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Cross-Talk Between Gonadotropin- Releasing CROSS-TALK BETWEEN GONADOTROPIN- RELEASING HORMONES AND PROGESTERONE RECEPTOR IN NEUROENDOCRINE CELLS by BEUM-SOO AN D.V.M., Chung-buk National University, 2000 M.Sc, Chung-buk National University, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Reproductive & Developmental Sciences) THE UNIVERSITY OF BRITISH COLUMBIA March 2007 © Beum-soo AN, 2007 ABSTRACT Hypothalamic gonadotropin-releasing hormone (GnRH) is a decapeptide that plays a pivotal role in mammalian reproduction. It is hypothesized that progesterone (P4) may regulate GnRH I, GnRH II (a second form of GnRH) and GnRH I receptor (GnRH I R) at the transcriptional level. Alternatively, GnRHs may stimulate transactivation of the progesterone receptor (PR), thereby, modulating gonadotropin subunit gene expression. Treatment of human neuronal cells with P4 suppressed GnRH I R promoter activity. This P4-stimulated inhibition was enhanced when PR A was over-expressed. With respect to the two GnRHs, P4 increased GnRH I mRNA levels, but did not significantly affect GnRH II gene expression. Regulation of gonadotropin production involves interplay between steroids and neuro• peptides, thus we have examined the effects of GnRHs on PR activation in pituitary cells. Treatment with GnRHs increased a progesterone response element (PRE)-luciferase reporter gene activity. PR was phosphorylated at Ser294 and translocated into nucleus after GnRH treatment in the absence of P4. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR: Steroid Receptor Coactivator-3 (SRC-3) interaction. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 to the PRE reporter gene was also increased by GnRHs. The knockdown of GnRH I R and SRC-3 levels by siRNA treatment reduced GnRH-induced PR transactivation. Gonadotropin subunit gene expression was evaluated following treatment with GnRHs, and common a-subunit and FSHjS transcription were upregulated by GnRHs. We used siRNA for PR to examine the involvement of PR in GnRH I-induced FSH/3 gene expression. The effect of GnRH I on FSH/3, but not a-subunit gene expression was reduced when siRNA targeting PR was introduced. In summary, these results indicate that P4 is a potent regulator of GnRH I R and GnRH I at ii the transcriptional level, and this distinct effect of P4 on the GnRH system may be derived from the differential action of PR A or PR B. Conversely, GnRHs can activate PR-mediated transcription in the absence of P4, and this ligand-independent mechanism of PR additionally regulates FSH/3 subunit gene expression. in TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES viii LIST OF ABBREVIATIONS xi 1. INTRODUCTION 1 1.1 The hypothalamic-pituitary gonadal axis 1 1.2 GnRH I and its receptor 4 1.3 GnRH I R-induced signaling 7 1.4 GnRH II and its receptor 10 1.5 Regulation of the GnRH system 13 1.6 Classification and structure of nuclear receptor superfamily 14 1.7 Structure and mechanism of action of PR 18 1.8 Phosphorylation of PR 22 1.9 Interactions between PR and coregulators 25 1.10 Transactivation of PR in the absence of P4 27 1.11 Transactivation of PR by GnRH I in the absence of P4 30 1.12 Pituitary gonadotropin hormones 30 1.13 Regulation of gonadotropin subunit genes 31 1.13.1 GnRH regulation of common a-subunit (q-GSU) gene expression.... 31 iv 1.13.2 GnPvH regulation of LH/3-subunit gene expression 32 1.13.3 GnRH regulation of FSH /3-subunit gene expression 32 1.13.4 P4 regulation of j3-subunit gene transcription 33 1.14 Hypothesis 33 1.15 Specific Obj ectives 33 2. MATERIALS AND METHODS 35 2.1. Materials 35 2.2 Cell cultures 35 2.3 Plasmids 35 2.4 PPvE-luciferase reporter gene assays 36 2.5 Transient transfection of GnRH I R promoter and over-expressing vectors for PR isoforms 37 2.6 In vitro transfection with small interference RNAs 38 2.7 Western blot analysis 39 2.8 Immunoprecipitation 40 2.9 Immunocytochemistry 41 2.10 RNA extraction and reverse transcriptase-PCR 41 2.11 Real-time RT-PCR 42 2.12 Chromatin immunoprecipitation (ChIP) Assay 45 2.13 Statistical analysis 46 3. RESULTS 47 3.1 Regulation of the GnRH system by P4 47 3.1.1 P4 regulates human GnRH I R promoter activity 47 v 3.1.2 PR A but not PR B mediates P4 induced repression of GnRH IR promoter... ! 