臺灣博碩士論文加值系統

雌性個體中的類固醇荷爾蒙黃體酮與雌激素主要由卵巢顆粒細胞製造。在顆粒細胞中，黃體酮由前驅物膽固醇所衍生而成，而雌激素由苯環化酶催化雄性素(源自於鞘膜細胞)而得。實驗室過去的研究發現腦下腺分泌之卵泡促素(FSH)可刺激顆粒細胞合成類固醇荷爾蒙，而卵巢內的細胞激素轉型乙型生長因子(TGFbeta1)可以更強化FSH的作用。在一般非類固醇生成細胞(non-steroidogenic cells)中，胞內膽固醇含量受恆定性調節──高量的膽固醇致使HMG-CoA reductase (膽固醇生合成之關鍵酶)以及低密度脂蛋白受體(LDLR, 用以攝入低密度脂蛋白)表現下降。而類固醇生成細胞卻額外表現了高密度脂蛋白受體SR-BI。這些瞭解引發此論文之研究動機，並分為兩個主要研究目的：第一個研究目的是調查FSH和TGF1如何調控獲取膽固醇之相關分子HMG-CoA reductase, LDLR 與SR-BI，此涵括調查這些分子之表現量以及參與類固醇生合成之活性，以及高量的固醇(sterol)如何影響這些分子的表現與功能。另外，我們近期發現了FSH和TGFbeta1透過calcineurin活化CRTC2 (一個CREB共轉錄因子)以提升黃體酮生成相關之基因表現，承接此發現，第二個研究目的為調查FSH和TGFbeta1如何透過calcineurin和CRTC2調控苯環化酶基因(Cyp19a1)表現。
實驗發現，HMG-CoA reductase抑制劑 simvastatin大幅降低了FSH±TGFbeta1所促進之黃體酮生合成，並且，FSH+TGFbeta1增加了細胞內HMG-CoA reductase的表現。此顯示細胞需要膽固醇生合成的增加以製造類固醇荷爾蒙。TGFbeta1亦促進FSH的效應而增加了脂蛋白受體SR-BI和LDLR的表現。給予細胞hHDL3和hLDL皆增加了黃體酮生合成，而SR-BI抑制劑BLT-1完全抑制了hHDL3和部分抑制hLDL的效果，證實SR-BI和LDLR確實具有獲取膽固醇的功能，並用以增加黃體酮生合成。使用具中性脂肪親和力之螢光染劑BODIPY493/503，可觀察到攝入之膽固醇亦儲存於胞內脂肪小滴。格外重要的是，LDLR與SR-BI對於固醇的調節有不同的敏感性。給予細胞脂蛋白或25-hydroxycholesterol (細胞通透之膽固醇類似物)降低了Ldlr的表現，而Scarb1 (SR-BI基因)的表現僅有在25-hydroxycholesterol和aminoglutethimide (類固醇生成抑制劑)共同處理造成固醇大量累積的狀況下才開始降低。最後，我們使用染色質免疫沉澱法證明了固醇對於Ldlr和Scarb1的影響是透過轉錄因子SREBP-2和LRH-1的作用。
我們接著探討calcineurin和CRTC2如何參與在FSH和TGF1對Cyp19a1基因轉錄之調控。首先，FSH經24小時作用後增加Cyp19a1蛋白表現，而此效應在48小時後減弱，TGFbeta1則可增加FSH作用而維持Cyp19a1高表現量。前處理calcineurin auto-inhibitory peptide (CNI)可抑制FSH與TGFbeta1共同增加之苯環化酶活性，但不影響FSH單獨處理之效應，而在Cyp19a1基因表現也可觀察到相似的調節現象。卵巢細胞的Cyp19a1 PII啟動子含有CREB和核受體LRH-1/NR5A2、SF-1/NR5A1的結合位，而Nr5a2啟動子亦含有CREB結合位。在FSH和TGF1刺激下，CRTC2結合Cyp19a1 PII啟動子和Nr5a2啟動子的程度增加了(control < FSH < FSH+TGFbeta1),，但CREB的結合卻不受影響。LRH-1和SF-1的表現在FSH與TGF1共同處理下被提升而此受CNI所抑制，FSH單獨處理則不具效應。FSH與TGFbeta1亦促使LRH-1和SF-1結合至Cyp19a1 PII啟動子。這些結果告訴我們FSH+TGFbeta1透過calcineurin活化CRTC2以增加Cyp19a1表現，而calcineurin亦可以間接透過先增加NR5A核受體再提升Cyp19a1表現量。
總結而論，我們首先發現了卵巢顆粒細胞可自行製造膽固醇，並如非類固醇生成細胞一般具有膽固醇恆定調節之機轉，而其又另用SR-BI來規避此恆定調節機轉，以攝入高量膽固醇來提供給大量黃體酮生合成所需。我們亦發現，FSH和TGFbeta1可透過calcineurin和CRTC2來活化Cyp19a1基因表現以增加雌激素生合成。而NR5A核受體在這些過程中都扮演了重要的調節角色。

