ESTROGEN REGULATION OF THE RAT LH BETA GENE

促黄体生成素(Lh)是一种脑下垂体糖蛋白激素，在体内起着至关重要的作用。 在性腺发育和功能中的作用，并调节 产生性类固醇，如雌二醇(E2)。黄体生成素包括 两个亚基，所有糖蛋白共有的阿尔法亚基 激素，以及独特的LHBeta亚单位，它在更多的 严格的生理调节。体内促黄体生成素的产生低于 雌二醇的正负调节，但目前尚不清楚 这种反馈是发生在下丘脑还是在脑下垂体 级别或两者兼而有之。我们已经证明了基因的转录速度 KLHBeta基因在体内受E2负调控，但初步 实验表明，E2刺激了脑下垂体的转录 片段和瞬时表达分析。第一个目标是 通过测量E_2-来确定E_2对LHBeta基因的直接作用 转录调控不受下丘脑影响。时间 而E2反应的剂量依赖性将在单独的 在细胞培养中不存在和存在时的脑垂体碎片 促性腺激素释放激素(GnRH)。第二个目标是 确定LHBeta基因中与E2反应相关的区域。 用标记基因片段和纯化的E2进行筛选结合分析 受体复合体表明至少有一个可能的受体结合 位于LHBeta基因5‘侧翼区。这一地区和 其他潜在的受体结合位点将通过 核酸外切酶III和DNase I足迹研究，以及 突变分析，以确定受体结合的序列。 生物学上的E2反应所需的基因区域将是 通过瞬时表达分析鉴定，其中上游LHBeta 胸苷激酶或催乳素旁边插入的基因区域 将控制报告基因表达的启动子导入 转化为GH3细胞或正常脑垂体细胞。预计 该反应需要受体结合区，但 其他DNA元素和结合蛋白可能是必要的，也将是 被指认出来。特定基因区域的能力赋予 E2对报告基因的反应，以及序列和定位 这些元素中的哪些将通过缺失分析来确定。这个 这些序列中特定核苷酸的重要性将是 通过定点突变进行评估。其他人的能力 将测试调节E2反应的荷尔蒙。这个 抗雌激素药物他莫昔芬与LHBeta基因E2的相互作用 调节区将由他莫昔芬-E2受体测量 足迹分析，以及他莫昔芬和孕酮调节 将通过转录来研究E2的影响，并在 瞬时表达分析。报告基因构建体包含 同源的LHBeta基因启动子和5‘侧翼区将 转染腺垂体细胞并检测其表达和E2水平 在没有或存在促性腺激素释放激素的情况下进行监管。这些研究将 增加我们对E2对黄体生成素产生的直接影响的理解 并澄清一些复杂的荷尔蒙调节在 生殖过程。

Lutropin (LH), a pituitary glycoprotein hormone, plays a critical role in gonadal development and function, and regulates the production of sex steroids such as estradiol (E2). LH consists of two subunits, the alpha subunit common to all glycoprotein hormones, and the unique LHBeta subunit which is under more stringent physiological regulation. LH production in vivo is under both positive and negative regulation by E2, but it is unclear whether this feedback occurs at the hypothalamic or pituitary level, or both. We have shown that the transcription rate of the kLHBeta gene is negatively regulated by E2 in vivo, but preliminary experiments indicate E2 stimulation of transcription in pituitary fragments and in transient expression assays. The first aim is to define the direct actions of E2 on the LHBeta gene by measuring E2- regulated transcription free of hypothalamic influences. The time and dose dependence of the E2 response will be assessed in isolated pituitary fragments and in cell culture in the absence and presence of gonadotropin releasing hormone (GnRH). The second aim is to identify regions of the LHBeta gene which confer an E2 response. Filter-binding assays with labeled gene fragments and purified E2 receptor complex indicate at least one putative receptor binding site in the 5'-flanking region of the LHBeta gene. This region and other potential receptor binding sites will be studied by Exonuclease III and DNAse I footprinting studies, as well as by mutational analysis, to identify the sequence for receptor binding. Gene regions necessary for the biological E2 response will be identified by transient expression assays, in which upstream LHBeta gene regions inserted next to a thymidine kinase or prolactin promoter controlling expression of a reporter gene are transfected into GH3 cells or normal pituitary cells. It is anticipated that the receptor binding region will be required for the response, but other DNA elements and binding proteins may be necessary, and will be identified. The ability of specific gene regions to confer the E2 response to the reporter gene, and the sequence and positioning of these elements will be determined by deletion analysis. The importance of specific nucleotides within these sequences will be evaluated by site-directed mutagenesis. The ability of other hormones to modulate the E2 response will be tested. The interaction of the antiestrogen tamoxifen with LHBeta gene E2 regulatory regions will be measured by tamoxifen-E2 receptor footprint analysis, and tamoxifen and progesterone modulation of the E2 effects will be investigated by transcription, and in transient expression assays. Reporter gene constructs containing the homologous LHBeta gene promoter and 5'flanking regions will be transfected into pituitary cells and tested for expression and E2 regulation in the absence or presence of GnRH. These studies will add to our understanding of the direct E2 effects on LH production and clarify some of the complex hormonal regulation in the reproductive process.