50 3.1.3 Over-expression of PR A or PR B has distinct effects on PRE promoter activity 50 3.1.4 Effects of P4 on human GnRH I and GnRH II inRNA levels 54 3.1.5 Effects of PR A and PR B on human GnRH I and GnRH II rnRNA levels 58 Ligand-independent activation of PR by GnRHs 61 3.2.1 Transactivation of PR by GnRH I and GnRH II in o/T3-l cells 61 3.2.2 Treatment with GnRHs affects PR phosphorylation 65 3.2.3 Treatment with GnRHs affects PR sub-cellular distribution 71 3.2.4 Interaction between SRC-3 and PR increases in aT3-l cell after treatment with GnRHs or P4 73 3.2.5 Recruitment of PR and SRC-3 to PREs is promoted by GnRHs 76 3.2.6 GnRH I R and SRC-3 are required for GnRH-mediated PR activation 78 3 GnRH-induced FSH/3 subunit gene transcription involves the ligand-independent transactivation of PR 84 3.3.1 Transactivation of PR by GnRH I in oT3-l and L(8T2 cells 84 3.3.2 Transcriptional regulation of gonadotropin subunit genes by GnRH I and GnRH II in pituitary cells 87 3.3.3 Effects of signaling pathway inhibitors on GnRH I-induced trans• activation of PR and gene expression of gonadotropin subunits 93 3.3.4 PR mediates GnRH I-induced FSH{3 gene expression 99 vi 4. DISCUSSION 102 4.1 Regulation of the GnRH system by P4 102 4.2 Ligand-independent activation of PR by GnRHs 106 4.3 Ligand-independent transactivation of PR mediates GnRH-induced FSH(3 subunit gene transcription 109 4.4 Clinical implications Ill 5. SUMMARY AND FUTURE STUDIES 115 5.1 Summary 115 5.1.1 Different regulation of GnRH system by PR isoforms 115 5.1.2 Ligand-independent activation of the PR by GnRHs 116 5.1.3 PR mediates GnRH-induced FSH(3 gene transcription via a ligand-independent transactivation 117 5.2 Future Studies 120 6. REFERENCES 122 7. APPENDICES 138 vii LIST OF TABLES Table. 1. Primers for Real-time PCR genes i viii LIST OF FIGURES Figure 1. The hypothalamic-pituitary-gonadal axis 3 Figure 2. Two-dimensional representation of the GnRH I R 6 Figure 3. Schematic representation of GnRH I signaling in oT3-l, COS7 and DU145 cells.... 9 Figure 4. Schematic representation of the human GnRH I and GnRH II genes 11 Figure 5. Shared functional domains of the nuclear receptor superfamily and SRC/pl60 family members 17 Figure 6. Structures of PR A and PR B 19 Figure 7. Model of the mechanism of action of PR in the presence of P4 21 Figure 8. Phosphorylation sites in human PR 24 Figure 9. Model of molecular mechanism of action of PR in the absence of P4 29 Figure 10. Dose- and time-dependent effects of P4 on GnRH I R promoter activity 48 Figure 11. Effects of RU486 on P4-induced GnRH I R promoter activity 49 Figure 12. Effects of PR A or PR B over-expression on GnRH I R or PRE promoter activities.... 53 Figure 13. Time- and dose-dependent effects of P4 on GnRH I mRNA levels 56 Figure 14. RU486 reverses P4-induced GnRH I mRNA levels 57 Figure 15. Effects of P4 on GnRH I and GnRH II mRNA levels after PR over-expression.... 59 ix Figure 16. Effects of GnRH I and II on PR-mediated trans-activation of a PRE-reporter gene in aT3-l cells 62 Figure 17. PKC and PKA inhibitors reverse GnRH-induced PR-mediated transactivation of a PRE-luciferase reporter gene in o;T3-l cells, but RU486 does not 64 Figure 18. Alignment of the amino acid sequences and phosphorylation sites of PR in mouse, rat and human 68 Figure 19. Regulation of PR phosphorylation at Ser294 by GnRHs 69 Figure 20. Cytoplasmic to nuclear translocation of PR in rxT3-l cells following treatments with GnRHs 72 Figure 21. Interaction between SRC-3 and PR increases in <xT3-l cells after treatment with GnRHs or P4 74 Figure 22. Recruitment of PR and SRC-3 on the PREs is promoted by GnRHs 77 Figure 23. GnRH I R mediates both GnRH I - and GnRH II-induced ligand-independent activation ol PR 79 Figure 24. SRC-3 is essential for the ligand-independent activation of PR by GnRH I and GnRH II, and the synergistic amplification of this effect by P4 80 Figure 25. Effects of GnRH I on PR-mediated transactivation of a PRE-reporter gene in 0.T3-1 and L{3T2 cells 86 Figure 26. The effects of GnRH I on a-GSU, FSH|3 and LHJ3 mRNA levels 88 Figure 27. P4 does not have synergistic effects with GnRH I and GnRH II at the level of gonadotropin subunit gene expression 91 Figure 28. PKC and PKA inhibitors, but not RU486, reduce GnRH-induced PR-mediatec transactivation of a PRE-luciferase reporter gene in <xT3-l or L/3T2 cells 94 Figure 29.
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