ESTROGEN REGULATION OF THE RAT LH BETA GENE

促黄体生成素（LH）是一种垂体糖蛋白激素， 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）。 LH包括 两个亚基，所有糖蛋白共有的α亚基 激素，和独特的LHBeta亚基，这是下更多 严格的生理调节。 体内LH的产生低于 E2的正、负调节作用，但尚不清楚 这种反馈是否发生在下丘脑或垂体 水平，或两者。 我们已经表明，转录速率的 kLHBeta基因在体内受E2的负调控，但初步 实验表明E2刺激垂体中的转录 片段和瞬时表达测定。 第一个目标是 通过测量E2-β来确定E2对LHBeta基因的直接作用， 不受下丘脑影响的调节转录。 的时间 E2反应的剂量依赖性将在隔离的 垂体碎片和在细胞培养中的存在和不存在 促性腺激素释放激素（GnRH）。 第二个目标是 鉴定赋予E2应答的LHBeta基因的区域。 用标记的基因片段和纯化的E2进行的滤膜结合试验 受体复合物表明至少有一个推定的受体结合 在LHBeta基因的5 '-侧翼区域中的位点。 本地区和 其他潜在的受体结合位点将通过 核酸外切酶III和DNA酶I足迹法研究，以及 突变分析，以鉴定受体结合的序列。 生物学E2反应所必需的基因区域将是 通过瞬时表达试验鉴定，其中上游LHBeta 插入胸苷激酶或催乳素旁边的基因区域 转染控制报告基因表达的启动子 GH 3细胞或正常垂体细胞。 预计各国 受体结合区是应答所必需的，但是 其它DNA元件和结合蛋白可能是必需的， 被识别。 特定的基因区域能够赋予 E2应答报告基因，并进行序列和定位 将通过缺失分析来确定这些元件的类型。 的 这些序列中特定核苷酸的重要性将被 通过定点诱变进行评价。 其他人的能力 将测试调节E2反应的激素。 的 抗雌激素他莫昔芬与LHBeta基因E2的相互作用 调节区域将通过他莫昔芬-E2受体来测量 足迹分析，以及他莫昔芬和孕酮调节 E2效应将通过转录来研究， 瞬时表达测定。 报告基因构建体，其包含 同源LHBeta基因启动子和5 ′侧翼区将 转染入垂体细胞并检测表达和E2 在缺乏或存在GnRH的情况下调节。 这些研究将 增加了我们对E2对LH产生的直接影响的理解 并阐明了一些复杂的激素调节， 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.