Pei L, Dodson R, Schoderbek W E, Maurer R A, Mayo K E
Department of Biochemistry, Northwestern University, Evanston, Illinois 60208.
Mol Endocrinol. 1991 Apr;5(4):521-34. doi: 10.1210/mend-5-4-521.
Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. To examine further the transcriptional regulation of the alpha inhibin gene by FSH and cAMP, we have isolated and characterized a genomic clone that contains the entire rat alpha inhibin gene. Sequence analysis of the alpha inhibin promoter region revealed several potential cAMP response elements (CREs) and transcription factor AP2-binding sites that might mediate cAMP regulation. To determine the functional importance of these sequences, fusion genes including the alpha inhibin 5' flanking region linked to a luciferase reporter gene were transiently transfected into primary granulosa cells isolated from immature rats. These fusion genes were both expressed and regulated by the adenylyl cyclase activator forskolin in transfected granulosa cells. Analysis of a series of 5' deletion mutants indicated that a construct containing as little as 170 basepairs up-stream of the alpha inhibin start site, which includes a single imperfect CRE and no AP2 sites, was regulated by forskolin. DNAse footprinting was used to demonstrate that bacterially expressed CRE-binding protein (CREB) binds to this CRE located 122 basepairs up-stream of the alpha inhibin gene transcriptional start site. To investigate further the role of this CRE in alpha inhibin gene expression, site-specific mutagenesis of the CRE was performed. The alpha inhibin promoter containing a mutated CRE was not regulated by forskolin in granulosa cells and did not bind the CREB protein. Interestingly, mutation of the CRE also substantially reduced basal expression of the alpha inhibin promoter. Lastly, a gel mobility shift assay was used to examine CRE-binding proteins from granulosa cell extracts. Granulosa cells contain a protein that specifically interacts with CRE-containing oligonucleotides or with the alpha inhibin promoter and that is recognized by antibodies against the CREB protein. Our results suggest that CREB or related transcription factors play an important role in both basal and cAMP-regulated expression of the alpha inhibin gene in ovarian granulosa cells.
促卵泡激素（FSH）可刺激卵巢中抑制素基因的表达，FSH利用环磷酸腺苷（cAMP）作为细胞内第二信使。为了进一步研究FSH和cAMP对α抑制素基因的转录调控，我们分离并鉴定了一个包含完整大鼠α抑制素基因的基因组克隆。对α抑制素启动子区域的序列分析揭示了几个潜在的cAMP反应元件（CREs）和转录因子AP2结合位点，它们可能介导cAMP的调控。为了确定这些序列的功能重要性，将包括与荧光素酶报告基因相连的α抑制素5'侧翼区域的融合基因瞬时转染到从未成熟大鼠分离的原代颗粒细胞中。这些融合基因在转染的颗粒细胞中既表达又受腺苷酸环化酶激活剂福斯可林的调控。对一系列5'缺失突变体的分析表明，一个构建体包含α抑制素起始位点上游仅170个碱基对，其中包括一个单一不完全CRE且无AP2位点，受福斯可林调控。DNA酶足迹法用于证明细菌表达的CRE结合蛋白（CREB）与位于α抑制素基因转录起始位点上游122个碱基对处的该CRE结合。为了进一步研究该CRE在α抑制素基因表达中的作用，对CRE进行了位点特异性诱变。含有突变CRE的α抑制素启动子在颗粒细胞中不受福斯可林调控且不与CREB蛋白结合。有趣的是，CRE的突变也显著降低了α抑制素启动子的基础表达。最后，使用凝胶迁移率变动分析来检测颗粒细胞提取物中的CRE结合蛋白。颗粒细胞含有一种蛋白质，它能与含CRE寡核苷酸或与α抑制素启动子特异性相互作用，并且能被抗CREB蛋白的抗体识别。我们的结果表明，CREB或相关转录因子在卵巢颗粒细胞中α抑制素基因基础表达和cAMP调控表达中都起重要作用。
