis of variance, followed by Tukey's multiple comparison tests using the computer software PRISM (GraphPad Software, Inc., San Diego, CA). Data were considered significantly different from each other at P < 0.05. All other studies were carried out at least twice, and consistent results were obtained among experiments. 99 CHAPTER III Characterization of a New Upstream GnRH-I Receptor Promoter in Human Ovarian GL Cells 3.1 Introduction GnRH-I plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins via binding to the GnRH-I receptor on pituitary gonadotropes. However, we and others have previously demonstrated that the hormone and its receptor are also expressed in various ovarian compartments (Ohno et al, 1993; Kakar et al, 1994b; Peng et al, 1994; Minaretzis et al, 1995; Kang et al, 2000c), where functionally, the decapeptide has been shown to modulate steroidogenesis (Bussenot et al, 1993; Hori et al, 1998), to stimulate MAPK cascades and apoptosis (Zhao et al, 2000a; Kang et al, 2001b), as well as to inhibit FSH-induced cAMP-dependent responses and cell proliferation (Furger et al, 1996; Emons et al, 1998). These findings therefore strongly indicate that GnRH-I serves as an important autocrine and/or paracrine factor in regulating local ovarian functions. However, to date, the mechanisms governing GnRH-I receptor gene expression in human ovary are virtually unknown. As a first step to understand the mechanisms regulating human GnRH-I receptor gene transcription, our laboratory has isolated and partially characterized the 5'-flanking region of the gene (Fan et al, 1995). Our earlier data have shown that a downstream GSE, which is located in the 5'-UTR and acts via the nuclear receptor SF-1, is largely responsible for gonadotrope-specific expression of the GnRH-I receptor gene (Ngan et al, 1999). Consistently, a proximal promoter spanning from -707 to +1 is found to be most active in gonadotropes (Cheng et al, 2001b). On the contrary, the same authors 100 have demonstrated that a distal promoter residing between -1737 and -1346 is employed specifically by placental cells (Cheng et al, 2001b). The activity of this upstream promoter is mediated by a functional cooperation among four regulatory elements including a CRE, GATA, Oct-1, and AP-1 binding site, of which the CRE and GATA motifs are important for conferring placenta-specificity (Cheng et al, 2001b). Interestingly, a similar pattern of promoter usage has also been observed for the human GnRH-I gene in directing cell-specific expression in hypothalamic neurons and placental cells (Dong et al, 1997), indicating that differential usage of downstream and upstream promoters by neuronal and reproductive tissues may be a primary mechanism by which tissue-specific expression of the GnRH-I receptor and GnRH-I genes occurs. Nevertheless, it remains unclear if other reproductive tissues such as the ovary employ another but yet unidentified upstream promoter to direct GnRH-I receptor gene transcription. To address this issue, two immortalized human GL cell lines SVOG-4o and SVOG-4m were used to identify and characterize other putative promoter regions. Surprisingly, a new upstream promoter located between -1300 and -1018 was found to exhibit the highest activity among four other cell lines in the GL cells. DNA sequence analysis revealed the presence of two putative C/EBP and one GATA motifs in this promoter region, and their functional significance in mediating GL cell-specific GnRH-I receptor gene transcription was examined by site-directed mutagenesis and EMSAs. Our results clearly indicated that these motifs functioned cooperatively with each other to control GnRH-I receptor gene expression in human GL cells. 101 3.2 Results 3.2. J Expression of GnRH-I Receptor in the Immortalized Human GL Cell Lines SVOG-4o andSV0G-4m RT-PCR and Western blot analysis were performed to study the expression of GnRH-I receptor in the immortalized GL cell lines SVOG-4o and SVOG-4m as well as in primary-cultured GL cells. A single PCR product of the expected size (373 bp) was obtained from both cell lines and primary cultures, with the immortalized GL cells showing a relatively higher expression level than the primary cells (Figure 11 A). The authenticity of the PCR product was confirmed by DNA base sequencing and Southern blot analysis (Figure 11 A). A mouse monoclonal antibody (F1G4) specific for the human GnRH-I receptor was used in the Western blot analysis, and two bands of about 62 kDa were detected in both the immortalized and primary-cultured cells (Figure 1 IB). Consistent with the result from RT-PCR, a higher protein expression level was observed in the SVOG-4o and SVOG-4m cells (Figure 11B). Taken together, these findings indicate that GnRH-I receptor is expressed in the immortalized human GL cell lines at both the mRNA and protein levels. 3.2.2 Mapping of the Human GnRH-I Receptor Promoter in GL Cells To locate the active promoter regions in GL cells, a series of 5'- and 3'-deletion mutants were constructed and analyzed in SVOG-4o and SVOG-4m cells. Transient transfection studies revealed that these cell lines exhibited similar promoter activity profiles. The promoter activity of all the 5'-deletion mutants was comparable and was less than 5-fold when compared with the promoterless pGL2-Basic vector 102 (A) SVOG -4m SVOG -4o PC 3 7 3 bp (B) SVOG -4m SVOG -4o m PC 62 kDa Figure 11. Expression of GnRH-I receptor mRNA and protein in immortalized and primary-cultured human GL cells. A, Upper panel: RT-PCR amplification of GnRH-I receptor cDNAs from immortalized (SVOG-4m and SVOG-4o) and primary-cultured (PC) GL cells using primers GnRH-IR (F) and GnRH-IR (R). Lower panel: The authenticity of the PCR product was confirmed by nucleotide sequencing, and the verified product was used as a probe in Southern blot analysis. B, Western blot analysis to detect GnRH-I receptor protein expression in total cellular extracts (35 pg) from the GL cells using the F1G4 mouse monoclonal antibody. 103 (Figure 12). However, a strong promoter activity (SVOG-4o: 38 fold vs. pGL2-Basic; SVOG-4m: 35 fold vs. pGL2-Basic) was observed when the proximal 1018-bp fragment was deleted [i.e. the construct p(-2197/-1018)-Luc] (Figure 13), indicating that upstream promoters are used by the immortalized GL cells. Further 3'-deletion to -1346 reduced the promoter activity by 40 % and 45 % in SVOG-4o and SVOG-4m cells, respectively. Interestingly, inclusion of the region between -1018 and -771 in p(-2197/-771)-Luc completely abolished the transcriptional activity (Figure 13), indicating the presence of powerful repressive elements in this region. To delineate the core promoter region that is sufficient for producing the maximal transcriptional activity in the GL cells, a more detailed 200-bp deletion mapping was performed between -2197 and -1018. As shown in Figure 14, 5'-deletion of a 200-bp fragment from p(-2197/-1018)-Luc reduced the promoter activity by 71 % and 68 % in SVOG-4o and SVOG-4m cells, respectively, indicating the existence of important positive regulatory elements in this distal region. On the contrary, further 5'-deletion from p(-1900/-1018)-Luc increased the promoter activity by 2-fold in both cell lines. An additional increase in promoter activity (1.6-fold in SVOG-4o; 1.3-fold in SVOG-4m) was observed when a 200-bp fragment was removed from p(-l500/-1018)-Luc. Thus, it is apparent that the minimal promoter that is most active in the GL cells resides between -1300 and -1018. To examine if this upstream promoter is GL cell-specific, the construct p(-1300/-1018)-Luc was transiently transfected into six different cell lines including SVOG-4o, SVOG-4m, IOSE-29EC, OVCAR-3, HEK-293, and alphaT3-l. As depicted in Figure 15, the highest promoter activity (33- to 35-fold) was observed in the 104 Figure 12. Progressive 5'-deletion analysis of the human GnRH-I receptor 5'-flanking region in SVOG-4o and SVOG-4m cells. A nested family of 5'-deletion mutants was transiently transfected into the GL cells by LIPOFECTAMINE PLUS Reagent. The RSV-lacZ vector was also cotransfected to normalize the transfection efficiency. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.05 vs. pGL2-Basic. 105 Human GnRH-I receptor 5'-flanking region Figure 13. Progressive 3'-deletion analysis of the human GnRH-I receptor 5'-flanking region in SVOG-4o and SVOG-4m cells. A nested family of 3'-deletion mutants was transiently transfected into the GL cells by LIPOFECTAMINE PLUS Reagent. The RSW-lacZ vector was also cotransfected to normalize the transfection efficiency. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.05 vs. pGL2-Basic. 107 Human GnRH-I receptor 5'-flanking region 108 Figure 14. Fine mapping of the minimal human GnRH-I receptor promoter in SVOG-4o and SVOG-4m cells. A series of 200-bp deletion constructs between -2197 and -1018 was generated and then cotransfected with RSV-lacZ into the GL cells. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.01 vs. pGL2-Basic. 109 Human GnRH-I receptor 5'-flanking region h- O 05 O •<- a> CM T -o o o o o o in co I I I I L Luc p(-2197/-1018)-Luc Luc |p(-1900/-1018)-l_uc Luc p(-1700/-1018)-Luc Luc p(-1500/-1018)-Luc Luc |p(-1300/-1018)-Luc ' pGL2-Basic Luc • SV0G-4m • SVOG-40 Relative promoter activity (fold induction) 110 Figure 15. Transcriptional activity of the upstream human GnRH-I receptor promoter (from -1300 to -1018) in different cell lines. The GnRH-I receptor promoter-luciferase construct p(-1300/-1018)-Luc was cotransfected with RSV-lacZ into SVOG-4o, SVOG-4m, IOSE-29EC, OVCAR-3, HEK-293, and alphaT3-l cells. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.01 vs. pGL2-Basic. i l l • pGL2 -Basic SVOG-4o SVOG-4m IOSE-29EC O V C A R - 3 HEK-293 alphaT3-1 Cell line 112 imrnortalized GL cells, whereas various degrees of lower promoter activity (5- to 17-fold) were detected in other cell lines. These results thus indicate that the upstream promoter is primarily used by GL cells. 3.2.3 Identification of the Transcription Start Sites for the Human GnRH-I Receptor Gene in GL Cells To further confirm the usage of the upstream promoter by GL cells, a primer extension analysis was performed using two oligonucleotides located at different positions along the human GnRH-I receptor 5'-flanking region. One extension product was generated by PE-1, whereas two products were generated by PE-2 when RNA from SVOG-4o cells was used (Figure 16A). The transcription start sites deduced from these products were situated at -769, -1375, and -1397. No extension product was obtained from the control HDF cells (Figure 16A). Based on their intensity, the -769 site was assigned as the major transcription start site, of which a TATA and a CAAT box were identified 79 and 104 bp upstream (Figure 16B). 3.2.4 Two Putative C/EBP Motifs and a GATA Motif Function Cooperatively to Regulate the Upstream Human GnRH-I Receptor Promoter in GL cells Two putative C/EBP binding sites, namely dC/EBP (at -1244/-1232, 5' TCTGTGGT AAC A A3', with 91 % homology to the consensus C/EBP motif) and pC/EBP (at -1157/-1144, 5'ATATTTAGTAACCA3', with 82 % homology to the consensus C/EBP motif), as well as one GATA binding site (GATAa) (at -1176/-1168, 5' AAGATAATG3', with 89 % homology to the consensus GATA motif) were identified 113 Figure 16. Identification of the transcription start sites for the human GnRH-I receptor gene in SVOG-4o cells by primer extension analysis. A, Total RNA (60 pg) from SVOG-4o and HDF cells were hybridized and extended with primers PE-1 and PE-2. One extension product (141 bp) was obtained from the GL cells with PE-1, whereas two products (90 and 112 bp) were obtained with PE-2. Their sizes were determined by comparing with a sequencing reaction (T, G, C, A) generated from the M13mpl8 DNA. The transcription start sites deduced from these products were located at -769, -1375, and -1397, respectively. No extension product was detected from the control HDF cells. B, A diagrammatic representation of the human GnRH-I receptor 5'-flanking region. The upstream GnRH-I receptor promoter primarily used by GL cells is shown as a shaded box and the location of the TATA (T) and CAAT (C) boxes is indicated. The position of PE-1 and PE-2 as well as the identified transcription start sites are marked with horizontal and bent arrows, respectively. The -769 site was assigned as the major transcription start site because of its strongest intensity. 114 PE-1 (-651/-630) PE-2 (-1305/-1284) o T o A C G T co I 141 bp (-769) o o O £ A C G T W X 112 bp (-1397) 90 bp (-1375) PE-2 P E - 1 -873 -848 _ l l _ -769 -1300 -1018 115 in sense orientation in the upstream promoter. To examine the functional significance of these motifs in directing GnRH-I receptor gene transcription in GL cells, site-directed mutants were constructed and transiently transfected into SVOG-4o cells as well as three other cell lines including OVCAR-3, HEK-293, and alphaT3-l. Mutation of the dC/EBP motif resulted in a 35 % and 26 % reduction of promoter activity in SVOG-4o and alphaT3-l cells, respectively (Figure 17A). In contrast, no significant change in promoter activity was observed in OVCAR-3 and HEK-293 cells. However, mutation of the pC/EBP motif significantly decreased the transcriptional activity in SVOG-4o (45 % reduction), OVCAR-3 (24 % reduction), and HEK-293 (27 % reduction) cells but not in alphaT3-l cells (Figure 17A). Interestingly, mutation of the GATAa motif caused a significant reduction of promoter activity in SVOG-4o cells exclusively (33 % reduction) (Figure 17 A), indicating a specific role of this element in regulating GnRH-I receptor gene transcription in GL cells. To investigate if there is any functional cooperation among these motifs, constructs containing double or triple mutations were generated and analyzed in SVOG-4o cells. As depicted in Figure 17B, constructs carrying two mutations (dC/EBP + pC/EBP or GATAa + pC/EBP) had their luciferase activities reduced by 54 %. However, an almost 80 % decrease in promoter activity was observed in the immortalized GL cells (compared to 31 % reduction in OVCAR-3 cells; 33 % reduction in HEK-293 cells; 17 % reduction in alphaT3-l cells) when all these three motifs were mutated simultaneously (Figures 17B and 17C). 116 Figure 17. Mutational analysis of the putative C / E B P and GATA motifs on the promoter activity of p(-1300/-1018)-Luc. A, Upper panel: A diagrammatic representation of mutant promoter constructs carrying only single mutation. Each motif was mutated by a three-step mutagenesis reaction, which artificially introduced a Notl restriction site into the target sequence. Mutations are marked with black crosses. Lower panel: Wild-type p(-1300/-1018)-Luc or the mutant constructs were cotransfected with RSW-lacZ into SVOG-4o, OVCAR-3, HEK-293, and alphaT3-l cells. B, Upper panel: A diagrammatic representation of mutant promoter constructs carrying single, double, or triple mutations. Lower panel: Wild-type p(-1300/-1018)-Luc or the mutant constructs were cotransfected with RSV-lacZ into SVOG-4o cells. C, Construct containing triple mutations was analyzed in SVOG-4o, OVCAR-3, HEK-293, and alphaT3-l cells. The relative promoter activity is represented as the percentage of p(-1300/-1018)-Luc, of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.05 vs. p(-1300/-1018)-Luc; b, P < 0.05 vs. pC/EBP-mut; c, P < 0.05 V J . (dC/EBP + pC/EBP)-mut; d, P < 0.05 vs. (GATAa + pC/EBP)-mut; e, P < 0.001 vs. SVOG-4o. 117 GATAa p(-13007-1018)-Luc dC/EBP-mut GATAa-mut pC/EE !P LUC -• Luc HAT H Luc pC/EBP-mut — J Luc • p(-1300/-1018)-Luc D dC/EBP-mut S V O G - 4 o O V C A R - 3 HEK-293 alphaT3-1 Cell line 118 (B) GATAa dC/EBP 1 pC/EBP p(-13007-1018)-Luc dC/EBP-mut-GATAa-mut pC/EBP-mut (dC/EBP + pC/EBP)-mut—® Q ^ -(GATAa + pC/EBP)-mut — ( ^ J J — ^ -(dC/EBP + GATAa + pC/EBP)-mut—^ ]B [ "X ' •H Luc •H Luc •H Luc •H Luc •H Luc •H Luc • H Luc 1 1 9 (C) 120 3.2.5 Analysis of DNA-Protein Interactions of the Putative C/EBP and GATA Motifs EMSAs revealed that DNA-protein complexes of identical mobility were formed with the putative C/EBP and GATA motifs when nuclear extracts from primary GL cells and SVOG-4o cells were used. Two DNA-protein complexes, complex-A and -B, were produced with the dC/EBP motif (Figure 18 A). The formation of these complexes was completely abolished in the presence of a 200-fold excess of the unlabeled probe, while addition of unrelated sequences (NF-KB-C, C R E B - C , and AP-l-c) or a mutant dC/EBP sequence failed to inhibit the complex formation (Figure 18B). Unexpectedly, C/EBP-c also failed to compete with the dC/EBP probe even when a 600-fold excess of the competitor was used (Figures 18B and 18C). Conversely, a dose-dependent inhibition of complex formation was observed with Oct-l-c (Figure 18C). However, one complex (complex-C) was produced with the GATAa probe (Figure 19A), and its formation could be prevented by the addition of the unlabeled sequence (Figure 19B). Competitive EMSAs showed that GATA-c could also inhibit the complex formation, while a mutant GATAa sequence could not (Figure 19B). Like the GATAa motif, a single DNA-protein complex (complex-D), whose formation could be abolished by the unlabeled sequence but not by the corresponding mutant sequence, was formed with the pC/EBP probe (Figure 20). Intriguingly, the formation of this complex was unaffected in the presence of various oligonucleotides containing consensus sequences for the transcription factors C/EBP, N F - K B , Oct-1, AP-1, CREB, and GR (Figure 20). 