PIASy抑制人類腎上腺細胞類固醇荷爾蒙生成基因的表現

PIASy Represses Steroidogenic Gene Expression in Human adrenocortical Carcinoma Cell H295

戴予辰(Yu-Chen Tai)

指導教授 ： 胡孟君

國立臺灣大學/醫學院/生理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.03104

摘要

PIASy是PIAS蛋白家族中的一員，參與許多訊息傳導路徑的調控。其調控的機制大多為調控轉錄因子的活性，包含干擾轉錄因子與DNA的結合，或是招募一些輔因子。此外，PIASy具有SUMO E3 ligase活性，因此也可以透過Sumoylation調節轉錄因子活性。H295為人類腎上腺腫瘤細胞，可以表現腎上腺皮質中許多固醇類荷爾蒙生成酵素，也可以分泌許多固醇類荷爾蒙。我們發現，在H295細胞當中，PIASy會調控固醇類生成酵素基因的表現。PIASy會抑制CYP11A1及StAR啟動子的表現，而且這個現象具有劑量依存性。CYP11A1的蛋白質產物為P450scc，是催化固醇類荷爾蒙生成第一個步驟的酵素。我們將PIASy蛋白過量表現在H295中，發現其內生性P450scc的蛋白表現量下降，顯示PIASy會抑制H295內生性的CYP11A1基因表現。在H295細胞中，可以偵測到PIASy蛋白的表現，所以我們進一步使用RNAi，降低內生性PIASy的表現，結果顯示P450scc的表現有上升的趨勢，也再度證明的我們的假設。
當我們刪除PIASy蛋白的兩個抑制區 (Repression Domain) 中的第一抑制區 (RD1)，抑制CYP11A1啟動子的現象即消失，顯示RD1是負責PIASy對於CYP11A1啟動子的抑制作用。此外，我們發現PIASy的抑制作用與SF-1、LRH-1及AP1/CREB like protein等調控CYP11A1基因的轉錄因子無關。於是我們利用不同長度的CYP11A1啟動子，發現PIASy抑制作用的區域，可能在CYP11A1上游0.5 kb的啟動子之內。我們的結論是，PIASy可以抑制CYP11A1基因的表現，因此可能參與腎上腺皮質等內分泌器官中固醇類荷爾蒙生成的調控。

關鍵字

腎上腺細胞 ； 固醇類荷爾蒙生成

並列摘要

PIASy, a member of protein inhibitor of activated STAT (PIAS) family, is a coregulator involved in many signal pathways. PIASy modulates the transcriptional activity of various proteins though multiple mechanism, such as interfering the binding with DNA or recruiting cofactors. Also as a SUMO E3 ligase, PIASy can affect the trans-activity of transcriptional factors through sumoylation. H295 is a human adrenocortical carcinoma cell line which can express various steroidogenic enzymes and secrete steroid hormones. In the present study, we found out that PIASy regulates steroidogenic gene in H295. PIASy repressed the promoter activity of CYP11A1 and StAR in a dose-dependant manner. P450scc is encoded by CYP11A1 and catalyses the first step of steroidogenesis. Overexpression of PIASy in H295 cells leaded to decreased amount of endogenous P450scc. Moreover, we used RNAi to knock down endogenous PIASy in H295 and found that P450scc was slightly up-regulated. These results suggest that PIASy represses the endogenous CYP11A1 gene expression.
The repression ability vanished when one of the repression domain, repression domain 1 (RD1), was deleted from PIASy. Thus we conclude that RD1 is responsible for the PIASy-mediated repression effect on CYP11A1 promoter. The repression effect of PIASy is not related to the transcriptional factors including SF-1, LRH-1 or AP1/CREB protein. The deletion analysis reveals that the region of PIASy-mediated repression is lacated at 0.5-kb 5’-flanking sequence of CYP11A1 promoter. Taken together, PIASy represses the expression of CYP11A1 gene in H295 and may participate in regulating steroidogenesis in endocrine organs like adrenal gland.

並列關鍵字

PIASy ； H295 ； Steroidogenic

參考文獻

吳美伶. 2006. LRH-1轉錄活性調控之研究. 生理學研究所. 台灣大學.

謝祥燦. 2007. LRH-1特性及其轉錄活性受PIASy調控之探討. 生理學研究所. 台灣大學.

王志宏. 2008. mLRH-1 結構功能之探討：PIASy 作用區、Hinge 結構區及核定位訊號. 生理學研究所. 台灣大學.

Aravind L, Koonin EV. 2000. SAP - a putative DNA-binding motif involved in chromosomal organization. Trends Biochem Sci 25:112-114.

Ben-Zimra M, Koler M, Orly J. 2002. Transcription of cholesterol side-chain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element. Mol Endocrinol 16:1864-1880.