| Summary: | 碩士 === 國立臺灣大學 === 生理學研究所 === 93 === 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.
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