121 Figure 18. EMSAs to characterize the dC/EBP motif using nuclear extracts from human GL cells. Synthetic oligonucleotides containing the dC/EBP motif were annealed and end-radiolabeled with 3 2P, followed by incubation with nuclear extracts ( N E ) from primary-cultured GL cells (PC) or SVOG-4o cells in the absence or presence of different competitor oligonucleotides. A, Formation of two DNA-protein complexes (complex-A and -B) using NE from PC (10 pg) or SVOG-4o cells (2, 5, and 10 pg). B, Ten micrograms of NE from SVOG-4o cells were incubated with 50 finol of the radiolabeled probe in the presence of a 200-fold excess of the unlabeled sequence, five different competitor oligonucleotides (Oct-l-c, C/EBP-c, N F - K B - C , C R E B - C , and AP-l-c), or the corresponding mutant dC/EBP sequence (mdC/EBP). C, Ten micrograms of NE from SVOG-4o cells were incubated with 50 frnol of the radiolabeled probe in the presence of an increasing amount of Oct-l-c or C/EBP-c. 122 (A) (B) | CD O" <D (0 PC SVOG-4Q 1 o V V V o | -^^^^Am Competitor - D o O z o < - E NE (ug) 10 2 5 10 NE + + + + + + + + + I I I ' i IL I i jj^ j ^ ^ j ^ J ^—F r e e p r o b e — • jjL. it Ifclj Aiiii J^ j| 123 Figure 19. EMSAs to characterize the GATAa motif using nuclear extracts from human GL cells. Synthetic oligonucleotides containing the GATAa motif were annealed and end-radiolabeled with 3 2P, followed by incubation with nuclear extracts (NE) from primary-cultured GL cells (PC) or SVOG-4o cells in the absence or presence of different competitor oligonucleotides. A, Formation of a DNA-protein complex (complex-C) using NE from PC (10 pg) or SVOG-4o cells (2, 5, and 10 pg) in the presence of 50 finol of the radiolabeled probe. B, Five micrograms of NE from SVOG-4o cells were incubated with 50 finol of the radiolabeled probe in the presence of a 200-fold excess of the unlabeled sequence, the corresponding mutant GATA sequence (mGATAa), or an increasing amount of GATA-c. 1 2 4 (A) NE(ug) 10 (B) PC SVOG-4Q Competitor 2 5 10 WW* mm NE Complex-C Free probe • c <D §f GATA-c | l 1 | * | | c o o o • D T - C M ^ + + + + + ro < + + fa u Ik 125 Figure 20. EMSAs to characterize the pC/EBP motif using nuclear extracts from human GL cells. Synthetic oligonucleotides containing the pC/EBP motif were annealed and end-radiolabeled with 3 2P, followed by incubation with nuclear extracts (NE) from primary-cultured GL cells (PC) or SVOG-4o cells in the absence or presence of different competitor oligonucleotides. Formation of a DNA-protein complex (complex-D) using 10 pg of NE from PC or SVOG-4o cells in the presence of 50 fmol of the radiolabeled probe. For the competitive assays, the radiolabeled probe was incubated with 10 pg of NE from SVOG-4o cells in the presence of a 200-fold excess of the unlabeled sequence, consensus sequences for six different transcription factors including C/EBP, N F - K B , Oct-1, AP-1, CREB, and GR, or the corresponding mutant pC/EBP sequence (mC/EBP). 126 S V O G - 4 o c 03 ! PC ® O O , . y CD * D l CQ V V Competitor - - z> o z O < o O - E NE + + + ++ + + ++ + + I 8 I I I: :: I: w U U U u W U W « Complex-D • M w Free probe 127 3.3 Discussion The presence of GnRH-I and its receptor in human ovary and other reproductive tissues (Peng et al, 1994; Lin et al, 1995; Chegini et al, 1996; Wolfahrt et al, 1998; Kang et al, 2000c) strongly supports an autocrine and/or paracrine role of the hormone in modulating local reproductive functions. Although transcriptional regulation of the human GnRH-I receptor gene has been well studied in gonadotropes (Ngan et al, 1999) and placental cells (Cheng et al, 2001b), little is known in the ovary. In this report, two immortalized human GL cell lines SVOG-4o and SVOG-4m, which express the GnRH-I receptor at both the mRNA and protein levels, were employed to investigate the molecular mechanism governing GnRH-I receptor gene expression in the ovary. These cell lines have been shown to retain steroidogenic functions such as basal and stimulated P 4 secretion as well as to exhibit the characteristic rounding response of steroidogenic cells to cAMP (Lie et al, 1996). Using Western blot analysis, two bands of about 62 kDa were detected in both the immortalized cell lines (Figure 1 IB). This observation contradicts a previous finding using membrane fractions, in which only one band was detected (Karande et al, 1995). This discrepancy may be explained by the detection of unprocessed or unglycosylated receptor proteins in our analysis. The new upstream promoter identified in our present study (i.e. from -1300 to -1018) is unlikely to be required for placenta-specific expression of the GnRH-I receptor gene since deletion of this region does not significantly affect the transcriptional activity in either JEG-3 or IEVT cells (Cheng et al, 2001b). Unfortunately, we could not examine the function of this promoter in primary-cultured GL cells, because we had repeatedly failed to transfect the upstream promoter construct into these cells using 128 various transfection methods. The identification of this novel promoter in GL cells and the presence of multiple TATA boxes in the human GnRH-I receptor 5'-flanking region (Fan et al, 1995) strengthen the notion that cell-specific expression of the GnRH-I receptor gene is mediated by differential promoter usage. Nevertheless, this phenomenon is yet to be confirmed by analyzing different GnRH-I receptor transcripts in various cell types. Another important message obtained from the present study is that the usage of the upstream promoter appears to be restricted to GL cells (Figure 15). These results indicate that a unique transcriptional machinery is involved in GL cell-specific expression of the GnRH-I receptor gene. This differential gene regulation also implies that the local GnRH-I/GnRH-I receptor system may possess distinct biological functions among various ovarian compartments. Although the usage of the upstream promoter by GL cells was confirmed by primer extension analysis, we cannot rule out the possibility that a more distal regulatory region (between -2197 and -1900) also participates in controlling GnRH-I receptor gene expression, because deletion of this sequence results in a dramatic loss of promoter activity in the ovarian cells (Figure 14). On the one hand, the identification of two transcription start sites at -1375 and -1397 downstream of this element further indicates the existence of an additional distal GnRH-I receptor promoter for GL cells. On the other hand, results from our deletion analysis showed that inclusion of the region between -1018 and -771 completely abolished the promoter function in both SVOG-4o and SVOG-4m cells (Figure 13). In fact, a similar scenario has also been observed in alphaT3-l (Kang et al, 2000b), OVCAR-3 (Kang et al, 2000b), and JEG-3 (Cheng et 129 al, 2001b) cells, indicating that the repressor protein binding to this negative regulatory region is commonly expressed in gonadotropes, ovary, and placenta. DNA sequence analysis revealed the presence of two putative C/EBP and one GATA motifs in the upstream promoter, and mutational studies showed that alteration of either one of these motifs did not completely abolish the promoter function in the immortalized GL cell lines. Interestingly, these motifs were found to function cooperatively to regulate GnRH-I receptor gene transcription in the ovarian cells because only simultaneous mutation of all the motifs could drastically reduce the activity of the upstream promoter (Figure 17). The observation that this functional cooperation was not observed in OVCAR-3 cells (Figure 17C) further supports the existence of differential regulatory mechanisms in directing GnRH-I receptor gene expression in various ovarian compartments. Although we failed to assess the activity of the upstream promoter in primary GL cells, EMSAs revealed that identical DNA-protein complexes were formed when nuclear extracts from SVOG-4o and the primary cells were used (Figures 18-20). Surprisingly, competitive EMSAs showed that there was a possible cross-talk between the POU homeodomain transcription factor Oct-1 and the dC/EBP motif. This observation may be attributed to the ability of the transcription factor to recognize various degenerate sequences and adopt different conformations (Bendall et al, 1993; Walker et al, 1994). Accordingly, it has been demonstrated that Oct-1 can bind to an element overlapping that of C/EBP and acts as a transcriptional repressor of the IL-8 gene(Wuef al, 1997). A GATA protein is believed to play a specific role in transcriptional regulation of the GnRH-I receptor gene in GL cells via acting on the 130 GATAa motif. So far, GATA-4, GATA-6, and the GATA family cofactor FOG-2 have been shown to be expressed in human GL cells (Laitinen et al, 2000). Among these proteins, GATA-4 has been reported to differentially transactivate multiple gonadal promoters including those of the steroidogenic acute regulatory protein, aromatase, and inhibin a-subunit genes through synergistic interactions with other transcription factors such as SF-1 (Tremblay and Viger, 2001). The possible involvement of GATA-4 in controlling GnRH-I receptor gene transcription in human GL cells is currently under investigation. On the contrary, we do not have a clear idea on the identity of the transcription factor binding to the pC/EBP motif since the complex formation is unaffected by consensus oligonucleotides for various transcription regulators including C/EBP (Figure 20). Indeed, the sequence of pC/EBP motif is quite different from that of the consensus C/EBP oligonucleotide used in the competitive experiments. The failure of the consensus sequence to abolish complex formation may be due to the fact that C/EBP dimerization is prerequisite for DNA binding (Landschulz et al, 1989) and that dimerization of different C/EBP proteins can generate different DNA binding specificities. Therefore, it is possible that the C/EBP dimer binding to the pC/EBP motif does not recognize the consensus sequence. Further studies are required to determine the nature of the C/EBP isoforms interacting with the pC/EBP motif. In conclusion, we have identified a novel upstream promoter in which two putative C/EBP motifs and one GATA motif function cooperatively to direct GnRH-I receptor gene transcription specifically in human GL cells (Figure 21). Importantly, these findings strengthen the notion that tissue-specific expression of the human GnRH-I receptor gene is mediated by differential promoter usage in various cell types. 131 Upstream promoters Downstream promoter A. r -1737 -1346 -1300 -1018 Placenta GL cells -769 -707 r +1 Gonadotropes Structural gene • ATG 101 bp — ..dC/EBP...GATAa...pC/EBP. >• .. . > Specific functional cooperation to confer full promoter activity Figure 21. Diagrammatic representation of differential promoter usage of the human GnRH-I receptor gene in various cell types. Promoter regions (upstream and downstream) primarily used by the placenta, GL cells, and gonadotropes are boxed, and their corresponding locations are indicated. The major transcription start site (-769) determined in GL cells is shown with a bent arrow. The functional role of the putative C/EBP and GATA motifs in controlling GnRH-I receptor gene transcription in GL cells is also indicated. The diagram is not drawn to scale. 132 CHAPTER IV Oct-1 Is Involved in the Transcriptional Repression of the GnRH-I Receptor Gene 4.1 Introduction Recent studies on the transcriptional regulation of the human GnRH-I receptor gene have provided us some insights in the molecular mechanism underlying tissue-specific expression of the gene. Our findings have revealed that gonadotrope-specific expression of the GnRH-I receptor gene is primarily mediated by a downstream GSE, which works by interacting with SF-1 (Ngan et al, 1999). On the contrary, Cheng and colleagues have found that an upstream promoter residing between -1737 and -1346 is used predominantly by placental cells and that a CRE and a GATA motif are responsible for placenta-specific gene expression (Cheng et al, 2001b). More recently, we have characterized a novel GL cell-specific GnRH-I receptor promoter, in which two C/EBP and one GATA motifs function cooperatively to regulate the receptor transcription (Cheng et al, 2002). Taken together, these findings indicate that tissue-specific expression of the human GnRH-I receptor gene is mediated by differential usage of various promoters in different cell types. Apart from positive regulation by enhancers, gene transcription may be negatively controlled by silencer elements and their associated repressor proteins. Although it has been demonstrated that there is an interplay between PACAP and a NRE (between -1673 and -1351) to regulate the activity of the human GnRH-I receptor promoter (Ngan et al, 2001), our understanding of the transcriptional repression of the GnRH-I receptor gene remains limited. Interestingly, previous deletion analysis of the 133 GnRH-I receptor 5'-flanking region has revealed a very powerful NRE located between -1017 and -771. This NRE can completely abolish the basal activity of the native promoter in alphaT3-l (Kang et al, 2000b), OVCAR-3 (Kang et al, 2000b), JEG-3 (Cheng et al, 2001b), and immortalized GL cells (Cheng et al, 2002), indicating that the transcription factor interacting with this element is ubiquitously expressed. However, to date, the functional significance of this NRE is unknown. In the present study, we demonstrated that this element behaved as an orientation-dependent silencer. Also, we revealed for the first time that Oct-1 was a transcriptional repressor for the GnRH-I receptor promoter and might play a very crucial role in silencing GnRH-I receptor gene transcription. 4.2 Results 4.2.1 Silencing Effect of the NRE on a Heterologous TK Promoter In order to examine if the NRE can repress a heterologous promoter, we cloned the element immediately upstream of a TK promoter in the pTK-Luc vector in both orientations and then transfected the constructs into SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells. The activity of the TK promoter was found to be completely abolished in these cell lines when the NRE was cloned in a sense orientation (sNRE-pTK-Luc) (Figure 22A). However, no significant change in promoter activity (relative to pTK-Luc) was observed when the NRE was placed in the opposite direction (rNRE-pTK-Luc) (Figure 22A), indicating that the repressive element functions as an orientation-dependent silencer. The construct sNRE-pTK-Luc was further analyzed in SVOG-4o, IOSE-29EC, and HEK-293 cells so as to demonstrate its silencing activity in other cell 134 Figure 22. Silencing activity of the NRE on a heterologous TK promoter. The NRE (shaded box) was placed immediately upstream of a TK promoter (black arrow) in pTK-Luc in both orientations (sNRE-pTK-Luc and rNRE-pTK-Luc), and the constructs were cotransfected with KSV-lacZ into various cell lines. A, The constructs sNRE-pTK-Luc and rNRE-pTK-Luc were analyzed in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells. B, The construct sNRE-pTK-Luc was further examined in three additional cell lines including SVOG-4o, IOSE-29EC, and HEK-293 cells. The relative promoter activity is represented as the percentage of pTK-Luc, of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. pTK-Luc. 135 Heterologous TK promoter pTK-Luc sNRE -pTK -Luc rNRE-pTK-Luc a a a • SVOG-4m • OVCAR-3 • JEG-3 • alphaT3-1 50 100 150 Relative promoter activity (%) • pTK-Luc • sNRE-pTK-Luc SVOG-4o IOSE-29EC HEK-293 Cell line 136 types. Similarly, the activity of the TK promoter was found to be completely removed in these cell lines (Figure 22B). 4.2.2 Positional Effect of the NRE Silencing Activity on the Native Promoter To examine the positional effect of the NRE silencing activity, the element was subcloned into the BamHI site of the construct p(-13007-1018)-Luc such that it was situated at about 2.8 kb upstream of a minimal human GnRH-I receptor promoter (from -1300 to -1018). The activity of the minimal promoter was found to reduce by about 60 % in all the cell lines tested when the NRE was placed in a sense orientation [sNRE-p(-1300/-1018)-Luc]. In contrast, only about 28 % reduction of promoter activity was observed when the element was cloned in the opposite direction [rNRE-p(-1300/-1018)-Luc] (Figure 23). These data thus indicate that the NRE can function in a position-independent manner, but the repressive effect is weaker when it is located further apart from the native promoter. 4.2.3 Identification of Critical Nucleotide Sequences Mediating the Silencing Effect of the NRE Progressive 3'-deletion analysis was performed from -771 to -1017 in order to identify DNA sequences that constitute the silencing activity of the NRE. Deletion constructs were examined in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells, which were found to display similar promoter activity profiles. As shown in Figure 24, 3'-deletions from -771 up to -954 had no apparent influence on the silencing effect of the NRE, indicating that the core repressive activity resides in the distal 64-bp region (i.e. 137 Figure 23. The silencing activity of the NRE is position-independent. The NRE (shaded box) was subcloned in both orientations into the BamBI site of p(-1300/-1018)-Luc, which contains a minimal human GnRH-I receptor promoter (from -1300 to -1018, white arrow), to generate the constructs sNRE-p(-1300/-1018)-Luc and rNRE-p(-1300/-1018)-Luc. The luciferase constructs were transiently transfected into SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells. The relative promoter activity is represented as the percentage of p(-1300/-1018)-Luc, of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values represent the mean + SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. p(-1300/-1018)-Luc; b, P < 0.01 vs. p(-1300/-1018)-Luc; c, P < 0.05 vs. p(-1300/-1018)-Luc. 138 Human GnRH-I receptor promoter SamHI 2.8 kb -1017 -771 2.8 kb -771 -1017 2.8 kb Luc Luc p(-1300/-1018)-Luc sNRE-p(-1300/-1018)-Luc rNRE-p(-1300/-1018)-Luc • SVOG-4m • OVCAR-3 S3 JEG-3 n alphaT3-1 0 50 100 Relative promoter activity (%) 139 Figure 24. Progressive 3'-deletion analysis of the human GnRH-I receptor 5'-flanking region in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells. A nested family of 3'-deletion mutants was generated by progressive deletion from -771. The NRE is shown as a shaded box. The RSV-/acZ vector was also cotransfected into the cells in order to normalize the transfection efficiency. The relative promoter activity is represented as the percentage of p(-1300/-1018)-Luc, of which the activity is set as 100 % after being normalized by p-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 V J . p(-1300/-1018)-Luc. 140 Human GnRH-I receptor 5'-flanking region o o ro oo o Luc Luc m Luc §|Luc p(-1300/-771)-Luc p(-1300/-834)-Luc p(-1300/-894)-Luc p(-1300/-954)-Luc • SV0G-4m • OVCAR-3 H JEG-3 • alphaT3-1 p(-1300/-1018)-Luc s\\\\m\SS\S\\SS\\N\\N^^^^^ 1 1 0 50 100 Relative promoter activity (%) 141 from -1017 to -954). To further delimit the regulatory sequences required for the silencing effect, a more detailed 3'-deletion mapping was performed. Interestingly, deletion from -954 to -1009 did not significantly increase the transcriptional activity in all the cell lines tested, and a putative octamer regulatory sequence (at -1017/-1009, 5'AAGCAAACT3') alone could reduce the activity of the GnRH-I receptor promoter by 76, 78, 90, and 69 % in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells, respectively (Figure 25). These observations thus clearly indicate that most of the silencing activity of the NRE retains in this octamer sequence. To examine the functional significance of this octamer sequence, four site-directed mutants were generated and then analyzed in OVCAR-3, JEG-3, and alphaT3-l cells. On the one hand, mutation of AAGC in the octamer motif to TGTG (mut-a) restored the transcriptional activity by 64, 48, and 74 % in OVCAR-3, JEG-3, and alphaT3-l cells, respectively (Figure 26). On the other hand, mutation of AAAC to TGTG (mut-b) completely resumed the promoter activity in OVCAR-3 and alphaT3-l cells, while an 80 % recovery of activity was observed in JEG-3 cells (Figure 26). Alteration of the octamer sequence into the consensus Oct-1 binding motif (5'ATGCAAAT3') (mut-cOct-1) alleviated its repressive effect in all the three cell lines (Figure 26). Interestingly, a single point mutation (AAAC to AAAG) converting the octamer sequence into that of the rodent GnRH-I receptor promoter (mut-rOct-1) had no apparent effect on its silencing activity (Figure 26), indicating that the repressive role of the octamer sequence is evolutionarily conserved. 