LRH-1抗體製備及LRH-1調控CYP11A1之研究

Abstract

CYP11A1基因的產物為膽固醇側鍵截切酶(cholesterol side-chain cleavage enzyme, P450scc)。 P450scc酵素在類固醇荷爾蒙的合成途徑中，負責催化第一個步驟，也就是將膽固醇轉換成孕烯醇酮 (pregenelone)。CYP11A1普遍存在於腎上腺、性腺等所有類固醇生成組織中。Liver receptor homolog-1 (LRH-1)為 NR5A家族中的一員，為一轉錄因子，其大量存在於某些類固醇生成組織內，例如卵巢，並具有調控類固醇生成基因的能力。 我們首先使用大腸桿菌表現系統大量產生小鼠LRH-1 (mLRH-1)兩個不同片段的胜肽，分別是具有N端1~115胺基酸序列及C端169~560胺基酸序列。經電泳純化後，以此製備LRH-1抗體。在西方墨染法中，以所製備的LRH-1抗體，證實LRH-1存在於黃體的初代培養細胞中。293T細胞株經轉染mLRH-1表現質體後，亦能以此抗體偵測到mLRH-1的表現。我們利用共同轉染的方法研究LRH-1調控CYP11A1基因表現的能力。LRH-1對於1.5 kb長度的CYP11A1啟動子，即具有增進其轉錄的作用。CYP11A1基因上的兩個SF-1結合序列(-40和-1600)，經由點突變後發現，位於-40上的SF-1結合序列，可能是LRH-1在CYP11A1基因啟動子上的主要結合序列。轉染Dax-1後，LRH-1增進CYP11A1基因啟動子的作用，會受到抑制。LRH-1會和轉譯後修飾蛋白SUMO-1產生鍵結，而lysine 289為其主要的結合位置。SUMO-1和LRH-1結合後，會抑制LRH-1的轉錄活性，其抑制效果和Dax-1並無關聯。我們發現LRH-1在轉染細胞中的表現並不穩定，當加入proteasome抑制劑MG-132後，可提升其蛋白含量。

CYP11A1 gene encodes P450scc enzyme, which controls the first and rate-limiting step of steroidogenesis by conversion of cholesterol to pregnenolone. CYP11A1 gene is expressed in many steroidogenic tissues like adrenal and gonad. Liver receptor homolog-1 (LRH-1) is a transcription factor and belongs to a member of nuclear receptor NR5A family. LRH-1 is expressed in some steroidogenic tissues such as ovary and may play a role in the regulation of steroidogenic gene. To study the function of LRH-1, two peptides of mouse LRH-1 were produced in E. coli for the generation of antibody. This antibody was used to detect the presence of LRH-1 in rat primary luteal cells by Western blotting. Transfection of LRH-1 expression vector in 293T cell line also can be detected by Western blotting. To examine the ability of LRH-1 in the regulation of CYP11A1 gene expression, we cotransfected LRH-1 expression vector and human CYP11A1 promoter construct into 293T cell line. LRH-1 can enhance the shortest 1.5 kb CYP11A1 promoter activity. Two functional SF-1 binding sites were located at -40 and -1600 in CYP11A1 promoter. By mutating the SF-1 binding sites, we found that SF-1 binding site located at -40 is the major site of LRH-1 binding to CYP11A1 promoter. Induction of CYP11A1 promoter activity by LRH-1 was inhibited by Dax-1. LRH-1 can be conjugated with post-translation modification protein SUMO-1 and we found that lysine 289 is the major conjugation site of SUMO-1. When LRH-1 was conjugated with SUMO-1 the ability of LRH-1 inducing CYP11A1 promoter activity was inhibited and that was independent of Dax-1 function. We found that LRH-1 may not be stable in the transfected cell.The proteasome inhibitor, MG-132, can increase the amount of LRH-1 protein in transfected cell.