Molecular Regulation of Progesterone Synthesis

描述（由申请人提供）： 为了有效地治疗与超过180万女性退伍军人患者和越来越多的女性在武装部队服役（14%）相关的疾病，必须了解调节类固醇生成的机制。本研究的长期目标是充分了解促性腺激素的细胞作用机制和类固醇生成的调节。本研究的短期目标是发现由LH启动的新的蛋白激酶A（PKA）信号传导事件，并确定这些新机制如何促进孕酮合成。这项研究涉及第二信使途径和原癌基因产物的组成部分，这些成分对性类固醇分泌的调节至关重要。这项研究旨在研究一种新的细胞内信号事件序列，介导卵巢细胞中的类固醇生成。所提出的目的是为了测试整体假设，即蛋白激酶A的激活抑制细胞内信号传导中间体糖原合成酶激酶3（GSK 3），这有助于通过增加2-连环蛋白的可用性，促进类固醇生成急性调节蛋白（星星）的表达的转录共激活剂，类固醇生成反应LH。这一假设得到了我们令人兴奋的观察结果的支持，这些观察结果表明，在黄体细胞中，来自cAMP/PKA通路的细胞内信号与Wnt/GSK 3/2-连环蛋白通路会聚。提出了三个具体的目标来检验这一假设：一）为了确定蛋白激酶A（PKA）是否降低GSK 3活性，从而增加2-连环蛋白和类固醇生成的水平，我们将使用信号中间体的表达和siRNA敲低的方法来验证它们对孕酮合成的贡献。这些研究将补充检查PKA，GSK 3和2-连环蛋白的亚细胞定位。2）为了确定LH/PKA依赖性磷酸化是否调节2-连环蛋白的稳定性和活性，我们将评估PKA的刺激是否增加2-连环蛋白上C-末端PKA磷酸化位点的磷酸化，并确定这是否有助于2-连环蛋白的稳定性和/或核定位。为了确定2-连环蛋白是否与已知调节星星表达和孕酮合成的关键转录因子LRH-1（肝受体同源物-1）合作，我们将使用突变蛋白进行免疫共沉淀和共定位研究。我们的假设是，2-连环蛋白作为核受体LRH-1的转录共激活剂，以增加类固醇生成急性调节蛋白（星星）的表达，因此，增加孕酮的合成。三）确定在星星表达和孕酮合成的调节过程中，2-连环蛋白与LRH-1的相互作用是否募集额外的转录共激活因子，如CBP（CREB结合蛋白）。我们的假设是，LH和PKA依赖2-catenin，LRH-1，CBP之间的相互作用是必需的类固醇反应。该研究计划整合了多种方法[表达或突变分子，siRNA敲除，免疫定位，酶测定，基因表达，蛋白降解，蛋白质-蛋白质和蛋白质-DNA相互作用，ChIP分析]，以鉴定LH激活的新型细胞内信号传导组分及其增强孕酮合成的潜力。了解这些细胞过程的信号传导对于了解控制内分泌细胞中类固醇生成的信号转导途径的整合至关重要。 公共卫生相关性： 与妇女健康有关的研究是D.V.A.的一个领域。关注为了有效地治疗与超过180万女性退伍军人患者和越来越多的女性在武装部队服役（14%）相关的疾病，必须了解调节类固醇生成的机制。这项研究涉及第二信使途径和原癌基因产物的组成部分，这些成分对类固醇分泌的调节至关重要。在男性和女性中，类固醇控制许多重要过程，包括碳水化合物，脂质和蛋白质代谢以及性功能。一些与卵巢类固醇分泌异常相关的代谢紊乱包括高血压、糖尿病、肥胖症、骨质疏松症、多囊卵巢综合征以及结肠、乳腺、卵巢和子宫肿瘤。考虑到性类固醇在健康和疾病中的重要性，我们必须更好地了解控制类固醇激素合成的机制。

DESCRIPTION (provided by applicant): PROJECT SUMMARY In order to effectively treat disorders associated with the over 1.8 million female veteran patients and the growing proportion of women serving in the armed forces (14%), it is imperative to understand the mechanisms that regulate steroidogenesis. The long-term objectives of this research are to fully understand the cellular mechanisms of action of gonadotropins and the regulation of steroidogenesis. The short-term goals of this research are to discover new protein kinase A (PKA) signaling events initiated by LH, and to determine how these novel mechanisms contribute to progesterone synthesis. This research involves components of second messenger pathways and proto-oncogene products that are central to the regulation of sex steroid secretion. The proposed research is designed to investigate a novel sequence of intracellular signaling events that mediate steroidogenesis in ovarian cells. The proposed aims are designed to test the overall hypothesis that activation of protein kinase A inhibits the intracellular signaling intermediate glycogen synthase kinase 3 (GSK3), which contributes to the steroidogenic response to LH by increasing the availability of a 2-catenin, a transcriptional co-activator that promotes the expression of the steroidogenic acute regulatory protein (StAR). This hypothesis is supported by our exciting observations showing that intracellular signals from the cAMP/PKA pathway converge with the Wnt/GSK3/2-catenin pathway in luteal cells. Three specific objectives are proposed to test this hypothesis: One) To determine whether protein kinase A (PKA) reduces