142 Figure 25. Fine deletion mapping of critical nucleotide sequences that mediate the silencing activity of the NRE in SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells. A nested family of 3'-deletion mutants was generated by progressive deletion from -954. The NRE is shown as a shaded box. The RSV-lacZ vector was also cotransfected into the cells in order to normalize the transfection efficiency. The relative promoter activity is represented as the percentage of p(-l 3007-1018)-Luc, of which the activity is set as 100 % after being normalized by p-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. p(-1300/-1018)-Luc. 143 Human GnRH-I receptor 5'-flanking region o o CO 00 o in W. Luc Luc Luc Luc p(-13007-954)-Luc p(-1300/-964)-Luc p(-1300/-979)-Luc p(-1300/-994)-Luc k ^ a r 3<a D a 7/A Luc p(-1300/-1009)-Luc ha • SVOG-4m • OVCAR-3 S JEG-3 El alphaT3-1 p(-1300/-1018)-Luc >^\\\\\\\\\\\\\\\\\\\^^^^ 50 100 Relative promoter activity (%) 144 Figure 26. Mutational analysis of the putative octamer regulatory sequence. Upper panel: A diagrammatic representation of the mutant promoter constructs. Four mutants (mut-a, mut-b, mut-cOct-1, and mut-rOct-1) were constructed by PCR using the forward primer p-1300 and reverse primers containing the desired mutations. The AAGC residues of the putative octamer sequence were mutated to TGTG in mut-a, whereas the AAAC residues were mutated to TGTG in mut-b. The octamer sequence was mutated to the consensus Oct-1 binding motif and that of the rodent GnRH-I receptor promoter in mut-cOct-1 and mut-rOct-1, respectively. Lower panel: Wild-type [p(-1300/-1018)-Luc and p(-1300/-1009)-Luc] or the mutant constructs were transiently transfected into OVCAR-3, JEG-3, and alphaT3-l cells. The relative promoter activity is represented as the percentage of p(-1300/-1018)-Luc, of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. p(-1300/-1018)-Luc; b, P < 0.05 vs. p(-1300/-1018)-Luc. 145 Human GnRH-I receptor 5'-flanking region o o CO L p(-1300/-1018)-Luc-p(-1300/-1009)-Luc. mut-a mut-b mut-cOct-1 mut-rOct-1 CO o •H Luc -•AAGCAAACT Luc TGTG AAACT -•AAGCTGTGT •• ATG C AAATT -•AAGCAAAGT Luc Luc Luc Luc p(-1300/-1018)-Luc p(-1300/-1009)-Luc mut-a mut-b mut-cOct-1 mut-r Oct-1 ^ Ta 1-Ha }na Da Ha > a |na • OVCAR-3 H JEG-3 m alphaT3-1 i i i 0 50 100 150 Relative promoter activity (%) 146 4.2.4 Binding of the POU Domain Transcription Factor Oct-1 to the Putative Octamer Regulatory Sequence Since the octamer sequence could repress the GnRH-I receptor promoter in OVCAR-3, JEG-3, and alphaT3-l cells, we sought to examine if the motif bound to the same transcription factor in these cell lines. As depicted in Figure 27A, common DNA-protein complexes of identical mobility (complex-F) were formed in a dose-dependent manner when nuclear extracts from the gonadotropes, ovarian, and placental cells were used. However, the intensity of this complex was much weaker and an additional DNA-protein complex (complex-E) was produced when the gonadotrope extracts were used (Figure 27A). Competitive EMSAs showed that the formation of these complexes could be dose-dependently inhibited by the unlabeled probe but not by other unrelated sequences including N F - K B - C , AP-2-c, and TFIID-c (Figure 27B), indicating that the interactions are specific for the octamer sequence. Consistent with the observation that Oct-l-c could selectively prevent complex-F formation (Figure 27C), addition of anti-Oct-1 but not anti-GATA-4 antibody in the supershift assays produced the same inhibitory effect (Figure 27D). Western blot analysis of nuclear extracts from OVCAR-3, JEG-3, and alphaT3-l cells revealed that the endogenous Oct-1 expression level was much lower in the gonadotropes (Figure 28A), and this finding was in agreement with the weaker Oct-1 binding signal detected in both Southwestern blot (Figure 28B) and EMSA studies (Figure 27). Taken together, these results clearly indicate that the POU homeodomain transcription factor Oct-1 binds to the putative octamer sequence. To assess the functional relevance of Oct-1 in transcriptional repression of the GnRH-I receptor gene, an Oct-1 expression plasmid was cotransfected with p(-1300/-147 Figure 27. EMSAs to characterize the octamer binding sequence using nuclear extracts from different cell lines. Synthetic oligonucleotides containing the putative octamer sequence were annealed and end-radiolabeled with 3 2 P , followed by incubation with nuclear extracts (NE) from OVCAR-3, JEG-3, or alphaT3-l cells in the absence or presence of competitor oligonucleotides (or antibodies). A, Formation of two DNA-protein complexes (complex-E and -F) with an increasing amount of NE from OVCAR-3, JEG-3, or alphaT3-l cells in the presence of 50 frnol of the radiolabeled probe. B , NE from OVCAR-3 (2 pg), JEG-3 (2 pg), or alphaT3-l (10 pg) cells was incubated with 50 frnol of the radiolabeled probe in the presence of an increasing amount of the unlabeled sequence or a 500-fold excess of N F - K B - C , AP-2-C, or TFIID-c. C, NE from OVCAR-3 (2 pg), JEG-3 (2 pg), or alphaT3-l (10 pg) cells was incubated with 50 frnol of the radiolabeled probe in the presence of an increasing amount of Oct-l-c. D, NE from JEG-3 (2 pg) or alphaT3-l (10 pg) cells was preincubated with 4 pg of anti-Oct-1 or anti-GATA-4 antibody for 30 min at room temperature before addition of the radiolabeled probe. 148 (A) OVCAR-3 JEG-3 alphaT3-1 i i 1 r NE (ug) 0 2 510 15 0 2 5 10 15 0 2 5 10 15 -« • < Complex-E • Complex-F Free probe 149 (B) Competitor N E OVCAR-3 JEG-3 alphaT3-1 jquence 11 jquence 1 1 squence ilabeled s< ilabeled s< ilabeled si z> i— z> 1 1 o 1 I 1 o 1 1 o 1 i i o 100X 250X 500X NF-KB-AP-2-c TFIID-100X 250X 500X NF-KB AP-2-c TFIID-100X 250X 500X NF-KB AP-2-( TFIID-+ + + + + + + + + + + + + + + + + + + + + 0 MMMw *— Complex-E mm* mm-m y compiex-F | I I | J 1,1 I | I i 150 (C) Competitor (fold excess) NE OVCAR-3 JEG-3 alphaT3-1 o o o O LO o O * - CN LO + + + + o o o o m <=> o ^- CNJ io + + + + o o o o m o O T— CN LO + + + + • Complex-E • Complex-F 151 Figure 28. Western and Southwestern blot analyses to confirm the binding of Oct-1 to the octamer sequence. Details of these analyses are described in "Materials and Methods". A, Western blot analysis of nuclear extracts (30 pg) from OVCAR-3 (O), JEG-3 (J), and alphaT3-l (a) cells using an anti-Oct-1 antibody. B, A single protein band of about 100 kDa was detected by Southwestern blot analysis from OVCAR-3, JEG-3, and alphaT3-l cells with the end-radiolabeled probe used in EMSAs. 152 1009)-Luc into OVCAR-3, JEG-3, and alphaT3-l cells. Although overexpression of Oct-1 in the ovarian and placental cells did not augment the silencing activity of the octamer sequence, a further 31 % reduction (P < 0.05 vs. control) of the GnRH-I receptor promoter activity was observed in the gonadotropes (Figure 29). The lack of response to Oct-1 overexpression in the reproductive cell types may be due to their high endogenous levels of Oct-1 protein (Figure 28A). Nevertheless, these results strongly support a role of Oct-1 as a repressor for the GnRH-I receptor promoter. 4.3 Discussion Recent studies on the transcriptional regulation of the human GnRH-I receptor gene in gonadotropes (Ngan et al, 1999), placenta (Cheng et al, 2001b), and ovarian GL cells (Cheng et al, 2002) have revealed that tissue-specific expression of the gene is mediated by differential promoter usage in various cell types. In contrast, the process of transcriptional repression of the GnRH-I receptor gene in humans or in other species remains poorly understood. An earlier study has found that there is a putative repressor element at -343/-335 of the mouse GnRH-I receptor promoter and that deletion of this sequence significantly increases the promoter activity in both basal and GnRH-I agonist-, phorbol ester-, and forskolin-stimulated somatolactotrope GGH3 cells (Maya-Nunez and Conn, 1999). Recently, Ngan and colleagues have shown that there is a functional interplay between PACAP and an upstream repressive element in the human GnRH-I receptor 5'-flanking sequence, and the authors have localized the PARE to a region between -1671 and -1644 (Ngan et al, 2001). Nevertheless, so far, none of these reports 153 Figure 29. Overexpression of Oct-1 augments the silencing activity of the octamer sequence in alphaT3-l cells. The construct p(-1300/-1009)-Luc was cotransfected with either pcDNA3-HA-Oct-l or pcDNA3-HA (control) into OVCAR-3, JEG-3, and alphaT3-l cells. The relative promoter activity is represented as the percentage of the control, of which the activity is set as 100 % after being normalized by p-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.05 vs. control. 154 150 i 100 A £ 50 • with pcDNA3-HA (control) • with pcDNA3-HA-Oct-1 I OVCAR-3 JEG-3 alphaT3-1 Cell line 155 has determined the nature of the repressor proteins interacting with the corresponding regulatory elements. Previous deletion analysis of the human GnRH-I receptor 5'-flanking region has also revealed a very powerful NRE residing between -1017 and -771, which exhibits constitutive and ubiquitous silencing activity (Kang et al, 2000b; Cheng et al, 2001b; Cheng et al, 2002). In the present study, four GnRH-I receptor-expressing cell lines including SVOG-4m, OVCAR-3, JEG-3, and alphaT3-l cells were used as model systems to identify the DNA motifs and transcription factors that mediate the NRE action. The ability of the NRE to completely abolish the activity of a heterologous TK promoter and to silence the native promoter in a position-independent manner indicates that the element behaves as a transcriptional silencer. However, unlike other classical silencers, the action of the NRE is orientation-dependent (Figure 22A). In addition, the NRE displayed some degrees of promoter-dependence since a 25 % reduction of the native promoter activity was observed even when the element was cloned in an opposite direction (Figure 23). To date, only a small number of silencers have been identified as orientation-dependent (Albert et al, 1994; Ye et al, 1994; Givogri et al, 2000), and their functional significance is still unclear. One putative mechanism for the functioning of this type of silencer is that these elements present their specific binding factors in a particular position or direction relative to other regulatory sequences or factors. For instance, DNA bending as a result of silencer complex binding has been shown to repress gene transcription by physically hindering upstream elements (Natesan and Gilman, 1993). Therefore, it is possible that inversion of the silencer element can produce a bend in the opposite direction and eliminate the steric hindrance of the 156 upstream enhancer elements. Since the NRE works ubiquitously and independent on its position, it is tempting to speculate that this element may functionally interact with different cell-specific GnRH-I receptor promoters (or enhancers) to tightly regulate the transcription of the GnRH-I receptor gene. Progressive deletion analysis indicated that most of the silencing activity of the NRE residing in a 9-bp sequence (5'AAGCAAACT3') is located at its distal end. This sequence shares a high level of identity with the consensus octamer regulatory element (5'ATGCAAAT3'). Antibody supershift assay and Southwestern blot analysis confirmed that the repressor protein binding to this element was the widely expressed transcription factor Oct-1. Importantly, mutation of this motif into the corresponding octamer sequence in the rodent GnRH-I receptor promoter had no significant effect on its silencing activity (Figure 26), indicating that the repressive function of the octamer element is evolutionarily conserved. In fact, the role of Oct-1 as a transcriptional repressor has been well studied in the thyrotropin P-subunit (Kim et al, 1996), Pit-1 (Delhase et al, 1996), von Willebrand factor (Schwachtgen et al, 1998), as well as the immunoglobulin P-chain (Malone et al, 2000) promoters, and the silencing activity of the homeodomain protein has been mapped to its alanine-rich carboxyl-terminal domain (Kim etal, 1996). In our present study, the octamer sequence was found to repress the GnRH-I receptor promoter in a wide variety of cell types, a phenomenon that could be explained by the ubiquitous expression pattern of Oct-1. However, it should be noted that Oct-1 has also been shown to participate in tissue-specific expression of the GnRH-I receptor gene via cooperation with other transcription factors. For instance, it has been 157 demonstrated that Oct-1 binds to an AT-rich octamer sequence (5'ATTTGTAT3') and functions cooperatively with CREB, GATA-2, GATA-3, and AP-1 to positively regulate the placenta-specific GnRH-I receptor promoter (Cheng et al, 2001b). However, we have recently found that there is a possible cross-talk between Oct-1 and a putative C/EBP binding site, which cooperates with another C/EBP and a GATA motif to direct GnRH-I receptor gene transcription in ovarian GL cells (Cheng et al, 2002). An important message obtained from these studies is that Oct-1 possesses dual transcriptional functions (both activator and repressor) for the GnRH-I receptor promoter. These observations may be explained by the fact that Oct-1 can adopt different conformations depending on the precise nature of the binding and flanking sequences, which in turn determines its transcriptional activity (Walker et al, 1994). This notion is supported by our mutational analysis, which revealed that alteration of the octamer sequence into the consensus Oct-1 binding motif could significantly reduce its repressive effect on the GnRH-I receptor promoter (Figure 26). It has been well demonstrated that Oct-1 stimulates gene transcription via interaction with other transcriptional regulators including Oct-binding factor 1, TBP, TFIIB, and high mobility group protein 2 (Zwilling et al, 1994; Luo and Roeder, 1995; Nakshatri et al, 1995; Strubin et al, 1995; Zwilling et al, 1995; Gstaiger et al, 1996). Although a recent study has shown that Oct-1 physically interacts with the silencing mediator for retinoid and thyroid hormone receptors (SMRT) to mediate its repressor function (Kakizawa et al, 2001), the mechanism underlying Oct-1 repression of the GnRH-I receptor promoter is obscure. Nonetheless, the finding that Oct-1 can bend DNA through its POU-specific domain (Verrijzer et al, 1991) may provide an 158 explanation for the orientation-dependent functioning of the NRE. Also, it is conceivable that the partial elimination of silencing activity observed after mutation of the AAGC nucleotides of the octamer sequence (Figure 26), which are presumably recognized by the POU-specific domain, is due to impairment of the DNA bending activity of Oct-1. Interestingly, Oct-1 has been implicated as a downstream transcriptional regulator of the glutamate/nitric oxide/cyclic guanosine 5'-monophosphate (cGMP) signal transduction pathway such that its phosphorylation and DNA binding affinity is controlled by the cGMP-dependent protein kinase (Belsham and Mellon, 2000). Likewise, there are several lines of evidence indicating that the DNA binding specificity of Oct-1 can be modulated by different kinases in vitro (Segil et al., 1991; Grenfell et al., 1996). In addition, the transcriptional activity of Oct-1 has been shown to be regulated by nuclear receptors (Kutoh et al., 1992; Prefontaine et al., 1998; Chandran and DeFranco, 1999), and for the case of GR, it has been demonstrated that the receptor inhibits Oct-1 function by a mechanism involving direct protein-protein interactions in a hormone-dependent manner (Kutoh et ai, 1992). Collectively, these findings indicate that Oct-1-regulated GnRH-I receptor gene transcription is possibly under the control of a variety of extracellular stimuli and depends strictly on the physiological status of the cell. Results from EMSAs revealed that an additional DNA-protein complex (complex-E), which was unlikely to contain Oct-1, was formed only with nuclear extracts from alphaT3-l cells (Figure 27). Since this complex was not observed with extracts from reproductive cell types, we speculate that the interacting transcription 159 factor is gonadotrope-specific. However, we had repeatedly failed to detect any additional signals (other than the Oct-1 band) in Southwestern blot analysis using the same gonadotrope extracts. The identity of this nuclear factor remains to be elucidated, and we cannot rule out the possible existence of a different (or additional) negative regulatory mechanism in controlling GnRH-I receptor gene expression in pituitary gonadotropes. In summary, we have identified an octamer sequence as the core cw-acting silencing element in the human GnRH-I receptor 5'-flanking region and revealed that the POU domain transcription factor Oct-1 is a transcriptional repressor for the GnRH-I receptor promoter. 