Ovarian granulosa cells are the major producer of female sex steroids progesterone and estrogen. Granulosa cells synthesize progesterone from the steroid precursor cholesterol, whereas estrogen is produced through aromatase catalysis of theca cell-derived androgen. Ovarian local factor TGFbeta1 facilitates the action of pituitary hormone FSH to promote granulosa cell steroidogenesis. It is known that in non-steroidogenic cells, cellular cholesterol is under homeostatic control involving feedback inhibition of HMG-CoA reductase (key enzyme for cholesterol de novo synthesis) and LDLR (receptor for LDL uptake), while steroidogenic cells additionally express the HDL receptor SR-BI. There are two objectives in this study. The first objective was to investigate how FSH and TGFbeta1 regulate HMG-CoA reductase, LDLR, and SR-BI in granulosa cells. This includes their 1) expression and functional involvement in steroidogenesis, and 2) responsiveness to sterol challenge and identification of potential transcription regulators. We have recently identified that FSH and TGFbeta1 induce calcineurin-dependent activation of CREB coactivator CRTC2 to upregulate steroidogenic gene expression. The second objective was to investigate how calcineurin and CRTC2 are involved in FSH and TGFbeta1 stimulation of estrogen synthesis through controlling aromatase gene (Cyp19a1) expression. Primary culture of granulosa cells from ovarian antral follicles of gonadotropin-primed immature rats was used.
HMG-CoA reductase inhibitor simvastatin suppressed the basal and FSH±TGFbeta1-stimulated progesterone synthesis, and additionally, treatment with FSH+TGFbeta1 increased HMG-CoA reductase mRNA and protein level, suggesting the involvement of cholesterol de novo synthesis during steroid synthesis in granulosa cells. Moreover, TGFbeta1 potentiated FSH to upregulate SR-BI and LDLR, and both are functional in uptaking cholesterol as hHDL3 and hLDL supplementation enhanced progesterone production, and the effect of each lipoprotein was completely or partially blocked by SR-BI selective inhibitor BLT-1. Uptaken cholesterol could also be stored in lipid droplets, and this was evidenced by utilization of the fluorescent dye BODIPY 493/503 that stains for neutral lipid. Importantly, LDLR and SR-BI responded to sterol with different sensitivity. Giving cells lipoproteins or 25-hydroxycholesterol downregulated Ldlr but not Scarb1; Scarb1 was ultimately downregulated by excessive sterol accumulation under 25-hydroxycholesterol and aminoglutethimide (inhibitor of steroidogenesis) cotreatment. Finally, ChIP analysis indicated that transcription factors SREBP-2 and LRH-1 crucially mediate Ldlr and Scarb1 differential response to sterol challenge.
Next, we asked whether FSH and TGFbeta1 regulate Cyp19a1 through calcineurin and CRTC2. We first observed FSH increased aromatase protein at 24 h which subsided by 48 h, while FSH+TGFbeta1 exerted a greater and longer effect. Further, pretreatment with calcineurin inhibitor (CNI) abolished the FSH+TGFbeta1- but not FSH-upregulated aromatase activity at 48 h, and corresponding changes of Cyp19a1 mRNA was observed at 24 h. Ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptors LRH-1/NR5A2 and SF-1/NR5A1, and Nr5a2 promoter has a CREB-binding site. Chromatin immunoprecipitation analysis revealed FSH and TGFbeta1 increased CRTC2 binding to Cyp19a1 PII-promoter and Nr5a2 promoter at 24 h post-treatment (control < FSH < FSH+TGFbeta1), while CREB was constitutively bound. Simultaneously, FSH+TGFbeta1 but not FSH alone increased the expression of LRH-1 and SF-1 and their binding to Cyp19a1 PII-promoter, and expression of both was suppressed by CNI. These results implicate that calcium-dependent calcineurin importantly mediates FSH and TGFbeta1 upregulation of Cyp19a1, acting directly through CRTC2 to enhance CREB transcriptional activity, and indirectly through upregulation of NR5As.
In conclusion, this thesis study first uncovers that ovarian granulosa cells are capable of de novo synthesizing cholesterol and retain the cholesterol homeostatic control machinery like non-steroidogenic cells, while during active steroidogenesis utilize SR-BI to evade such feedback control. Secondly, calcineurin and CRTC2 are critically involved in FSH and TGFbeta1 upregulation of Cyp19a1 transcription for estrogen synthesis. Remarkably, the NR5A nuclear receptors play important regulatory roles in both events.