GSK3 activity and thereby increases levels of 2-catenin and steroidogenesis we will use approaches for the expression and siRNA knockdown of signaling intermediates to validate their contribution to progesterone synthesis. These studies will be complimented by examining the subcellular localization of PKA, GSK3 and 2-catenin. Two) To determine whether LH/PKA-dependent phosphorylation regulates the stability and activity of 2-catenin we will evaluate whether stimulation of PKA increases the phosphorylation of C-terminus PKA phosphorylation sites on 2-catenin and determine if this contributes to the stability and/or nuclear localization of 2-catenin. To determine whether 2-catenin cooperates with a critical transcription factor LRH-1 (liver receptor homolog-1) known to regulate StAR expression and progesterone synthesis we will perform co-immunoprecipitation and co-localization studies using mutant proteins. Our hypothesis is that 2-catenin serves as a transcriptional co-activator for the nuclear receptor LRH-1 to increase the expression of steroidogenic acute regulatory protein (StAR) and, as a result, increase progesterone synthesis. Three) To determine whether 2-catenin interaction with LRH-1 recruits additional transcriptional co-activators, such as CBP (CREB-binding protein), during the regulation of StAR expression and progesterone synthesis. Our hypothesis is that LH- and PKA-dependent interactions among 2-catenin, LRH-1, and CBP are required during the steroidogenic response. This research plan integrates a variety of approaches [expression or mutant molecules, siRNA knockdown, immuno-localization, enzyme assays, gene expression, protein degradation, protein-protein and protein-DNA interactions, ChIP analysis] to identify novel intracellular signaling components activated by LH, and their potential for enhancing progesterone synthesis. Understanding signaling these cellular processes is vital to understand the integration of signal transduction pathways that control steroidogenesis in endocrine cells. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Research relevant to women's health is an area of D.V.A. attention. In order to effectively treat disorders associated with over 1.8 million female veteran patients and growing proportion of women serving in the armed forces (14%), it's imperative to understand the mechanisms that regulate steroidogenesis. This research involves components of second messenger pathways and proto-oncogene products that are central to the regulation of steroid secretion. In both men and women steroids control many vital processes, including carbohydrate, lipid, and protein metabolism, and sexual function. Some metabolic disorders associated with aberrant ovarian steroid secretion are hypertension, diabetes, obesity, osteoporosis, polycystic ovary syndrome and neoplasms of the colon, breast, ovary, and uterus. Considering the importance sex steroids in health and disease, we must have a better understanding of the mechanisms controlling the synthesis of steroid hormones.