160 CHAPTER V An AP-l-Like Motif Mediates E 2 Repression of the Human GnRH-I Receptor Promoter via an ERa-Dependent Mechanism in Ovarian and Breast Cancer Cells 5.1 Introduction In addition to its pivotal role in stimulating gonadotropin secretion, GnRH-I has been shown to function as an autocrine and/or paracrine regulator in certain extrapituitary tissues (Bussenot et al, 1993; Lin et al, 1995; Emons et al, 1998; Kang et al, 2000c; Chen et al, 2002a). One of the well-established autocrine actions of GnRH-I is to suppress the proliferation of hormone-sensitive tumors derived from various reproductive tissues such as the ovary, endometrium, and prostate gland (Emons et al, 1998). Since the growth of these cancer cells can be significantly inhibited by nanomolar concentrations of GnRH-I analogs, it is believed that the antiproliferative action of the hormone is mediated via the high-affinity GnRH-I receptor. Modulation of the mitogenic signal transduction pathways has been implicated to be a major mechanism by which GnRH-I exerts its growth-inhibitory effect in these tumors (Emons et al, 1998). For instance, it has been demonstrated that leuprolide mediates its antiproliferative effect in ovarian carcinoma Caov-3 cells via a sustained stimulation of the ERK signaling cascade, leading to dephosphorylation of the retinoblastoma protein (Kimura et al, 1999). Alternatively, GnRH-I agonists have been shown to be capable of antagonizing growth factor-induced mitogenic signaling in certain ovarian and endometrial cancer cell lines, and thereby suppressing their proliferation (Emons et al, 1996). Other mechanisms in mediating the antiproliferative action of GnRH-I or its 161 analogs in cancer cells include inhibition of phosphatidylinositol kinase activity (Takagi et al, 1995) and downregulation of telomerase reverse transcriptase and vascular permeability factor expression (Olson et al, 1995; Nagai et al, 2002). Estrogen has been implicated in the pathogenesis and positive growth regulation of carcinomas arising from the ovary, breast, and uterus (Hoover et al, 1977; Galtier-Dercure et al, 1992; Chien et al, 1994; Langon et al, 1994; Persson, 2000; Clemons and Goss, 2001). Previous studies from our laboratory have shown that E 2 can dose- and time-dependently inhibit GnRH-I receptor mRNA expression in OVCAR-3 and human GL cells via a receptor-mediated mechanism (Nathwani et al, 2000; Kang et al, 2001a). Concomitantly, cotreatment with E 2 antagonizes the antiproliferative effect of GnRH-I in the ovarian cancer cell line (Kang et al, 2001a), indicating the existence of a functional interaction between the GnRH-I/GnRH-I receptor and E 2/ER systems such that the balance of antiproliferative and proliferative signals from these pathways may play an important role in regulating tumor cell growth. However, the molecular mechanism underlying this E2-mediated inhibition of the GnRH-I receptor gene expression has not yet been clearly addressed. The human GnRH-I receptor 5'-flanking region is characterized by the presence of multiple promoter elements, which direct tissue-specific expression of the gene (Ngan et al, 1999; Cheng et al, 2001b; Cheng et al, 2002). In addition, a number of hormone-response c/s-acting motifs have been identified in the flanking region (Fan et al, 1995; Kakar, 1997). Recently, we have demonstrated that a putative PRE at -535/-521 is responsible for mediating the differential activity of PR-A and PR-B isoforms in transcriptional regulation of the human GnRH-I receptor gene (Cheng et al, 2001a). On 162 the contrary, search on the 5'-regulatory region of the mammalian GnRH-I receptor genes has revealed no consensus ERE (Albarracin et al, 1994; Fan et al, 1995; Campion et al, 1996), which is a palindrome of the A/GGGTCA motif separated by 3 nucleotides. Until now, transcriptional regulation of the GnRH-I receptor genes by estrogen has been a far-from-understood issue. Earlier studies on the proximal 1.9-kb 5'-flanking region of the mouse GnRH-I receptor gene have failed to demonstrate any E2 regulation in vivo (McCue et al, 1997). Intriguingly, it was later on shown that E 2 responsiveness of the ovine GnRH-I receptor promoter was undetectable in vitro but was only revealed in transgenic mice (Duval et al, 2000), indicating that the in vivo model may represent one of the few viable avenues for defining the mechanism mediating E 2 regulation of the GnRH-I receptor gene expression. Nevertheless, it remains unclear if this phenomenon is also applicable to other mammalian GnRH-I receptor promoters. In the present study, we demonstrated that E 2 could repress human GnRH-I receptor gene transcription in OVCAR-3 and MCF-7 cells via an AP-1-like motif located in the proximal promoter region. Also, we showed that the repression was mediated by ERa and involved the transcriptional coactivator CBP. 5.2 Results 5.2.1 E2 Repression of the Human GnRH-I Receptor Promoter Requires ER a To investigate whether E 2 downregulates GnRH-I receptor gene expression at the transcriptional level, the human GnRH-I receptor promoter-luciferase construct p(-1671/+l)-Luc was transiently cotransfected with expression plasmids encoding ERa, ERp\ ERa and ERp, or PR-B into OVCAR-3 cells, followed by 163 stimulation with 100 nM E 2 for 24 h. As shown in Figure 3 OA, E 2 treatment had no apparent effect on the GnRH-I receptor promoter when either an empty pCMV5 vector or pSG5-PR-B was introduced into the cells. However, a 46 % reduction (P < 0.001 vs. control) of promoter activity was observed when ERa was forced expressed in the cancer cells. In contrast, overexpression of ERP produced an insignificant reduction of promoter activity, indicating the involvement of ERa but not ERP in mediating the repression. Since it has been demonstrated that ERp can serve as a transdominant repressor of ERa transcriptional activity (Hall and McDonnell, 1999), we cotransfected both ER subtypes simultaneously into OVCAR-3 cells in order to investigate any functional interference of ERa activity by ERp or vice versa. Our present data revealed that an elevated expression of ERp did not significantly alter the overall sensitivity of the ERa-overexpressing cells to E 2 (Figure 3 OA) though it has been reported that coexpression of ERa and ERP results in the preferential formation of receptor heterodimers instead of homodimers that may possess unique transcriptional activities (Cowley, et al, 1997; Pettersson et al, 1997; Hall and McDonnell, 1999). The specificity of the E 2 action was demonstrated by the observation that repression of the GnRH-I receptor promoter could be attenuated by tamoxifen, which by itself had no apparent effect on the promoter activity (Figure 30B). The functional importance of ERa but not ERP in mediating the repressive effect of E 2 on the GnRH-I receptor promoter was further examined by using the MCF-7 breast adenocarcinoma cell line, which was found to express a significantly higher level of ERa protein than OVCAR-3 cells (Figure 30C). In stark contrast, the expression level of ERP in the breast cancer cells was virtually undetectable (Figure 30C), and similar 164 Figure 30. ERa but not ERP mediates E 2 repression of the human GnRH-I receptor promoter. A, The human GnRH-I receptor promoter-luciferase construct p(-1671/+l)-Luc was transiently transfected with expression plasmids encoding various steroid hormone receptors into OVCAR-3 cells by LIPOFECTAMINE Reagent. The RSV-/acZ vector was also cotransfected in order to normalize the transfection efficiency. Twenty four hours after transfection, the cells were treated with 100 nM E 2 under serum- and phenol red-free conditions for an additional of 24 h before harvest. Lane 1: 1 pg of empty pCMV5; lane 2: 1 pg of pCMV5-ERa; lane 3: 1 pg of pCMV5-ERP; lane 4: 0.5 pg each of pCMV5-ERa and pCMV5-ERp; lane 5: 1 pg each of pCMV5-ERa and pCMV5-ERp; lane 6: 1 pg of pSG5-PR-B. B, The ER antagonist tamoxifen attenuates E2-dependent repression of the GnRH-I receptor promoter. The construct p(-1671/+l)-Luc was cotransfected with pCMV5-ERa and RSV-/acZ into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with 100 nM E 2 , 100 nM tamoxifen (T), or 100 nM E 2 and 100 nM T for 24 h. 165 C, Upper panel: Western blot analysis of the endogenous ERa and ERp proteins in OVCAR-3 (O) and MCF-7 (M) cells. Lower panel: Quantitative comparison of the ERa protein level in the ovarian and breast cancer cell lines by densitometry. D, The construct p(-1671/+l)-Luc was transiently cotransfected with RSV-lacZ into OVCAR-3 and MCF-7 cells. Twenty four hours after transfection, the cells were treated with 100 nM E 2 for 24 h. E, As a control experiment, the reporter plasmid ERE2-tkl09-Luc containing two copies of ERE was cotransfected with 1 pg of either pCMV5-ERa or pCMV5-ERp into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with 100 nM E 2 for 48 h. The relative promoter activity is represented as the percentage of the respective control group (vehicle-treated), of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values in all panels represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control; b, P < 0.05 vs. control; c, P < 0.001 vs. E 2 ; d, P < 0.05 vs. OVCAR-3 cells. 166 (A) (C) <D O C ra •o c 3 ra CD > _ra a: 200 150 100 M <-OVCAR-3 MCF-7 Cell line (D) > o ra o E o k . Q. Cj> > _ra a> 150 ™ 100 I vehicle • E2 (100 nM) OVCAR-3 MCF-7 Cell line 168 (E) 250 -, I* 200 -I o ro k_ <D •+-> O E o i _ Q. 150 A 100 A •S 50 H « 8. • vehicle • E2(100 nM) ER overexpression 169 observations have also been reported elsewhere (Shanmugam et al, 1999). Treatment of MCF-7 cells with 100 nM E 2 for 24 h resulted in a 26 % reduction (P < 0.001 vs. control) of promoter activity, whereas the same treatment exerted no apparent effect in OVCAR-3 cells in the absence of ERa overexpression (Figure 30D). These findings thus implicate that a threshold level of ERa may be required to confer negative E 2 regulation of the GnRH-I receptor promoter. In support of this speculation, we found that the repressive effect of E 2 on the GnRH-I receptor promoter in OVCAR-3 cells was ERa dose-dependent and that the optimal repression was achieved when 1 pg of pCMV5-ERa was cotransfected (data not shown). This amount was then used in all subsequent experiments. To rule out the possibility that E 2 nonspecifically suppressed the GnRH-I receptor promoter, we cotransfected the reporter plasmid ERE2-tkl09-Luc with either pCMV5-ERa or pCMV5-ER|3 into OVCAR-3 cells and then treated the transfected cells with or without the estrogen. The reporter plasmid ERE2-tkl09-Luc contains two copies of the vitellogenin gene ERE upstream of a 109-bp fragment of the TK promoter and has been described previously (Jakacka et al., 2001). The activity of the TK promoter was found to increase by about 2.1- and 1.5-fold in response to E 2 when ERa and ERp were overexpressed in the cells, respectively (Figure 30E). These results thus clearly indicate that E 2 selectively represses the GnRH-I receptor promoter via an ERa-dependent mechanism. 13 11 At low E 2 concentrations (10" and 10" M), no significant decrease in the transcriptional activity of the GnRH-I receptor promoter could be observed in the ERa-overexpressing OVCAR-3 cells (Figure 31 A). However, a dose-dependent inhibition of 170 Figure 31. Dose- and time-dependent effects of E 2 on the human GnRH-I receptor promoter activity in ERct-overexpressing OVCAR-3 cells. The construct p(-1671/+l)-Luc was transiently cotransfected with pCMV5-ERa and RSV-/acZ into the cancer cells. Twenty four hours after transfection, the cells were treated with different concentrations of E 2 for 24 h (A) or with 100 nM E 2 for different time periods (B). The relative promoter activity is represented as the percentage of the control group (vehicle-treated), of which the activity is set as 100 % after being normalized by (3-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control; b, P < 0.05 vs. control. 171 (A) o 10-13 10-1 10-9 10-8 10-7 E2 concentration (M) (B) ^ 150 > u (0 CD o E o CD > CD a: 100 vehicle • E2 (100 nM) 1 7 2 promoter activity was detected when the cells were treated with an increasing concentration of the steroid hormone (10"9 to 10"7 M) (Figure 31A). Repression of the GnRH-I receptor promoter by E 2 was also time-dependent. Short-term E 2 treatment (4 and 8 h) had no apparent effect on the GnRH-I receptor promoter (Figure 3IB). Significant inhibition of promoter activity was only evident after 12 h, and repression was found to increase with time, with a maximal of 80 % reduction being observed after 48 h of the treatment (Figure 3 IB). 5.2.2 Domains of ERa Required for Repression of the Human GnRH-I Receptor Promoter Human ERa is a 595-amino acid protein that has been studied extensively by mutational analysis (Kumar and Chambon, 1988; Mader et al, 1989; Tora et al, 1989). It has been demonstrated that the transcriptional activity of ERa is mediated by two transactivation functions (AFs) located at the amino-terminus (AF-1) and carboxyl-terminus (AF-2). Although both of these AFs work in a synergistic manner in most circumstances, they can also function independently in a cell- and promoter-specific manner (Berry et al, 1990; Tzuckerman et al, 1994). To define the ERa domains required for repression of the GnRH-I receptor promoter, expression vectors encoding a series of 5'- and 3'-deletion mutants of the steroid hormone receptor were constructed (Figure 32A) and cotransfected with p(-1671/+l)-Luc into OVCAR-3 cells. Cotransfection of the wild-type receptor (ERa-wt) resulted in a 50 % repression of promoter activity in response to E 2 (Figure 32B). Deletion of the ligand-binding domain (LBD) created a mutant (ERa-mutl) that was not able to repress the GnRH-I receptor 173 Figure 32. ERa domains required for human GnRH-I receptor promoter repression overlap those for classical transactivation at ERE. A, Structural domains of the wild-type and mutant human ERa. Numbers represent the amino acid (aa) positions. B, The construct p(-1671/+l)-Luc was transiently cotransfected with the wild-type or mutant ERa expression plasmids into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with 100 nM E 2 for 24 h. C, For studies of the classical mode of ER transactivation, the reporter plasmid ERE 2-tkl09-Luc was cotransfected with the wild-type or mutant ERa expression plasmids. Twenty four hours after transfection, the cells were treated with 100 nM E 2 for 48 h. The relative promoter activity is represented as the percentage of the respective control group (vehicle-treated), of which the activity is set as 100 % after being normalized by p-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control; b, P < 0.05 vs. control. 174 3 1 38 180 263 302 A B (AF-1) C (DNA) D E (Hormone, AF-2) F A B (AF-1) C (DNA) C (DNA) D E (Hormone, AF-2) F D E (Hormone, AF-2) F 553 595 ERa-wt ERcx-mut1 ERoc-mut2 A B (AF-1) D E (Hormone, AF-2) F ERcc-mut4 175 promoter (Figure 32B). In contrast, an ERa mutant with a deletion of the amino-terminal AP-1 (ERa-mut2) still mediated the E2-dependent repression (Figure 32B). Further deletion of the ammo-terminus up to amino acid 262 (ERa-mut3) or internal deletion of the DNA-binding domain (DBD) (ERa-mut4) generated mutants that lose the ability to suppress the GnRH-I receptor promoter (Figure 32B). Collectively, these data indicate that both the DBD and LBD are required for the E2-mediated repression of GnRH-I receptor gene transcription. In parallel, we also examined the ability of these ERa mutants to activate the reporter plasmid ERE2-tkl09-Luc. As shown in Figure 32C, the AF-1 was dispensable for the activation (ERa-mut2), whereas both the DBD and LBD were essential for transactivation at the ERE since deletion of either of these regions nearly abrogated transactivation of the reporter gene (Figure 32C). Therefore, these findings indicate that the same ERa domains are required for mediating the classical mode of transactivation and transrepression of the GnRH-I receptor promoter. 5.2.3 An AP-l-Like Motif at -130/-124 Mediates Both Basal Activity and E2 Repression of the Human GnRH-I Receptor Promoter A progressive deletion analysis was performed between -1671 and +1 in order to locate the regions that confer negative E 2 regulation of the GnRH-I receptor promoter. Deletion of the distal region from -1671 to -1018 did not abolish the E 2 sensitivity of the GnRH-I receptor promoter (Figure 33), indicating that the core E2-response elements reside in the proximal 1-kb region. This observation was supported by the finding that removal of this proximal region [i.e. the construct p(-1700/-1018)-Luc] could completely 177 Figure 33. Localization of the E2-response region to -266/-117 of the human GnRH-I receptor 5'-flanking region. A panel of 5'- and 3'-deletion mutants of the GnRH-I receptor 5'-flanking region was cotransfected with pCMV5-ERa and RSW-lacZ into OVCAR-3 cells by LIPOFECTAMINE Reagent. Twenty four hours after transfection, the cells were treated with 100 nM E 2 or vehicle (control) for 24 h. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control. 