行政院國家科學委員會專題研究計畫期中進度報告:CYP11A1基因於卵巢黃體細胞內之調控(1/2)

類固醇由膽固醇衍生而成，主要於腎上腺、性腺與胎盤產生。CYP11A1 基因 (P450scc) 是控制所有類固醇生成的第一個步驟，亦是決定步驟。黃體為一重要 的類固醇荷爾蒙生成組織，人類黃體生產助孕酮最高日產量可達40 豪克。 CYP11A1 基因將膽固醇轉換為孕烯醇酮，再經由HSD3B 基因轉換而生成助孕 酮。其主要的作用為孕期之建立與維持。多數類固醇生成基因的調控機制類似， 目前已知轉錄因子SF-1 (steroidogenic factor 1) 為其中一個重要的調控因子， SF-1 同時亦為腎上腺與性腺發育所必需。排卵後，CYP11A1 大量表現於卵巢黃 體組織，但SF-1 的表現量卻很低。轉錄因子LRH-1 (liver receptor homologue-1) 與 SF-1 結構非常相似，且LRH-1 mRNA 大量存在於黃體組織。近來的研究顯示， LRH-1 可能為一重要因子調控黃體細胞類固醇的生成。 為了研究黃體類固醇生成之調控機制，我們首要建立一個黃體細胞培養系統 以探討CYP11A1 基因表現的調控。我們自懷孕三天大鼠的卵巢分離出黃體細 胞，並且可以偵測到CYP11A1 和HSD3B 基因表現的mRNA 與蛋白質。利用酵 素活性分析也可偵測到很強的HSD3 基因產物的活性，但卻只存在於小部份的細 胞中。相反的是，免疫組織染色分析結果顯示LRH-1 存在於大部份黃體細胞的 細胞核中。我們還利用一個來自山羊黃體之細胞株GLC 進行啟動子活性分析， 結果顯示人類CYP11A1 基因的啟動子在GLC 細胞中具有轉錄活性。若將啟動子 上兩個SF-1 結合序列突變後，明顯地會降低CYP11A1 啟動子的轉錄活性。另外， 我們將LRH-1 蛋白的兩個片段分別大量表現於大腸桿菌，正進一步將其纯化以 產生抗LRH-1 抗體。Steroids are synthesized from cholesterol in adrenal, gonads and placenta. CYP11A1 gene encodes cytochrome P450scc that catalyzes the first and rate-limiting step in all steroids synthesis pathway. Corpus luteum is a remarkable steroidogenic gland that is able to synthesize 40 mg of progesterone daily in humans. P450scc converts the cholesterol to pregnenolone, which is then converted to progesterone by 3-hydroxysteroid dehydrogenase type 2 (HSD3B). Luteal progesterone is essential for establishment and maintenance of pregnancy. Steroidogenic factor-1 (SF-1) is a nuclear receptor expressed in steroidogenic tissues and is responsible for transcriptional activation of steroidogenic genes. After ovulation, CYP11A1 expression increases in the corpus luteum. However, only low levels of SF-1 expression are present in the luteal tissue. Liver receptor homologue-1 (LRH-1) is closely related to SF-1 and is abundantly expressed in the luteal tissues. Recent studies showed that LRH-1 may play an important role in regulation of luteal steroidogenesis. To study the controlling mechanism of CYP11A1 expression in corpus lutem, we attempted to establish a luteal cell culture system. The primary luteal cells were isolated from corpora lutea of day 3 pregnant rats. The CYP11A1 and HSD3B mRNA and protein could be detected in the primary luteal cells. However, 3-HSD activity only presents in a small amount of cell population.We further examined LRH-1 expression in the primary luteal cells and found that it was present in most of the cells. The human CYP11A1 promoter was tested in a goat luteal cell line GLC. The 1.5 kb promoter fragment was sufficient to active transcription in GLC cells. In addition, mutation in two putative SF-1 binding sites reduced the promoter activity, respectively. In addition, we have overproduced two fragments of LRH-1 protein in E. coli. The overproduced protein will be further purified for the generation of antibody