Begeot M, Shetty U, Kilgore M, Waterman M, Simpson E
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1993 Aug 15;268(23):17317-25.
This study examines the transcriptional regulation of the bovine CYP11A (P450scc) gene by activators of protein kinase A and protein kinase C in bovine ovarian luteal cells. Cells were transfected with reporter gene constructs containing deletion mutations of the 5'-flanking region of the bovine CYP11A gene linked to the minimal beta-globin gene. A construct containing -118/-101 base pairs of CYP11A sequence retains the same degree of stimulation by forskolin and inhibition by co-treatment with phorbol 12-myristate 13-acetate as larger constructs. This sequence contains two putative binding sites for nuclear proteins, an AP1-like sequence and an overlapping GA box element. Gel shift analysis using nuclear extracts of bovine ovarian luteal cells demonstrated that both the wild-type -118/-101-base pair sequence and a consensus GC box bound Sp1 or Sp1-like proteins. Mutation of the GA box element completely suppressed stimulation by forskolin. Absence of binding using the same mutated sequence correlated with the reporter gene transcription results. Mutation of the AP1-like site had little effect on forskolin induction of phorbol 12-myristate 13-acetate inhibition. These results indicate that both stimulation by forskolin and inhibition by phorbol esters are mediated by the same GA box element, which binds Sp1 or an Sp1-like protein.
本研究检测蛋白激酶A和蛋白激酶C激活剂对牛卵巢黄体细胞中牛CYP11A（P450scc）基因转录的调控。用含有与最小β-珠蛋白基因相连的牛CYP11A基因5'-侧翼区缺失突变的报告基因构建体转染细胞。含有CYP11A序列-118 / -101碱基对的构建体与较大构建体相比，保留了相同程度的毛喉素刺激和佛波醇12-肉豆蔻酸酯13-乙酸酯共处理抑制作用。该序列包含两个假定的核蛋白结合位点，一个AP1样序列和一个重叠的GA框元件。使用牛卵巢黄体细胞核提取物进行的凝胶迁移分析表明，野生型-118 / -101碱基对序列和共有GC框均结合Sp1或Sp1样蛋白。GA框元件的突变完全抑制了毛喉素的刺激作用。使用相同突变序列时缺乏结合与报告基因转录结果相关。AP1样位点的突变对毛喉素诱导佛波醇12-肉豆蔻酸酯13-乙酸酯抑制作用影响很小。这些结果表明，毛喉素的刺激作用和佛波酯的抑制作用均由相同的GA框元件介导，该元件结合Sp1或Sp1样蛋白。