178 Human GnRH-I receptor 5'-flanking region O T - CD 0 0 0 1 S - -<r i - r- S S (D N t^CD CO O Is- 0 0 CO CD T— T - T - T - T - h- " t n C \ | T - Y I I i i i i i i i + U I I I I L Luc - » | Luc | p(-1671/+1 •»|Luc| p(-1346/+1 •H L u c I p(-1018/+1 p(-1700/-1018 ••I Luc | p(-777/+1 • • f u l c l P(-487/+1 ^[Lucl p(-367/+1 Luc] p(-266/+1 -•TLTJCI p{-117/+1 Luc pGL2-Basic • vehicle • E2(100nM) 0 2 4 6 Relative promoter activity (Fold induction) 179 abrogate the E 2 response (Figure 33). To further delineate the location of the negative E2-response elements, a more detailed 5'-deletion mapping was performed. As shown in Figure 33, although the basal GnRH-I receptor promoter activity was found to decrease when the region between -1018 and -266 was removed, no significant change in the E 2 responsiveness was detected. However, further deletion to -117 totally abolished the basal activity as well as the E 2 sensitivity of the GnRH-I receptor promoter, indicating that the core E2-response elements lie in the 150-bp region between -266 and -117. Sequence analysis revealed a number of putative transcription factor binding sites including an ERE-like element in the 150-bp E2-response region (Figure 34). These elements have been demonstrated to mediate diverse transcriptional responses triggered by the ERs (Blobel et al, 1995; Stein and Yang, 1995; Holth et al, 1997; Ray et al, 1997; Xing and Archer, 1998; Wang et al, 1999; Homma et al, 2000; Farsetti et al, 2001; Jones et al, 2002). Site-directed mutagenesis indicated that alteration of an AP-1-like motif [AP-l-like-mut (a), from 5'TG AC AT A3' to 5'TAGGCCT3'1 resulted in an almost complete loss of the E 2 suppression in OVCAR-3 cells (Figure 34). Interestingly, this mutation also abolished the basal promoter activity, indicating that the motif may concomitantly involve in basal transcription of the GnRH-I receptor gene. In contrast, mutations (or deletion) of other regulatory elements neither significantly affected basal activity nor impaired the E 2 responsiveness of the GnRH-I receptor promoter (Figure 34). To further examine the role of the AP-l-like motif in mediating the suppression, we generated a mutant construct in which the AP-l-like motif was changed to the consensus AP-1 sequence [AP-l-like-mut (b), from 5'TGACATA3' to 5'TGACTCA3']. As shown in Figure 34, this nucleotide substitution had no obvious effect on the sensitivity of the 180 Figure 34. An AP-l-like site at -1307-124 mediates both basal activity and E 2 responsiveness of the human GnRH-I receptor promoter. Upper panel: A diagrammatic representation of the mutant promoter constructs. Nucleotide positions of various potential transcription factor binding sites in the core E2-response region are indicated. Each motif was mutated by introducing a restriction enzyme recognition site into the core binding sequence. The AP-l-like-mut (b) was prepared by mutating the wild-type sequence into the consensus AP-1 site (5'TGACTCA3'). Mutations are marked with black crosses. Lower panel: Wild-type [p(-266/+l)-Luc and p(-214/+l)-Luc] or the mutant constructs were transiently cotransfected with pCMV5-ERa and RSV-/acZ into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with 100 nM E 2 or vehicle (control) for 24 h. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control. DRE, dioxin-response element; NF-Y, nuclear factor-Y. 181 LU LU O or «. LU CD < p ( - 2 6 6 / + i ) - L u c i-^HHK) E-box-1-mut E-box-2-mut p(-214/+1)-Luc r^JJH^-Q NF-Y-mut ^-^y}H>0 ERE-like-mut GATAb-mut ^ - | ^ | ^ { ) AP-1-like-mut(a) ^ f ^ J | - ( > § Ap-1-|ike-mut^ i-W^iHK} TGACATA I TGACTCA Luc Luc Luc Luc Luc Luc j * \ , Luc ) * T h Luc L * Luc p(-266/+1)-Luc E-box-1-mut E-box-2-mut p(-214/+1)-Luc NF-Y-mut ERE-like-mut GATAb-mut AP-1-like-mut (a) AP-1-like-mut (b) pGL2-Basic m n -r— 2 3 • vehicle • E2(100n - i — 4 0 1 Relative promoter activity (Fold induction) 5 182 GnRH-I receptor promoter to E 2 , but rather, it strongly stimulated the basal activity by 2.3 fold. Therefore, it is apparent that a consensus AP-1 sequence and the AP-l-like motif possess opposing effects on the GnRH-I receptor promoter. 5.2.4 Multiple Transcription Factors Including c-Jun and c-Fos Form Complexes with the AP-1-Like Motif To examine if E 2 represses the GnRH-I receptor promoter via altering DNA-binding activity of transcription factors at the AP-l-like site, we performed EMSAs using nuclear extracts prepared from E 2 - or vehicle-treated OVCAR-3 cells transfected with pCMV5-ERoc. Three DNA-protein complexes (complex-G, -H, and -I) were formed with the AP-l-like motif, and we found that the intensity of these complexes was not significantly altered after E 2 treatment (Figure 35A). Formation of these complexes could be inhibited by the unlabeled probe or AP-l-c but not by other nonspecific sequences (NF-KB-C and AP-2-c), indicating specific interactions of AP-1 transcription factors with the AP-l-like site (Figure 35A). The specificity of these complexes was further confirmed by the observation that mutation of the AP-l-like site completely abolished complex formation (Figure 35A). Antibody supershift assays showed that c-Jun and c-Fos were present in complex-I but not in the two upper complexes (Figure 35B). The identity of complex-G and -H remains to be determined as antibody targeted against the CREB family transcription factors, which also recognize AP-1 motif, did not affect complex formation (Figure 35B). Despite the finding that the DBD of ERa was required for repression of the GnRH-I receptor promoter, we found that addition of anti-ERa antibody (or anti-ER(5, data not shown) also had no effect on 183 Figure 35. Binding of the AP-1 transcription factors c-Jun and c-Fos but not ERa to the AP-l-like motif. Synthetic oligonucleotides containing the wild-type (wt) and mutant (mut) AP-l-like motifs were annealed and end-radiolabeled with 3 2P, followed by incubation with nuclear extracts (NE) from OVCAR-3 cells in the absence or presence of competitor oligonucleotides (or antibodies). A, Five micrograms of NE from vehicle-treated or E2-treated ERa-overexpressing OVCAR-3 cells were incubated with 50 frnol of the radiolabeled probes in the presence of the unlabeled sequence, AP-l-c, NF-K B - C , or AP-2-c. B, Five micrograms of NE from the vehicle-treated cells were preincubated with 3 pg of anti-GATA-4, anti-c-Jun, anti-c-Fos, anti-ERa, or anti-CREB antibody for 30 min at room temperature before the addition of the wild-type radiolabeled probe. Similar binding results were obtained from E2-treated cells (data not shown). C, In vitro translated human ERa proteins (2.5 pi) were incubated with the wild-type AP-l-like probe or three copies of ERE in the presence or absence of 100 nM E 2 . 184 (A) wt-probe vehicle-treated E2-treated CD SI o 3 E X X o o LO O CO X o LO X o o co CD O c CD 3 CT CD </> ~o CD Competitor NE Complex-G • Complex-H • Complex-I • CL < CL < CD J5 ro c ID CD o c CD c r CD </> T3 CD CD . O ro C X o o co CO X o o CO CL < X o LO CL < X o o co CL < X o LO 8 c CD •D CT CD C/3 TO 03 CD - O TO c 3 X o o co CD O c CD C J 03 en T3 03 03 X3 ro c 3 X o o co o i CO Y X o O CO-o I C\l i CL < • a 03 ro 03 I 03 O  > ~C 03 ro 0) LL) H " HHH HH Free probe -185 (B) I § 8 8 S < -=i u_ or or Antibody CD 6 6 L U O N E U y u y y . Complex -G • Complex -H • Complex-I Free probe (C) AP-1- l ike E R E E2 (100 nM) E R a + + + I I + + + Free probe 186 complex formation (Figure 35B). Moreover, although in vitro translated ERa proteins could interact with a consensus ERE sequence in the presence or absence of E2, they did not bind to the AP-l-like motif (Figure 35C). These results thus indicate that ERa represses the GnRH-I receptor gene transcription via a mechanism that does not rely on direct binding to the GnRH-I receptor promoter. 5.2.5 PMA Antagonizes E2-Dependent Repression of the Human GnRH-I Receptor Promoter An essential feature of AP-1 binding sites is their ability to confer phorbol ester responsiveness to the promoter. To examine whether the AP-l-like motif at -130/-124 behaves as a classical AP-1 element, we transiently transfected the GnRH-I receptor promoter-luciferase construct p(-266/+l)-Luc into OVCAR-3 cells and then treated the transfected cells with various concentrations of PMA. A dose-dependent stimulation of promoter activity was observed, with a maximal of 1.8-fold increase being detected at 10 nM PMA (Figure 36A). Higher doses (100 nM and 1 uM) reduced the PMA responsiveness of the promoter (Figure 36A). In contrast, under the conditions that have been shown to generate the optimal forskolin response of the GnRH-I receptor promoter in gonadotropes and placental cells (Cheng and Leung, 2001; Cheng and Leung, 2002), forskolin treatment had insignificant stimulatory effect on the activity of the construct in OVCAR-3 cells (Figure 36A). Importantly, mutation of the AP-l-like motif significantly attenuated the PMA sensitivity of the GnRH-I receptor promoter in the cancer cells (Figure 36B), indicating that it functions as a typical PMA-response element. 187 Figure 36. Functional antagonism between the ER and AP-1 signaling pathways in regulating human GnRH-I receptor gene transcription. A, The human GnRH-I receptor promoter construct p(-266/+l)-Luc was transiently cotransfected with pCMV5-ERoc and RSV-lacZ into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with various concentrations of PMA for 24 h or forskolin for 6 h. B, The construct p(-266/+l)-Luc or AP-l-like-mut (a) was cotransfected with pCMV5-ERa and RSV-/acZ into OVCAR-3 cells, and the transfected cells were treated with 100 nM PMA for 24 h. C, The construct p(-266/+l)-Luc was cotransfected with pCMV5-ERcc and RSV-/acZ into OVCAR-3 cells, and the transfected cells were treated with 100 nM E 2 , 100 nM PMA, or 100 nM E 2 and 100 nM PMA for 24 h. The relative promoter activity is represented as the percentage of the control group (DMSO-treated), of which the activity is set as 100 % after being normalized by (3-galactosidase activity. Values in all panels represent the mean + SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control; b, P < 0.05 vs. control. EtOH, Ethanol. 188 D, Western blot analysis of the phosphorylation (P) and total expression (T) levels of c-Jun and c-Fos in OVCAR-3 cells following transient transfection and pharmacological treatment as described in panel C. E, Transcription factor binding to the AP-l-like site is not affected by PMA or cotreatment with PMA and E 2 . Five micrograms of nuclear extracts (NE) from ERoc-overexpressing OVCAR-3 cells (treated for 24 h with DMSO, 100 nM PMA, or 100 nM E 2 and 100 nM PMA) were incubated with 50 frnol of the radiolabeled AP-l-like probe for 15 min at room temperature before analyzed by a 6 % native gel. 189 (A) 200 i 5 150 H o ro i _ a 4-1 o E o a. > _ro 0) Qi 100 PMA X a X Forskolin Treatment (B) 200 • vehicle • PMA (100 nM) p(-266/+1)- AP-1-like-Luc mut (a) Promoter construct 190 (C) 250 >% > 200 U re *5 150 CD > ro 100 EtOH DMSO E2 PMA E2 + PMA Treatment E2+ (D) EtOH DMSO E 2 PMA PMA I 1 « — P-c-Jun T-c-Jun .» II niiiiiiiMir ^0000m''^ ^ T~C"' FOS ' '''™ ••^•r'"-^ ^^ pr 191 192 Since it is apparent that both the negative E 2 and positive PMA responses of the GnRH-I receptor promoter converge at the AP-l-like motif, we sought to investigate whether PMA stimulation could antagonize the repressive effect of E 2 on the GnRH-I receptor promoter. As shown in Figure 36C, cotreatment of OVCAR-3 cells with PMA completely abolished the E 2 repression, supporting the existence of a functional antagonism between the ER and AP-1 signaling pathways in regulating the GnRH-I receptor gene transcription. To elucidate the mechanism by which this antagonism occurs, we examined the phosphorylation and expression levels of the AP-1 components c-Jun and c-Fos in OVCAR-3 cells after the transfection and pharmacological treatment as described in Figure 36C. Treatment with E 2 alone had no apparent effect on AP-1 protein expression and c-Jun phosphorylation, as revealed by Western blot analysis (Figure 36D). Similarly, stimulation with PMA or PMA in the presence of E 2 did not affect AP-1 expression significantly (Figure 36D). Interestingly, we found a strong stimulation of c-Jun phosphorylation on serine-63 after 24 h of PMA treatment and that this change was unaffected by cotreatment with E 2 (Figure 36D). To examine the functional consequence of this enhanced c-Jun phosphorylation in terms of AP-1 binding to the AP-l-like motif, we performed EMSAs using nuclear extracts prepared from ERa-transfected OVCAR-3 cells treated with PMA or PMA in the presence of E 2 . Consistent with an earlier finding from Baker and coworkers (Baker et al, 1992), we found that the enhanced phosphorylation did not increase the binding of the c-Jun/c-Fos heterodimer to the AP-l-like site (Figure 36E), indicating that the ER-AP-1 antagonism occurs independent on the AP-1 DNA binding activity. 193 5.2.6 Overexpression of the Transcriptional Coactivator CBP Attenuates E2-Dependent Repression of the Human GnRH-I Receptor Promoter So how does ERa suppress GnRH-I receptor gene transcription in cancer cells? It has been proposed that nuclear receptor-AP-1 antagonism can occur through direct competition for limiting transcriptional coactivators that are commonly required to induce gene expression (Arias et al, 1994; Bannister et al, 1995; Chakravarti et al, 1996; Kamei et al, 1996). A potential candidate of these cofactors is CBP, which has been reported to interact with both nuclear receptors and AP-1 transcription factors (Kamei et al, 1996). It could be imagined that under the condition of coactivation, AP-1 and ligand-activated ER would compete for a limiting amount of CBP, leading to suppression of AP-1 transcriptional activity. To examine whether this mechanism is involved in repression of the GnRH-I receptor promoter, we cotransfected the construct p(-1671/+l)-Luc with an increasing amount of a CBP expression plasmid into OVCAR-3 cells and then examined the degree of repression after 24 h of E2 treatment. As shown in Figure 37, overexpression of CBP resulted in a dose-dependent reduction of the E2-induced repression of the GnRH-I receptor promoter such that a significant decrease (51 % to 28 % repression) was observed when 1.5 pg of pRc/RSV-CBP-HA were cotransfected, and the decrease was even more evident (to 12 % repression) when 2 pg of the expression plasmid were used. These findings thus implicate that CBP is involved in transcriptional repression of the GnRH-I receptor gene by ligand-activated ERa. 194 Figure 37. Overexpression of the transcriptional coactivator CBP attenuates the E2-dependent repression of the human GnRH-I receptor promoter. The construct p(-1671/+l)-Luc was cotransfected with pCMV5-ERoc and RSV-/c7cZ in the presence of an increasing amount of pRc/RSV or pRc/RSV-CBP-HA into OVCAR-3 cells. Twenty four hours after transfection, the cells were treated with 100 nM E 2 for 24 h. Data are expressed as the percentage of repression relative to each respective control group (vehicle-treated). Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. no CBP overexpression. 195 o ^  1 1 1 1 1 0 0.5 1 1.5 2 3 pRc/RSV-CBP-HA (pg) 196 5.3 Discussion There is convincing evidence that GnRH-I functions as an important autocrine and/or paracrine factor in some extrapituitary tissues (Bussenot et al, 1993; Lin et al, 1995; Emons et al, 1998; Kang et al, 2000c; Chen et al, 2002a), in addition to its essential role in stimulating gonadotropin secretion. A well-known example of these extrapituitary functions is to serve as a negative growth regulator in tumors derived from the reproductive tract of both sexes (Emons et al, 1998). Although previous studies from our laboratory have shown that E 2 downregulates the GnRH-I receptor mRNA level in human ovary and antagonizes the growth-inhibitory effect of GnRH-I in ovarian cancer via an ER-mediated process (Nathwani et al, 2000; Kang et al, 2001a), the mechanism underlying these negative E 2 actions is obscure. In this report, we demonstrated for the first time that an AP-l-like motif was responsible for mediating E 2 repression of the GnRH-I receptor promoter via an ERoc-dependent mechanism. Also, we showed that this repression was apparently independent of direct ER binding to the promoter and involved the transcriptional coactivator CBP, which is recruited by both nuclear receptors and AP-1 proteins for activating gene expression. In the present study, we found that cotransfection of a wild-type ERa expression piasmid into OVCAR-3 cells was necessary to restore the estrogen response. This phenomenon is probably due to downregulation of the receptor expression in cultures as the ER gene is known to locate in an unstable chromosomal region (Stein and Yang, 1995). This scenario would be advantageous as we could definitely dissect the role of the ER subtype in mediating the E 2 effect in the cancer cells. On the one hand, the data obtained from MCF-7 cells (without ERa overexpression) (Figure 30D) indicate that 197 estrogen repression of the GnRH-I receptor promoter does occur at a physiological level of ERa protein. On the other hand, we do not have a clear explanation for the modest transactivation response observed for ERE2-tkl09-Luc in OVCAR-3 cells (Figure 30E), but we speculate that the cancer cells may lack certain transcription factors, which can interact with the vitellogenin gene ERE besides the ER (Jost et al, 1990) and may be essential for full expression of the estrogen response. Despite the fact that ERa and ERp share a high degree of homology in their DBDs and LBDs so that they recognize the same consensus ERE and have similar affinities to estrogens, they do respond differentially to E2 in controlling gene transcription in various cellular systems (Scheidegger et al, 2000; Shapiro et al, 2000; Liu et al, 2002). Furthermore, gene-knockout studies have also revealed that ERa possesses biological functions distinct from those of ERp as evidenced with the different phenotypes of the aERKO and PERKO mice (Krege et al, 1998; Couse et al, 1999). Our data showed that ERa, but not ERP, was able to repress the GnRH-I receptor promoter in OVCAR-3 and MCF-7 cells, providing an indication that ERa may play a predominant role in controlling cell proliferation in ovarian and breast cancers, where both receptor subtypes are expressed (Dotzlaw et al, 1997; Branderberger et al, 1998; Lau et al, 1999; Shaw et al, 2002). In fact, these observations are supported by previous findings, which have demonstrated an upregulation of ERa relative to ERp gene expression during ovarian and mammary carcinogenesis (Leygue et al, 1998; Pujol et al, 1998). The human ER belongs to the nuclear receptor superfamily of ligand-inducible transcription factors (Evans, 1988), whose members include the receptors for steroids, thyroid hormone, retinoic acid, vitamin D, and orphan receptors for which no ligands 198 have been identified. In the classical mode of ER action, the receptor binds to ERE as homodimers (Kumar and Chambon, 1988) or heterodimers (Pace et al, 1997; Ogawa et al, 1998; Tremblay et al, 1999) in