POWRE: Estrogen Receptor-Beta Activation of Prolactin Gene Expression

项目名称：雌激素受体- β激活催乳素基因表达雌激素通过影响雌激素应答基因的激活或失活来调节许多身体组织的生长和分化。激活的第一步，也是最关键的一步是雌激素与其内源性细胞内受体（ER）的结合。ER- α (ER - alpha, ERa)是第一个被发现和鉴定得最好的内质网，它由几个区域或功能域组成。这些结构域中的每一个都执行特定的功能，这对于内质网诱导基因激活是绝对必要的。最近，一种新的ER变异ER- β (ER- b)被发现。ER-B与ERa在大小和组成它的氨基酸序列上都不同。这些差异可能导致这种新的受体与新的基因或新的配体相互作用，或者允许它以不同于ERa的方式与已经确定的雌激素敏感基因相互作用。关于这种新的受体如何与它所控制的基因相互作用，人们知之甚少。本研究的总体目标是确定雌激素反应性催乳素基因激活所需的ER-B功能域。基于对ER-B结构及其与ERa同源性的了解，第一个具体目标是构建一系列表达截断或突变ER-B受体的表达载体。利用这些突变体，在体外转染试验中，我们将能够确定ER-B分子的哪个结构域与各种雌激素化合物结合，哪些是基因激活所必需的。ER-B已在包括垂体在内的多种组织中检测到。催乳素是一种垂体激素，对雌激素非常敏感。催乳素参与许多生理功能，包括生殖、泌乳、性发育、细胞免疫和行为。关于ER-B对催乳素基因的调控，目前所知甚少。雌激素与内质网结合，并与垂体特异性因子Pit-1协同作用，刺激催乳素的合成和释放。虽然Pit-1与ERa变体之间相互作用的性质已被很好地表征，但Pit-1与ER-B之间的相互作用尚未确定。本研究的第二个具体目的是表征Pit-1和ER-B之间参与催乳素基因激活的分子相互作用。这将通过在基因转移研究中使用制备的突变受体来完成。每个突变受体将与Pit-1和催乳素报告基因结合引入异源细胞培养。这些细胞将被选定的雌激素化合物处理，泌乳素报告基因的转录激活将被量化。这些研究将确定ER-B的哪些区域与Pit-1协同工作，哪些区域是雌激素化合物诱导催乳素所必需的。这些研究的结果将开始确定这种新的受体异构体与它控制的基因之间的物理相互作用，以及扩展我们现有的关于调节催乳素稳态机制的知识。这些结果将促进我们对这种新发现的受体的作用的认识。

Rosemary Steinmetz Project Title: Estrogen Receptor-Beta Activation of Prolactin Gene Expression Estrogens modulate the growth and differentiation of many body tissues by influencing the activation or deactivation of estrogen-responsive genes. The first, and most critical, step that is involved in this activation is the binding of estrogen to its endogenous intracellular receptor (ER). ER- alpha (ERa), the first identified and best characterized ER, is composed of several regions or functional domains. Each of these domains carry out a specific function which is absolutely essential for the ER to induce gene activation. Recently, a new ER variant ER-Beta (ER-B) has been discovered. ER-B differs from ERa both in size and the sequence of the amino acids which comprise it. These differences may cause this new receptor to interact with new genes or new ligands or allow it to interact with already identified estrogen-sensitive genes in a manner different from that of ERa. Little is known concerning how this new receptor interacts physically with the genes it governs. The overall objective of this study is to determine the functional domains of ER-B that are required for the activation of the estrogen-responsive prolactin gene. Based on information concerning the structure of ER-B and its homology to ERa, the first specific aim is to construct a series of expression vectors which will express either truncated or mutated ER-B receptors. Using theses mutants, for in vitro transfection assays, we will be able to identify which domain(s) of the ER-B molecule bind various estrogenic compounds and which are required for gene activation. ER-B has been detected in a variety of tissues including the pituitary. Prolactin is a pituitary hormone that is exquisitely estrogen-sensitive. Prolactin is involved in many physiologic functions including reproduction, lactation, sexual development, cellular immunity and behavior. Little is known concerning the regulation of the prolactin gene by ER-B. Estrogens bind to the ER and in synergy with the pituitary-specific factor Pit-1 stimulate prolactin synthesis and release. While the nature of the interaction between Pit-1 and the ERa variant has been well characterized, the interaction between Pit-1 and ER-B has not been determined. The second specific aim of this study is to characterize the molecular interactions between Pit-1 and ER-B that are involved in prolactin gene activation. This will be accomplished by using the prepared mutant receptors in gene transfer studies. Each of the mutant receptors will be introduced into heterologous cell cultures in combination with Pit-1 and a prolactin reporter gene. These cells will then be treated with selected estrogenic compounds and the transcriptional activation of the prolactin reporter gene will be quantified. These studies will determine which regions of the ER-B work synergistically with Pit-1 and which are required for induction of prolactin by estrogenic compounds. Results of these studies will begin to determine the physical interactions between this new receptor isoform and the genes that it controls, as well as, expanding our existing knowledge concerning the mechanisms which govern prolactin homeostasis. These results will advance our knowledge concerning the actions of this newly discovered receptor.