Contents
Abstract...................................................i
中文摘要.................................................iii
Abbreviations..............................................v
Study background...........................................1
1. The ovary..............................................2
A. Ovarian follicle development..........................2
B. Ovarian steroidogenesis...............................4
C. FSH signaling in ovarian granulosa cells..............8
D. TGFbeta action in ovarian follicle development........9
E. Calcineurin and CRTC2 regulation of CREB activity....11
F. Role of NR5A nuclear receptors in ovarian development11
G. Role of aromatase and ovarian-specific PII-promoter..13
2. Control of cholesterol utilization and implications in the ovary................................................14
A. Cholesterol uptake from lipoproteins.................14
B. Cellular cholesterol homeostasis control.............18
3. Objectives............................................19
Materials and methods 21
Part 1. Ovarian granulosa cells utilize SR-BI to evade cholesterol homeostatic control for steroid synthesis.....29
Introduction.............................................29
Results..................................................31
Discussion...............................................37
Part 2. Calcineurin and CREB coactivator CRTC2 importantly mediate FSH and TGFbeta1 collateral upregulation of Cyp19a1 and Nr5a expression in rat ovarian granulosa cells........58
Introduction.............................................58
Results..................................................59
Discussion...............................................62
Overview..................................................73
Acknowledgment............................................76
References................................................77
Appendix: publication containing parts of this work.......90

List of figures and table
Figure 1. Ovarian steroidogenesis..........................7
Figure 2. Effect of simvastatin on FSH and TGFbeta1-induced progesterone production in granulosa cells................42
Figure 3. FSH and TGFbeta1 regulation of HMG-CoA reductase mRNA and protein expression in granulosa cells............43
Figure 4. FSH and TGFbeta1 regulation of SR-BI and LDLR protein expression in granulosa cells.....................44
Figure 5. FSH and TGFbeta1 regulation of SR-BI and LDLR subcellular localization in granulosa cells...............45
Figure 6. Effect of BLT-1 on hHDL3 supplementation in FSH and TGFbeta1-induced steroidogenesis......................46
Figure 7. Effect of BLT-1 on hLDL supplementation in FSH and TGFbeta1-induced steroidogenesis......................47
Figure 8. Fluorescent staining of neutral lipids in steroidogenic granulosa cells.............................48
Figure 9. Effect of hHDL3 supplementation on FSH and TGFbeta1-induced expression of cholesterogenic genes in granulosa cells...........................................49
Figure 10. Effect of hLDL supplementation on FSH and TGFbeta1-induced expression of cholesterogenic genes in steroidogenic granulosa cells.............................50
Figure 11. Effect of 25-OHC and AMG on FSH and TGFbeta1-induced expression of cholesterogenic genes in granulosa cells.....................................................51
Figure 12. FSH and TGFbeta1 regulation of SREBP-2 in granulosa cells...........................................52
Figure 13. FSH and TGFbeta1 regulation of LRH-1 in granulosa cells...........................................53
Figure 14. FSH and TGFbeta1 regulation of SREBP-2 and LRH-1 to Scarb1 and Ldlr promoters in granulosa cells...........54
Figure 15. Effect of 25-OHC and AMG on FSH and TGFbeta1 upregulation of lipoprotein receptors and key transcription factors SREBP-2, LRH-1, SR-BI, and LDLR in granulosa cells.....................................................55
Figure 16. A proposed working model for FSH and TGFbeta1 regulation of cholesterol uptake in steroidogenic granulosa cells.....................................................56
Figure 17. Effect of calcineurin inhibitor on FSH±TGFbeta1 stimulation of aromatase in granulosa cells...............66
Figure 18. Effect of FSH and TGFbeta1 regulation of CRTC2 and CREB binding to Cyp19a1 PII-promoter..................67
Figure 19. FSH and TGFbeta1 regulation of SF-1 in granulosa cells.....................................................68
Figure 20. Effect of FSH and TGFbeta1 regulation of LRH-1 and SF-1 binding to Cyp19a1 PII-promoter..................69
Figure 21. Effect of calcineurin inhibitor (CNI) on FSH and TGFbeta1 regulation of NR5A expression....................70
Figure 22. Effect of FSH and TGFbeta1 on CRTC2 and CREB binding to Nr5a2 promoter.................................71
Figure 23. A proposed working model of FSH and TGFbeta1 regulation of Cyp19a1 expression through calcineurin and CRTC2.....................................................72
Table 1. Primer pairs used in RT-PCR and ChIP analyses....28

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