estrogen-responsive promoters and then recruits an array of transcriptional cofactors, via which the nuclear receptor interferes with other transcription factors including components of the general transcription factor machinery (McKenna et al, 1999). In addition, some of the cofactors possess chromatin-remodeling activities or yet recruit additional proteins to the nuclear receptor-cofactor complex to mediate transcription regulation of the target genes (McKenna et al, 1999). However, it has also been well reported that EREs are absent in many genes that are regulated by ERs. Multiple lines of evidence indicate that this nonclassical mode of ER action occurs in the absence of direct ER binding to D N A and is achieved via protein-protein interactions with other transcription factors that bind to their response sequences. For instance, it has been demonstrated that physical interactions of ERa with N F - K B and C/EBP transcription factors can lead to transcriptional repression of the N F - K B - and C/EBP-dependent IL-6 promoter (Stein and Yang, 1995; Ray et al, 1997). Also, repression of erythropoiesis by estrogen has been demonstrated to involve estrogen-dependent inhibition of the transcriptional activity of GATA-1 by direct interaction with ERa (Blobel et al, 1995). Furthermore, repression of the IGF-1 receptor gene transcription has been shown to be mediated by ERa inhibition of Spl binding to the target promoter (Scheidegger et al, 2000). Our finding that mutation of the AP-l-like site reduced the promoter activity to the basal level does not oppose its involvement in mediating the E 2 suppression. Rather, it implies that the estrogen exerts its repressive effect via interfering with the basal 199 transcription of the GnRH-I receptor gene. The fact that the AP-l-like site is quite different from the consensus sequence may contribute to their opposing effects on the GnRH-I receptor promoter (Figure 34). Other promoters that are also repressed by E2 via AP-1 sites include those of the human hepatic lipase (Jones et al, 2002), murine lipoprotein lipase (Homma et al, 2000), ovine FSH(3 (Miller and Miller, 1996), as well as human choline acetyltransferase (Schmitt et al, 1995) genes. It should be noted that ERa has been shown to physically interact with promoter-bound c-Jun to mediate the nonclassical pathway and that the DBD of the receptor is dispensable for its activity at AP-1 sites (Gaub et al, 1990; Webb et al, 1995; Jakacka et al, 2001; Teyssier et al, 2001). Accordingly, our present data revealed that E2 repressed the GnRH-I receptor promoter in the absence of direct DNA binding (Figure 35). However, our data also indicated that ERa was unlikely to be tethered to the promoter via interaction with c-Jun as we had repeatedly failed to interfere with c-Jun binding by using an anti-ERa antibody. In addition, our data also rule out the possibility that E 2 repressed the GnRH-I receptor promoter via altering AP-1 binding activity, as reported in some instances (Homma et al, 2000; Jones et al, 2002). Furthermore, we found that the DBD together with the LBD, which are necessary for gene activation at a classical ERE, were required for transrepression of the GnRH-I receptor promoter. The LBD harbors the ligand-dependent AF-2, which mediates activation of gene transcription by recruiting a large coactivator complex composed of pi 60, CBP/p300, as well as p300 and CBP-associated factor (Shibata et al, 1997; Torchia et al, 1998; Webb et al, 1998). Since the antiestrogen tamoxifen has been shown to prevent the formation of an active AF-2 surface (Shiau et al, 1998), the observation that tamoxifen could antagonize E 2 -200 mediated suppression of the GnRH-I receptor promoter indicates that the AF-2 surface is involved in the repression process. Our present findings indicate that c-Jun phosphorylation is involved in regulating GnRH-I receptor gene transcription via a pathway independent on its DNA binding activity. There are several lines of evidence indicating that this posttranslational modification may affect c-Jun interaction with other protein factors such as CBP (Bannister et al, 1995; Inada et al, 1997; Zeiner et al, 1997). The transcriptional coactivator CBP has been proposed as an integrator of multiple signal transduction pathways by virtue of its ability to enhance transcriptional activation by CREB, AP-1, and nuclear receptors (Arias et al, 1994; Kwok et al, 1994; Kamei et al, 1996). Recruitment of CBP by c-Jun requires phosphorylation of the AP-1 protein on serines-63 and 73 by JNKs (Pulverer et al, 1991; Smeal et al, 1991). In contrast to the finding that ligand-activated nuclear receptors including those for glucocorticoids, retinoic acid, and thyroid hormone can antagonize AP-1 activity by inhibiting c-Jun phosphorylation (Caelles et al, 1997), our current data found that E2-bound ERa did not reduce c-Jun phosphorylation induced by PMA (Figure 36D). It is speculated that the ERa-AP-1 antagonism observed in E2 regulation of the GnRH-I receptor gene transcription may occur via competition for a limiting amount of CBP that is shared by both classes of the transcription factors for activating gene expression. Nonetheless, it is possible that other transcriptional cofactors may participate in this antagonism as it has been reported that steroid receptor coactivator-1 and CAPER (Coactivator of AP-1 and ERs) are also coactivators for ERa and AP-1 proteins (Lee et al, 1998b; Jung et al, 2002). In addition, it remains to be determined if other CBP-recruiting factors repress the GnRH-I 201 receptor promoter equivalently. Furthermore, one should note that as the E 2 effect could only be observed after 12 h of treatment (Figure 3 IB), the repression may be indirect and involve activation of certain early response genes, whose products can interfere with AP-1 induction. For instance, it has been demonstrated that the expression of the Egrl gene can be rapidly and strongly induced by E 2 via ERKs (de Jager et al, 2001). Since Egrl is also capable of physically and functionally interacting with CBP to activate gene transcription (Silverman et al, 1998), it is possible that an early response protein, instead of the ligand-activated ER, competes with AP-1 for the limiting CBP. Nevertheless, as cofactor sharing appears to be the major mechanism in mediating E 2 suppression of the GnRH-I receptor gene transcription, our data strengthen the notion that ERa and ERp have different preferences and affinities in cofactor recruitment (Wong et al, 2001; Bramlett and Burris, 2002) that may account for their differential transrepressing activities on the GnRH-I receptor promoter. In addition to the c-Jun/c-Fos heterodimer, two unidentified complexes (complex-G and -H) were formed with the AP-l-like motif (Figure 35). We found that the formation of these complexes was also unaffected by antibodies specific to other AP-1 proteins such as JunB, JunD, and FosB (data not shown). Indeed, a similar situation has also been observed in the lipoprotein lipase promoter, in which an AP-l-like sequence mediating E 2 repression is bound by an unknown AP-1-related protein (Homma et al, 2000). Whether these complexes are derived from other existing or unknown members of AP-1 proteins remains to be elucidated. Also, it is not known whether the failure to detect ERa binding to the GnRH-I receptor promoter is due to inappropriate experimental conditions such as dilution of transcription factors in the 202 nuclear extracts and the use of nonphysiological buffers. Importantly, the identification of these complexes indicates that additional transcriptional regulators, which may be more important than CBP, are involved in mediating the E 2 effect. Further studies are warranted to elucidate the complex cross-talk between the ER and AP-1 signaling pathways in transcriptional regulation of the GnRH-I receptor gene. It should be noted that pituitary GnRH-I receptor gene expression is also tightly regulated by estrogen. Like the ovary, E 2 has been shown to exert an inhibitory effect on GnRH-I receptor expression in the pituitary both in vitro and in vivo (Emons et al, 1988; Kaiser et al, 1993). However, E 2 also exerts a positive feedback action, presumably by enhancing hypothalamic GnRH-I secretion, on pituitary GnRH-I receptor gene expression, which is important for sensitizing gonadotropes to GnRH-I during the preovulatory gonadotropin surge (Bauer-Dantoin et al, 1995). Accordingly, a stimulatory estrogen response on pituitary luciferase expression has been observed for the ovine GnRH-I receptor promoter in vivo (Duval et al, 2000). In contrast, using the gonadotrope-derived alphaT3-l cells, our preliminary data have failed to reveal any E 2 regulation of the human promoter even with ER overexpression (data not shown). Whether this discrepancy is due to species differences of the GnRH-I receptor promoters or concomitant requirement of the hypothalamic input to mediate the estrogen action on pituitary GnRH-I receptor gene transcription requires further investigation. 203 CHAPTER VI Functional Cooperation between Multiple Regulatory Elements in the Untranslated Exon 1 Stimulates the Basal Transcription of the Human GnRH-II Gene 6.1 Introduction Until recently, GnRH-I was thought to be the sole hypothalamic regulator controlling the mammalian reproductive process. However, the identification of a second form of GnRH from chicken hypothalamus (termed GnRH-II) reveals that GnRH-II is the most widely expressed form of GnRH and that its structure is conserved among vertebrates from primitive fishes to humans (Miyamoto et al, 1984; King and Millar, 1986; Powell et al, 1986; Sherwood and Whittier, 1988; Lovejoy et al, 1992; King et al, 1994; Rissman et al, 1995; Kasten et al, 1996; Lescheid et al, 1997; Chen et al, 1998; White et al, 1998; Gestrin et al, 1999; Urbanski et al, 1999). This second GnRH form differs from GnRH-I by three amino acids at positions 5, 7, and 8 (His5Tip7Tyr8GnRH-I), and the gene encoding GnRH-II has been cloned from monkey and human brains (White et al, 1998; Urbanski et al, 1999). In contrast to GnRH-I, GnRH-II mRNA is expressed at significantly higher levels outside the human brain, particularly in the kidney, bone marrow, and prostate gland (White et al, 1998). The evolutionary conservation of GnRH-II and its wide distribution in tissues indicate that the neuropeptide has vital biological functions. For instance, it has been reported to suppress the proliferation of some reproductive tissue-derived cancer cells (Choi et al, 2001; Chen et al, 2002b; Grundker et al, 2002), to regulate hCG release from the placenta (Siler-Khodr and Grayson, 2001), and to inhibit ovarian steroidogenesis (Kang 204 et al, 2001c). In addition, GnRH-II has been shown to preferentially stimulate FSH release from cultured pituitary cells (Millar et al, 1986), thus supporting a notion for the existence of a specific FSH-releasing factor (Lumpkin et al, 1987; Padmanabhan and McNeilly, 2001). More recently, Chen and coworkers have demonstrated that GnRH-II can increase the expression of a 67-kDa laminin receptor in both normal and cancer T cells and stimulate their adhesion to laminin, in vitro chemotaxis, and in vivo homing to specific organs (Chen et al, 2002a). Several studies have revealed that the expression of the human GnRH-I and GnRH-II genes is regulated differentially. In the neuronal medulloblastoma TE-671 cells, transcription of the GnRH-II gene is strongly upregulated by cAJVIP, which only produces a modest stimulation of the GnRH-I promoter (Chen et al, 2001a). This cAMP-activated GnRH-II gene expression is mediated via a CRE located between -860 and -853, which is also crucial for the basal transcription of the gene (Chen et al, 2001a). Consistent with this finding, Kang and colleagues have demonstrated that the mRNA level of the GnRH-II gene is stimulated by gonadotropins in human GL cells (Kang et al, 2001c). Apart from cAMP, the expression of these neuropeptides has been shown to be differentially controlled by E 2 in such a way that the estrogen increases the transcription and the steady-state mRNA level of the GnRH-II gene but decreases those of the GnRH-I gene (Chen et al, 2002c). Together with their discrete tissue expression patterns, these observations strongly indicate that the two forms of GnRH play distinct biological roles in humans. Although the cAMP/PKA signaling pathway has been implicated to mediate full expression of the GnRH-II promoter activity (Chen et al, 2001a), the involvement of 2 0 5 other molecular mechanisms in controlling the GnRH-II gene transcription remains obscure. To address this timely issue, two human GnRH-II-expressing cell lines TE-671 and JEG-3 were used to identify and characterize additional transcriptional machinery important for GnRH-II gene expression. Interestingly, we found that two putative EBSs and one ELE in the untranslated first exon functioned cooperatively to stimulate the basal transcription of the GnRH-II gene. Also, we revealed for the first time that the ubiquitously expressed bHLH transcription factor AP-4 is an enhancer protein for the GnRH-II promoter. 6.2 Results 6.2.1 Identification of the Transcription Start Sites for the Human GnRH-II Gene A primer extension analysis was performed in order to identify the transcription start sites for the human GnRH-II gene. By using a primer (PE-GII) situated at exon 2 of the gene, six extension products were generated when poly(A) RNA from TE-671 and JEG-3 cells were used. The transcription start sites deduced from these products were located at -956, -912, -899, -882, -861, and -858 (Figure 38). No extension product was obtained from the control yeast transfer RNA (tRNA) (Figure 38). In addition, a 5'-RACE reaction was performed in order to examine any possible usage of upstream transcription start sites by the GnRH-II gene. Nucleotide sequencing revealed that all of the transcripts had their 5' ends located approximately from -950 to -796, with no evidence for utilization of upstream initiation sites by either the neuronal or placental cells (data not shown). 206 Figure 38. Identification of the transcription start sites for the human GnRH-II gene in TE-671 and JEG-3 cells by primer extension analysis. Five micrograms of poly(A) RNA from TE-671 and JEG-3 cells were hybridized and extended with the primer PE-GII. Six extension products (marked with arrows) were obtained from both cell lines, and their sizes were determined by comparing with a sequencing ladder (T, G, C, A) generated from the M13mpl8 DNA. The transcription start sites deduced from these extension products were located at -956, -912, -899, -882, -861, and -858. No extension product was detected from the control yeast tRNA. 207 T G C A *. ~» trass co 9 6 LU U J I - =3 < Z -t—i co CO CD >--269 bp (-956) -225 bp (-912) -212 bp (-899) -195 bp (-882) ^ 1 7 4 bp (-861) •171 bp (-858) 208 6.2.2 Transcriptional Activity of Various Regions of the Human GnRH-II Gene To identify regulatory sequences important for human GnRH-II gene transcription, a panel of promoter constructs containing various regions of the gene was generated and examined in TE-671 and JEG-3 cells. Transient transfection studies revealed that these cells displayed similar promoter activity profiles. The promoter activity was found to be completely abolished in both cell lines when intron 1 was present in constructs p(-2103/+l)-Luc-GII, p(-793/+l)-Luc-GII, and p(-749/+l)-Luc-GII (Figure 39A), indicating the presence of very strong NREs in this region. Deletion of intron 1 increased the promoter activity of p(-2103/-750)-Luc-GII drastically in TE-671 (210-fold vs. pGL2-Basic) and JEG-3 (198-fold vs. pGL2-Basic) cells (Figure 39A). On the contrary, removal of the untranslated exon 1 from p(-2103/-750)-Luc-GII [i.e. the construct p(-2103/-794)-Luc-GII] reduced the promoter activity by 4.3- and 4.5-fold in the neuronal and placental cells, respectively (Figure 3 9A), whereas lower levels of reduction (2.2-3.3-fold) were also observed in OVCAR-3, MCF-7, and COS-7 cells (Figure 39B). Collectively, these results indicate that the noncoding exon plays an important role in mediating full expression of the basal GnRH-II promoter activity and that the transcription factors interacting with this region are widely expressed. To delimit the core promoter region, a series of 5'-deletion mutants between -2103 and -750 was constructed and then analyzed in TE-671 and JEG-3 cells. Deletion of the 5'-flanking sequence from -2103 to -1124 had no significant effect on the promoter activity (Figure 40A). However, removal of the sequence to -924 decreased the activity by 1.5- and 2.6-fold in the neuronal and placental cells, respectively. Further 5'-deletion from -864 to -793 completely abolished the promoter function (Figure 40A), 209 Figure 39. Transcriptional activity of various regions of the human GnRH-II gene in TE-671 and JEG-3 cells. A, Upper panel: A diagrammatic representation of the human GnRH-II gene structure adopted from the published sequence (GenBank accession no.: AF036329). The location of the 5'-fianking region, exon 1 (Exl), intron 1, and exon 2 (Ex2) is indicated. Lower panel: A series of deletion constructs containing various regions of the GnRH-II gene was transiently transfected into TE-671 and JEG-3 cells by LIPOFECTAMINE Reagent. The RSV-/acZ vector was also cotransfected to normalize the transfection efficiency. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector. B, The constructs p(-2103/-794)-Luc-GII and p(-2103/-750)-Luc-GII were further analyzed in OVCAR-3, MCF-7, and COS-7 cells to examine the enhancer activity of the untranslated exon 1 in other cell types. The relative promoter activity is represented as the percentage of p(-2103/-794)-Luc-GII, of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. pGL2-Basic; b, P < 0.05 vs. p(-2103/-794)-Luc-GII. 210 (A) Human GnRH-II gene A T G -794 -750 Ex1 Ex2 -2103 -793 -749 +1 5 ' - f lank ing : -2103to -794 Exon 1: -793 to -750 Intron 1 : -749 t o - 8 Exon 2: -7 to +154 Luc p(-2103/+1)-Luc-GII W L u c p(-793/+1)-Luc-GII Luc Luc Luc Luc p(-749/+1)-Luc-GII p( -2103/-794)-Luc-GII p( -2103/-750)-Luc-GII p G L 2 - B a s i c • J E G - 3 • TE-671 1 1 1 1 1 0 50 100 150 2 0 0 250 Relative promoter activity (fold induction) 211 (B) > U rs a> o E o JS CD 400 -, 300 -J 200 4 • p(-2103/-794)-Luc-GII • p(-2103/-750)-Luc-GII » 100 4 0 OVCAR-3 MCF-7 COS-7 Cell line 212 Figure 40. Fine mapping of the minimal human GnRH-II promoter in TE-671 and JEG-3 cells. A, A panel of 5'-deletion constructs between -2103 and -750 was generated and then cotransfected with RSV-lacZ into TE-671 and JEG-3 cells. B, Comparison of the transcriptional activity of the construct p(-l 124/-750)-Luc-GII in GnRH-II-expressing (TE-671, JEG-3, and SK-OV-3) and nonGnRH-II-expressing (SH-SY5Y and HDF) cell lines. The relative promoter activity is represented as the fold induction when compared to the promoterless pGL2-Basic vector in the respective cell lines. