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.
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.
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