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. pGL2-Basic. 213 (A) Human GnRH-II 5'-flanking region Spel Nhe\ (-1962) (-1524) _l l _ -2103 -750 Ex1 Luc Luc -•I Luc |p(-2103/-750)-Luc-GII p(-1962/-750)-Luc-GII p(-1789/-750)-Luc-GII p(-1524/-750)-Luc-GII p(-1324/-750)-Luc-GII p(-1124/-750)-Luc-GII p(-924/-750)-Luc-GII Luc Luc Luc Luc ••I Luc | p(-864/-750)-Luc-GII p(-793/-750)-Luc-GII pGL2-Bas ic Luc Luc 100 200 300 Relative promoter activity (fold induction) 214 (B) GnRH-II- nonGnRH-ll-expressing expressing 400 -i •B 300 A o ^ (0 c i _ o O o ° E -o 200 -| o c L . — o o ~ - 100 H w 0) Cell line 215 indicating that positive regulatory elements exist between -1124 and -924 and that the untranslated first exon itself has no transcriptional activity. Taken together, these data indicate that the core promoter region resides between -1124 and -750 and that exon 1 is likely to serve as an enhancer element to stimulate the GnRH-II gene transcription. The functional relevance of this minimal promoter in directing GnRH-II gene transcription was confirmed by analyzing p(-1124/-750)-Luc-GII in the nonGnRH-II-expressing SH-SY5Y (Chen et al, 2001b) and HDF cells, which were found to exhibit much lower promoter activity than the GnRH-II-expressing TE-671, JEG-3, and SK-OV-3 (Choi et al, 2001) cells (Figure 40B). To ascertain whether the exon 1 can function as a stand-alone enhancer, four heterologous constructs, in which the exon was cloned in different positions (upstream and downstream) or orientations (sense and reverse) relative to a SV40 promoter, were prepared. As shown in Figure 41, no significant change in promoter activity was observed when the exon was cloned upstream of the viral promoter (UsExon l-pGL2-Promoter and UrExon l-pGL2-Promoter). However, a 2.9- and 3.5-fold induction of promoter activity was observed in TE-671 and JEG-3 cells, respectively when the exon was situated in a sense orientation and downstream of the viral promoter (DsExon 1-pGL2-Promoter) (i.e. in its native position and orientation in the human GnRH-II gene). In contrast, a significant reduction of promoter activity (69 % in TE-671 cells and 56 % in JEG-3 cells) was detected when the exon was cloned in the opposite direction (DrExon l-pGL2-Promoter) (Figure 41). Taken together, these data indicate that the first exon of the human GnRH-II gene can function in a promoter-independent manner, 216 Figure 41. The enhancer activity of exon 1 of the human GnRH-II gene is position- and orientation-dependent. Upper panel: A diagrammatic representation of the heterologous promoter constructs. The untranslated exon 1 of the human GnRH-II gene (shaded box) was cloned either upstream or downstream of a SV40 promoter (white arrow) in the pGL2-Promoter vector in either sense (UsExon l-pGL2-Promoter and DsExon l-pGL2-Promoter) or reverse (UrExon l-pGL2-Promoter and DrExon l-pGL2-Promoter) orientation. Lower panel: The heterologous constructs were transiently cotransfected with RSV-/acZ into TE-671 and JEG-3 cells. The relative promoter activity is represented as the percentage of the pGL2-Promoter vector, of which the activity is set as 100 % after being normalized by P-galactosidase activity, a, P < 0.01 vs. pGL2-Promoter. 217 SV40 promoter Luc -793 -750 Luc -750 -793 pGL2-Promoter UsExon 1 -pGL2-Promoter UrExon1-pGL2-Promoter DsExon1-pGL2-Promoter DrExon1-pGL2-Promoter pGL2-Promoter UsExon 1-pGL2-Promoter UrExon 1-pGL2-Promoter DsExon 1-pGL2-Promoter DrExon 1-pGL2-Promoter 0 100 200 300 400 Relative promoter activity (%) 218 but its enhancer activity is strictly dependent on its position and orientation relative to the target promoter. 6.2.3 Two Putative EBSs and One ELE in the Untranslated Exon 1 Function Cooperatively to Stimulate Basal Human GnRH-II Gene Transcription Two putative EBSs, designated as dE-box (at -790/-785, 5'CAGCTG3') and pE-box (at -1621-151, 5'CAGCTC3'), as well as one ELE (at -1191-116, 5'AGGA3') were identified in sense orientation in exon 1 of the human GnRH-II gene (Figure 42). The dE-box motif is identical to the consensus AP-4 binding sequence (5'CAGCTG3'), whereas the pE-box motif differs from the consensus sequence by one nucleotide and has been identified as a functional E-box element (Boulanger et al, 2000). Site-directed mutagenesis showed that mutation of either one of these motifs could only partially abolish the enhancer activity of the untranslated exon (Figure 43). In TE-671 cells, a single mutation at the dE-box, pE-box, or ELE motif resulted in a 33, 38, or 20 % decrease in promoter activity, respectively, whereas a 56, 49, or 38 % reduction was observed in JEG-3 cells. To examine whether there is any functional cooperation among these DNA elements, constructs containing double or triple mutations were prepared and analyzed. As depicted in Figure 43, an almost 60 % and 80 % removal of promoter activity were observed in the neuronal and placental cells when either two of the elements were mutated concurrently. Complete loss of the enhancer activity was only achieved when all the three motifs were altered simultaneously (Figure 43), indicating that these regulatory elements work cooperatively to stimulate basal GnRH-II gene transcription. 219 Figure 42. Nucleotide sequence of the minimal human GnRH-II promoter. Numbers along side the sequence refer to the positions relative to the ATG initiation codon, of which the position is designated as +1. Lowercase and uppercase letters represent nucleotides of the 5'-flanking region and exon 1, respectively. The six transcription start sites determined by primer extension analysis are marked by black inverted triangles. Two putative EBSs, namely dE-box and pE-box, as well as one ELE in the untranslated exon 1 are underlined. The CRE that was previously shown to be important for both the basal and cAMP-stimulated transcription of the human GnRH-II gene is boxed. 220 112 4 a a t t c t c t t t t g g g a t c a g g g a a g g c t g t g a g g g c t t t c t 10 84 a g g t c t c a a g a t c a g g a g c t t g a a g a t g c a g c c t g g g a a g 1044 t g g g a a g g t g a g a c c a g g a c a t a g g c c a g c c t a a a g c a a g 10 04 a g t c c t g g g c c t g a a g g c t c c t g g g a a g g t g g t g g g g a g g T 964 g a g c a t g t g t c g g t g g c c t c a g g g c a g c a g c t g c c t g g t g T • 924 a a t g t t c a t g g a c t g g a t g c t c t g g g a a g c g g g t t g g g t g • T • 884 g t g a g c t t c t c t c t t c c c c t c t g a f i g a c g t c a p t g g a g t c C R E 844 t g g g g g t g g a g c t g c c t g g t c t a t a a a t c c t g g g g c c a t c 8 04 aggctggggtcCTGCAGCTGCCTGAAGGAGCCATCTCATC d E - b o x E L E 764 CACAGCTCTTCCTTG p E - b o x 221 Figure 43. Mutational analysis of the putative EBS and ELE motifs on the enhancer activity of the untranslated first exon. Upper panel: A diagrammatic representation of the mutant promoter constructs. Each EBS was mutated by introducing a Hpal restriction site into the core binding site, whereas the ELE motif was mutated from 5'AGGA3' to 5'GTTT3'. Mutations are marked with black crosses. Lower panel: Wild-type [p(-1124/-750)-Luc-GII and p(-1124/-794)-Luc-GII] or the mutant constructs were transiently cotransfected with RSV-/acZ into TE-671 and JEG-3 cells. The relative promoter activity is represented as the percentage of the construct p(-1124/-750)-Luc-GII, of which the activity is set as 100 % after being normalized by P-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. p(-1124/-750)-Luc-GII; b, P < 0.05 vs. p(-1124/-750)-Luc-GII. 222 Exon 1 p(-1124/-750)-Luc-GII dE-box-mut ELE-mut pE-box-mut (dE-box + pE-box)-mut (dE-box + ELE)-mut (ELE + pE-box)-mut (dE-box + ELE + pE-box)-mut p(-1124/-794)-Luc-GII E L E C R E dE-box j pE-box 7* A Luc Luc Luc Luc Relative promoter activity (%) TE-671 JEG-3 p(-1124/-750)-Luc-GII 100 100 dE-box-mut 67.0±4.0a 44.2±1.5a ELE-mut 80.5±2.4b 61.9±5.5a pE-box-mut 61.7±2.1a 50.6±1.7a (dE-box + pE-box)-mut 43.6±0.8a 22.2±2.2a (dE-box + ELE)-mut 40.5±7.8a 22.6±1.1a (ELE + pE-box)-mut 40.7±2.9a 20.8±2.3a (dE-box + ELE + pE-box)-mut 28.7±3.4a 8.3±4.3a p(-1124/-794)-Luc-GII 25.9±5.4a 10.7±0.9a 223 6.2.4 The bHLH Transcription Factor AP-4 Binds to Both EBSs, whereas an Unknown Protein Binds to the ELE EMSAs showed that DNA-protein complexes of almost identical mobility were formed with the dE-box (complex-J) and pE-box (complex-K) motifs when nuclear extracts from TE-671 and JEG-3 cells were used (Figure 44A). The formation of these complexes was found to be dose-dependently inhibited by the corresponding unlabeled probes but not by unrelated sequences such as N F - K B - C and TFIID-c (Figure 44B), suggesting that the interactions are specific for the EBSs. Also, the formation of these complexes could be abolished by a consensus AP-4 sequence (Figure 44C). On the contrary, competitor oligonucleotides carrying a Hpal mutation in the EBSs failed to prevent the complex formation (Figure 44D). Antibody supershift assays revealed that the bHLH transcription factors E2A (El2 and E47), dHAND, eHAND, USF-1, and USF-2 were not present in either complex-J or complex-K (Figure 44E). By UV crosslinking (Figure 45A) and Southwestern blot analysis (Figure 45B), nuclear factors of 48 kDa [a size that is consistent with that of the bHLH transcription factor AP-4 (Mermod et al, 1988)] from both TE-671 and JEG-3 cells were found to interact with both EBSs specifically. Since antiserum against AP-4 is not yet commercially available to confirm its presence in the complexes, we performed EMSAs using in vitro translated human AP-4 proteins. As shown in Figure 46A, the in vitro translated products bound to both EBSs as single DNA-protein complexes, which share similar electrophoretic mobility with those formed with TE-671 nuclear extracts. The specificity of the complexes formed by the in vitro translated products was confirmed by the use of AP-4-c, which could dose-dependently inhibit the complex formation (Figure 46B). Collectively, these 224 Figure 44. EMSAs to characterize the two functional EBSs using nuclear extracts from TE-671 and JEG-3 cells. Synthetic oligonucleotides containing the dE-box and pE-box motifs were annealed and end-radiolabeled with 3 2P, followed by incubation with nuclear extracts (NE) from TE-671 or JEG-3 cells in the absence or presence of competitor oligonucleotides (or antibodies). A, Formation of DNA-protein complexes of almost identical mobility with the dE-box (complex-J) and pE-box (complex-K) probes using NE from TE-671 and JEG-3 cells. B, Five micrograms of NE from TE-671 cells were incubated with the radiolabeled probes in the presence of an increasing amount of the unlabeled sequences or a 500-fold excess of N F - K B - C or TFIID-c. C, Five micrograms of NE from TE-671 cells were incubated with the radiolabeled probes in the presence of an increasing amount of AP-4-c. D, Five micrograms of NE from TE-671 cells were incubated with the radiolabeled probes in the presence of a different amount of the corresponding mutant EBS (mEBS) sequences. E, Five micrograms of NE from TE-671 cells were preincubated with anti-dHAND, anti-eHAND, anti-E2A, anti-USF-1, anti-USF-2, or anti-GATA-4 antibody for 30 min at room temperature before the addition of the radiolabeled probes. Similar DNA binding results were observed when NE from JEG-3 cells were used (data not shown). ( A ) NE (jig) dE-box pE-box 1 TE-671 J E G - 3 TE-671 J E G - 3 I II 1 I II 1 0 5 10 5 10 0 5 10 5 10 Complex-J • Complex -K Free probe (B) dE-box pE-box Competitor NE Unlabeled sequence •2 2 LO o . CM LO Z |— Unlabeled sequence T- t\ m Complex-J • Ijijgijjjl WW w Complex-K Free probe 226 (C) dE-box pE-box AP-4-c (fold excess) o N E + o o o O LO o T- CM LO o + o o o o LO o CM LO + + Complex-J Complex-K Complex-J • * - ' - •* Complex -K Free probe 227 (E) dE-box pE-box Antibody NE < X3 < 0 LU l l l u c n 00 00 < CD < I T3 < CN CD LUT - CN i i L L L L 00 00 < CD Complex-J • » 4 »- W Complex-K Free probe 228 Figure 45. Identification of a 48 kDa-nuclear factor binding to the EBSs by UV crosslinking and Southwestern blot analysis. A, Twenty micrograms of nuclear extracts (NE) from TE-671 or JEG-3 cells were incubated with 100 fmol of the radiolabeled EBS probes in the absence or presence of a 500-fold excess of the unlabeled sequences. The binding reactions were crosslinked by exposure to UV for 30 min at 4 °C before analyzed by 10 % SDS-PAGE. NS represents nonspecific signals. B, One hundred micrograms of NE from TE-671 cells were resolved by 10 % SDS-PAGE and then transferred onto a nitrocellulose membrane. Transferred proteins were allowed to renature and then hybridized with the radiolabeled EBS probes. Binding signals were detected by autoradiography. 229 (A) Unlabeled sequence NE TE-671 JEG-3 I  1 + - - + + + - + + dE-box pE-box * 48 kDa < 48 kDa N S • 230 (B) dE-box pE-box < 48 kDa 231 Figure 46. In vitro translated human AP-4 proteins bind specifically to both EBSs. A, EMSAs were performed by incubating the radiolabeled EBS probes with 5 pg of TE-671 nuclear extracts (NE), 2 pi of reticulocyte lysate, or 2 pi of in vitro translated AP-4 proteins. Two independent preparations of the in vitro translated products [IVT AP-4 (1) and IVT AP-4 (2)] were used. The complexes formed with the in vitro translated proteins (white arrows) share similar electrophoretic mobility with those formed with TE-671 NE (black arrows). B, The radiolabeled probes were incubated with 2 pi of in vitro translated AP-4 proteins in the presence of an increasing amount of AP-4-c. 232 233 (B) data strongly support the notion that the bHLH transcription factor AP-4 is the nuclear factor binding to the EBSs. EMSAs indicated that a nuclear protein commonly expressed in TE-671 and JEG-3 cells interacted with the ELE motif (Figure 47A) and that the formation of this complex (complex-L) could be inhibited by the unlabeled probe but not by a mutant ELE sequence (Figure 47B). Although the complex formation could be dose-dependently prevented by a consensus Ets transcription factor binding oligonucleotide (Figure 47C), the identity of this ELE-binding factor remains to be determined since the use of five different antibodies targeted against Ets-1, Ets-2, Elk-1, Erg-1, and Erg-2 did not interfere with the complex formation (data not shown). 6.2.5 Effect of Overexpression of Sense and Antisense AP-4 cDNAs on Human GnRH-II Promoter Activity To investigate the functional significance of AP-4 in regulating human GnRH-II gene transcription, we constructed expression plasmids encoding the sense and antisense AP-4 mRNAs and then cotransfected them with the GnRH-II promoter construct p(-1124/-750)-Luc-GII. As shown in Figure 48A, overexpression of sense AP-4 mRNA resulted in an upregulation of the GnRH-II promoter activity in JEG-3 cells, with a maximum of 2-fold induction when 2 pg of the expression plasmid were cotransfected. Conversely, forced expression of antisense AP-4 mRNA suppressed the promoter in the placental cells. These changes in the GnRH-II promoter activity correlated with the endogenous levels of AP-4 transcript, as revealed by RT-PCR and Southern blot analysis (Figure 48B). The repressive effect of antisense AP-4 mRNA 235 Figure 47. EMSAs to characterize the ELE using nuclear extracts from TE-671 and JEG-3 cells. Synthetic oligonucleotides containing the ELE motif were annealed and end-radiolabeled with 3 2P, followed by incubation with nuclear extracts (NE) from TE-671 or JEG-3 cells in the absence or presence of competitor oligonucleotides. A, Formation of DNA-protein complexes (complex-L) of identical mobility with the radiolabeled probe using NE from TE-671 and JEG-3 cells. B , Five micrograms of NE from TE-671 cells were incubated with the radiolabeled probe in the presence of an increasing amount of the unlabeled sequence, a 500-fold excess of N F - K B - C or TFIID-c, or a different amount of a mutant ELE oligonucleotide (mELE). C, Five micrograms of NE from TE-671 cells were incubated with the radiolabeled probe in the presence of an increasing amount of Ets-c. Similar DNA binding results were observed when NE from JEG-3 cells were used (data not shown). 236 (B) TE-671 JEG-3 E(ug) 0 5 10 5 10 mm ««*r Competitor NE Unlabeled sequence r x o o 1 X o LO X o o CQ T- CN LO X X o o LO o o St S 9 !fj y fT LU LU I- E E Complex-L Free probe (C) Ets-c (fold excess) o NE + o o o O L O o T- C M in Complex-L §§; •*— Free probe 237 Figure 48. Endogenous expression level of AP-4 regulates human GnRH-II promoter activity. A, The construct p(-1124/-750)-Luc-GII was cotransfected with an increasing amount of pcDNA3.1, pcDNA3.1-AP-4 (sense), or pcDNA3.1-AP-4 (antisense) into JEG-3 cells. B, RT-PCR and Southern blot analysis of the endogenous AP-4 and GAPDH mRNA levels in JEG-3 cells transfected with 1 pg of pcDNA3.1, pcDNA3.1-AP-4 (sense), or pcDNA3.1-AP-4 (antisense). The AP-4 transcripts were hybridized with a [a-32P]-labeled full-length AP-4 cDNA probe. C, One microgram of p(-l 124/-750)-Luc-GII, p(-1124/-794)-Luc-GII, (dE-box + pE-box)-mut, the human GnRH-I receptor promoter-luciferase construct [p(-1300/-1018)-Luc], or the human secretin receptor promoter-luciferase construct [Sp(-223/-158)-Luc] was cotransfected with 1 pg of pcDNA3.1 or pcDNA3.1-AP-4 (antisense) into JEG-3 cells. 238 D, Upper panel: The endogenous level of AP-4 mRNA in JEG-3 and SH-SY5Y cells. Amplification of GAPDH mRNA was performed as a positive control. Lower panel: One microgram of p(-1124/-750)-Luc-GII was cotransfected with 1 pg of pcDNA3.1, pcDNA3.1-AP-4 (sense), or pcDNA3.1-AP-4 (antisense) into JEG-3 and SH-SY5Y cells. The RSV-/acZ vector was also cotransfected to normalize the transfection efficiency. The relative promoter activity in all panels is represented as the percentage of the control (cotransfection with pcDNA3.1), of which the activity is set as 100 % after being normalized by (3-galactosidase activity. Values represent the mean ± SEM of three independent experiments each performed in triplicate, a, P < 0.001 vs. control; b, P < 0.01 vs. control; c, P < 0.05 vs. control. 239 (A) • w/pcDNA3.1 • w/pcDNA3.1-AP-4 (sense) W w/pcDNA3.1-AP-4 (antisense) 250 500 750 1000 2000 DNA amount (ng) 240 241 (C) <?3 m AP-4 (304 bp) GAPDH (221 bp) 400 300 A 200 100 • w/pcDNA3.1 • w/pcDNA3.1-AP-4 (sense) M w/pcDNA3.1-AP-4 (antisense)l JEG-3 SH-SY5Y Cell line 243 expression on the GnRH-II promoter was found to be attenuated when the EBSs in the untranslated first exon were either deleted [p(-1124/-794)-Luc-GII] or mutated [(dE-box + pE-box)-mut] (Figure 48C). In addition, a weak but significant stimulation rather than inhibition of promoter activity was observed in promoters (those of the human GnRH-I receptor and human secretin receptor genes) lacking AP-4 binding sites when antisense RNA of the bHLH protein was introduced into the cells (Figure 48C). The significance of AP-4 in controlling human GnRH-II gene transcription was further investigated by examining the level of AP-4 mRNA in the nonGnRH-II-expressing SH-SY5Y cells. As shown in Figure 48D, the transcript level was found to be much lower in SH-SY5Y cells than in JEG-3 cells. In addition, we found that AP-4 overexpression could stimulate the activity of p(-1124/-750)-Luc-GH in SH-SY5Y cells by 2.8-fold, a degree that is greater than that observed in JEG-3 cells (1.9-fold) when an equal amount of the expression plasmid was cotransfected. Consistently, forced expression of antisense AP-4 mRNA caused an insignificant repression of the GnRH-II promoter in SH-SY5Y cells (Figure 48D), and this phenomenon may be due to their scanty level of AP-4 expression. Taken together, these findings clearly indicate that the bHLH protein AP-4 plays a specific positive role in regulating human GnRH-II gene transcription via the EBSs in the untranslated exon 1 of the gene. 6.3 Discussion In an attempt to identify additional regulatory sequences and transcription factors involved in the transcriptional regulation of the human GnRH-II gene, we have performed a detailed deletion analysis on the GnRH-II 5'-flanking region in two GnRH-244 II-expressing cell lines TE-671 and JEG-3. Our present data demonstrated that these cells (and also the ovarian carcinoma OVCAR-3 cells, data not shown) exhibited similar promoter activity profiles and that a minimal promoter region (from -1124 to -750) was sufficient to direct GnRH-II gene transcription in both the neuronal and reproductive cells. These observations are in stark contrast to previous findings, which have shown that tissue-specific expression of the human GnRH-I and GnRH-I receptor genes is mediated by alternative promoter usage such that downstream and upstream promoters are selectively used by neuronal and reproductive cell types, respectively (Dong et al, 1993; Dong et al, 1997; Ngan et al, 1999; Cheng et al, 2001b; Cheng et al, 2002). These differential regulatory mechanisms in controlling the basal expression of the two forms of human GnRH genes may help explain their distinct tissue distribution patterns (White et al, 1998). Sequence analysis revealed that neither TATA box nor Inr was present upstream of or overlapping with the six transcription start sites of the human GnRH-II gene. The absence of these regulatory motifs is not unique to the GnRH-II promoter as this phenomenon has also been reported in the mouse thymidylate synthase (Geng and Johnson, 1993), mouse protoporphyrinogen oxidase (Dailey et al, 2002), and human Spl (Nicolas et al, 2001) genes. In addition, we did not find any MED-1 (Multiple start site Element Downstream) element, which has been conserved in many TATA- and Zrcr-less promoters and proposed to be important for promoter function (Ince and Scotto, 1995), downstream of the initiation window of the GnRH-II gene. Further investigations are needed to elucidate the mechanism that directs transcription initiation of the human GnRH-II gene. 245 Our deletion analysis indicated that strong repressive elements exist in the first intron of the GnRH-II gene (Figure 39A). To date, a considerable number of genes containing intronic silencers have been identified (Haniel et al, 1995; Bossu et al, 1996; Donda et al, 1996; He et al, 1996; Dietrich-Goetz et al, 1997), and it has been suggested that this type of silencer represses gene transcription in a number of ways including physical blockage of transcriptional elongation, intronic splicing, and basal transcription apparatus assembly (Ogbourne and Antalis, 1998). Although the first intron of the GnRH-II gene appears to work constitutively, its silencing activity may be tightly controlled under different physiological or developmental conditions to allow timely expression of the GnRH-II gene. The functional significance of this intron in repressing GnRH-II gene transcription is currently under investigation, and surprisingly, our preliminary data have revealed that its activity can be modulated by certain signal transduction pathways (Hoo, unpublished data), which may have important implications in silencing human GnRH-II gene expression. The role of untranslated exons in regulating gene expression has been well reported (Fujimori et al, 2000; Hiroi et al, 2001; Wang et al, 2002a; Tomassetti et al, 2003). Unlike many classical enhancers, our present data indicated that the enhancer activity of the untranslated exon 1 of the human GnRH-II gene is strictly dependent on its position and orientation. Notably, a silencing activity was even detected when the exon was cloned in an aberrant orientation (DrExon l-pGL2-Promoter, Figure 41). These observations indicate that binding of transcription factors to the exon is not symmetrical and that efficient transcriptional activation requires a specific spatial and directional arrangement of protein factors along the sequence. In addition, it should be 246 mentioned in this context that although the primary structure of GnRH-II is conserved over all vertebrate classes, the size and the nucleotide sequence of their untranslated first exons vary dramatically, indicating that the exon may not possess similar regulatory roles in controlling GnRH-II gene transcription in other species as in humans. Results from EMSAs, UV crosslinking, and Southwestern blot analysis provided solid evidence that the bHLH transcription factor AP-4 is the candidate nuclear protein binding to the two EBSs. The wide expression pattern of AP-4 may contribute to the ubiquitous enhancer activity of the untranslated first exon. The transcription factor was originally identified as a cellular protein that bound to the SV40 enhancer and cooperated synergistically with AP-1 to stimulate both SV40 late and the human metallothionein-IiA gene transcription in vitro (Mermod et al, 1988). In addition, the bHLH protein has been shown to be involved in cAMP-inducible proenkephalin transcription (Comb et al, 1988), Tax-mediated transactivation of bovine leukemia virus long terminal repeat (Unk et al, 1994), as well as IGF-binding protein-2 gene transcription (Badinga et al, 1998). On the contrary, the transcription factor has been demonstrated to repress gene expression in some cases. For instance, binding of AP-4 to the human immunodeficiency virus type 1 (HIV-1) TATA element has been found to inhibit in vitro transcription from the virus long terminal repeat (Ou et al, 1994). Moreover, cotransfection of an AP-4 expression plasmid has been shown to downregulate the activity of the human angiotensinogen promoter (Cui et al, 1998). Similar to other members of the family, AP-4 contains a HLH motif and an adjacent basic domain that are necessary and sufficient for site-specific DNA binding. However, unlike other bHLH factors, AP-4 possesses two additional protein dimerization domains 247 consisting of leucine repeat elements, which have been shown to prevent heterodimer formation (Hu et al, 1990). This property of AP-4 is consistent with our current data, which indicated that other bHLH proteins were not present in DNA-protein complexes formed with the EBSs (Figure 44E). Interestingly, deletion analysis of the chromatin structure of the SV40 late promoter has uncovered a novel function of AP-4 in conferring significant levels of nuclease sensitivity, thus implicating it in the process of chromatin remodeling (Friez et al, 1999). Additionally, based on its considerable homology with other transcription factors such as Myc and Max, it has also been proposed that AP-4 may bind to its target sequence even when it is incorporated into nucleosomes, and thereby allow other factors to bind in a cooperative fashion (Friez et al, 1999). As noted below, our present finding is in agreement with the function of AP-4 in that the bHLH protein may serve as a "nucleating" factor to facilitate interactions with other transcription regulators. Given that AP-4 possesses multiple protein dimerization domains and that the two EBSs are juxtaposed with the ELE motif, it is logical to postulate that the cooperative action among these regulatory motifs involves direct protein-protein interactions, which may lead to synergistic transactivation of the GnRH-II gene. Evidence supporting this view comes from the finding that USF-1 can interact with the Ets domain of Ets-1 via its HLH domain to cooperatively activate HIV-l expression (Sieweke et al, 1998). In addition, the leucine zipper repeats of AP-4 may also participate in the formation of higher-order complexes because it has been reported that the Ets domain of PU.l can interact with the basic leucine zipper of C/EBPp, leading to synergistic activation of an artificial promoter containing both Ets and C/EBPP 248 consensus binding sequences (Nagulapalli et al, 1995). Interestingly, a similar arrangement of two EBSs and one ELE has also been found in the immunoglobulin p heavy-chain gene enhancer, and it has been shown that transcriptional synergy between the bHLH proteins E47 and TFE-3 requires Ets-1, which is believed to bridge the transactivation domains of the flanking bHLH proteins (Dang et al, 1998). Based on this phenomenon, it is tempting to speculate that the weak transactivation response observed when only AP-4 cDNA is overexpressed is due to the concomitant requirement of an intermediate Ets protein, which is essential to mediate functional cooperation between the neighboring AP-4 proteins. However, unlike the so-called "protein-tethered transactivation" (Yang et al, 2000), our EMSA results showed that both AP-4 and the unidentified Ets-related proteins were participated in DNA binding, indicating that the cooperative effect may be the result of an overall enhanced binding affinity to the target sequences. Nevertheless, whether the ELE-binding protein belongs to an existing or an unknown member of the Ets family transcription factors remains to be elucidated. Such investigations should greatly enhance our understanding on the mechanistic action of the exonic enhancer in stimulating GnRH-II gene transcription and facilitate the identification of potential heterodimerization partners for AP-4, which are so far unknown. Although we demonstrated in the present study that the untranslated exon 1 constitutively stimulated the basal transcription of the human GnRH-II gene, it is conceivable that its enhancer activity is modulated by certain extracellular stimuli. It has been well documented that the transcriptional activity of bHLH proteins is tightly regulated by phosphorylation both positively and negatively. Thus, the chromosome 3p 249 kinase and MAPK-activated protein kinase-2 have been shown to phosphorylate E47 and repress its transcriptional activity (Neufeld et al, 2000). Also, overexpression of casein kinase II has been found to stimulate myogenic regulatory factors MRF4 and MyoD in vivo through a mechanism involving phosphorylation of E47 (Johnson et al, 1996). Furthermore, Bain and coworkers have demonstrated a direct connection between the Ras-ERK signaling pathway and HLH proteins in a common pathway involved in thymocyte positive-selection (Bain et al, 2001). Likewise, many Ets-domain transcription factors have been shown to be nuclear targets of the MAPK cascades (Sharrocks et al, 1997), and in particular, all members of the ternary complex factor subfamily of the Ets proteins are ERK1/2 targets and respond differentially to the related JNK and p38 MAPK pathways (Wasylyk et al, 1998). Since MAPKs are known to response to a diverse array of extracellular stimuli, it is speculated that under different physiological conditions, the transcriptional activity of AP-4 and the ELE-binding protein will be modulated accordingly, which in turn regulates the rate of basal transcription of the human GnRH-II gene. 250 CHAPTER VII Conclusion and Future work GnRH-I plays a diverse functional role in human beings including stimulating gonadotropin secretion in the anterior pituitary, inhibiting cell proliferation in various reproductive tract-derived tumors, interfering with steroidogenesis in the gonads and placenta, as well as modulating immune functions in T lymphocytes. All these actions are mediated by the GnRH-I receptor, which belongs to a member of the rhodopsin-like GPCR family. Studying the transcriptional regulation of the human GnRH-I receptor gene is important for us to understand the tissue-specific and hormonal regulation of gene expression (Figure 49). Although it has been shown that two different upstream GnRH-I receptor promoters are employed by placental and GL cells to direct cell-specific gene expression, it remains to be determined if other extrapituitary tissues like the breast, prostate gland, and endometrium use additional (or common) promoter elements for GnRH-I receptor gene transcription. Further experiments such as deletion mapping and primer extension analysis should be carried out to define sequences of the GnRH-I receptor 5'-flanking region required for tissue-specific expression in these extrapituitary compartments. Despite the demonstration of Oct-1 as a repressor protein for the human GnRH-I receptor promoter, the mechanism underlying the constitutive repression has not been elucidated. Since it has been reported that Oct-1 can bend DNA via its POU-specific domain, circular permutation gel shift assays will be performed to measure the degree of DNA bending induced by the transcription factor. Also, as Oct-1 has been demonstrated 251 Figure 49. Summary of the transcriptional regulation of the human GnRH-I receptor gene. A diagrammatic representation of the human GnRH-I receptor 5 ' -flanking region. The location of important regulatory motifs (all numbers are relative to the ATG start codon, of which the position is assigned as +1) and their functional significance are indicated. Those elements identified in the present study are typed in bold. The diagram is not drawn to scale. 252 + 5 SS3U8AjSU0dS8J zj sjejuoo <- (^L-/0£L-) a>l!l-l.-dV - 1 ssauaAjSuodssj *d SJSJUOO <- (£<:g-/9£g-) 3yd—I ssdoj;opeuo6 u| sens f pe\s uojjduosuej) lewjxojd 8jB|n6aj ~\_ (^ 99-/069-) // JUI (869-/e09-) / Ml -m uojssajdaj eAjiniiisuoo sjejuoo <- (600V-IIV0V-) k-POH 8 > _i o i_ o ^ 1 s. u f (WH-/ZSH.-) da3/0<H (89H.-/9ZLL-)BV1V9-| (zczk/rai.-) daa/op-AjjAjpe J90U8IJS pue ssauaAjsuodsaj dVOVd sjejuoo <- utJH-/l.Z9|.-) 3dVd AjjAjpe jaiouuojd f- (ZZ91-/E891-) J " / —L |B)S!p SAnnmsuoo jajuoo \ (00/ 1-) 1WO — • — GSE (-143/-135) -» confers gonadotrope specificity - AP/CRE-2 (-341/-334) A h- AP/CRE-1 (-569A562) - AP-1-like (-1000/-994) -> confers GnRH-I responsiveness h-AP-1 (-1519/-1513) A ro • — GATA (-1603/-1598) I ro £ Q - O 7= 0 5 CRE (-1650/-1642) Oct-1 (-1718/-1711) J l O 253 to recruit the nuclear receptor corepressor SMRT to mediate its silencing activity, the histone deacetylase inhibitor trichostatin A will be used to examine the potential role of histone hypoacetylation in mediating the repressive effect of Oct-1 on GnRH-I receptor gene expression. Furthermore, chromatin immunoprecipitation assays will be done to investigate the in vivo binding of the transcription factor to the GnRH-I receptor promoter. Our preliminary data have found that the pure antiestrogen ICI 182780 exerts an even stronger repressive effect on the human GnRH-I receptor gene transcription than equimolar concentrations of E 2 in OVCAR-3 cells. Like E 2 , this repression requires ERa but not ERp, indicating that in the presence of various selective ER modulators (SERMs), ERa has differential actions on the GnRH-I receptor promoter. Further experiments should be carried out to elucidate the mechanistic action (e.g. cw-acting regulatory motifs and transcription factors) of the antiestrogen or other SERMs on GnRH-I receptor gene expression. The recent discovery of GnRH-II in humans and the demonstration of a growing number of extrapituitary functions of the hormone strongly indicate the existence of an additional GnRH/GnRH receptor system in our body. The observation that the tissue distribution of GnRH-II is significantly different from that of GnRH-I indicates that these decapeptides have distinct biological roles. Moreover, it also indicates to us that the mechanisms governing the expression of these hormones are dissimilar. Although it has been shown that the basal transcription of the human GnRH-II gene is mediated by a CRE and is enhanced by a cooperative action among multiple regulatory elements in the untranslated first exon of the gene in vitro, their significance in vivo is obscure. To 254 confirm our in vitro data, transgenic mice harboring various human GnRH-II promoter-luciferase constructs should be generated to definitely dissect the sequences essential for tissue-specific and hormonal regulation of the gene expression. In addition, experiments like mammalian one-hybrid assay, UV crosslinking, and Southwestern blot analysis will be carried out to determine the exact nature of the ELE-binding protein. 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