17 alpha-Hydroxylase Expression in Human Ovarian Cells

描述(申请人提供)：拟议的实验旨在阐明正常周期妇女和多囊卵巢综合征(PCOS)妇女卵巢膜内膜细胞雄激素生物合成的调节机制。在过去的资助期间，我们获得了令人兴奋的数据，表明PCOS卵泡膜细胞中雄激素的增加和P450 17α-羟基酶(CYP17)基因表达的增加是MEK1/ERK途径激活状态的降低和MKK3途径激活状态的刺激共同作用的结果。我们已经观察到，用表达显性阴性MEK1和结构性活性MKK3的腺病毒感染正常卵泡膜细胞，可以重现PCOS卵泡膜细胞的表型。我们现在有了工具来充分探索MAPK信号通路的其他组成部分的贡献。在这项提案中，我们将检验这一假设，即卵巢雄激素产生的增加是PCOS卵泡膜细胞中丝裂原激活蛋白激酶(MAPK)信号变化的结果。目的1研究ERK和p38信号通路上游信号成分(S)和p38信号通路(S)(即Raf-1、MEKK1、MEKK3)的失调是否会直接影响每条同源通路的下游信号成分，从而上调细胞色素PYP17和Cyp11a1基因的表达和雄激素的生物合成。我们还将研究一条通路(S)信号成分的失调是否会导致并行通路(S)信号的代偿性变化。在目标2中，我们将研究ERK和p38信号的改变在多大程度上有助于PCOS卵泡膜细胞中CYP17和Cyp11a1基因转录和mRNA稳定性的提高。在整个文献中，有数据支持肿瘤坏死因子a(TNF)抑制卵泡膜细胞色素P17基因的表达和雄激素的生物合成。然而，这些数据与多囊卵巢综合征妇女循环血浆和卵泡液中肿瘤坏死因子水平升高的观察结果相矛盾。在目标3中，我们将研究PCOS卵泡膜细胞中MAPK信号的缺陷在多大程度上影响依赖于肿瘤坏死因子的雄激素生物合成调节。这些研究的结果将为卵巢雄激素分泌增加的原因和分子基础提供新的见解，并导致开发治疗多囊卵巢综合征妇女的新靶点。

DESCRIPTION (provided by applicant): The proposed experiments are designed to elucidate the regulatory mechanisms underlying androgen biosynthesis in theca interna cells from ovaries of normal cycling women and women with polycystic ovary syndrome (PCOS). During the past funding period, we obtained exciting data demonstrating that increased androgen production and augmented P450 17alpha-hydroxylase (CYP17) gene expression in PCOS theca cells results from a combined reduction in the activation state of the MEK1/ERK pathway and a stimulation in the activation state of the MKK3 pathway. We have observed that infection of normal theca cells with adenovirus expressing dominant negative MEK1 and constitutively active MKK3 recapitulates a PCOS theca cell phenotype. We now have the tools to fully explore the contributions of the other components of the MAPK signaling pathways. In this proposal we will test the hypothesis that increased ovarian androgen production is a consequence of altered mitogen activated protein kinase (MAPK) signaling in PCOS theca cells. Aim 1 studies are designed to investigate whether dysregulation of upstream signaling component(s) of the ERK and p38 signaling pathway(s) (i.e., Raf-1, MEKK1, MEKK3) directly affect the down stream signaling components of each homologous pathway to the extent that CYP17 and CYP11A1 gene expression, and androgen biosynthesis are up-regulated in PCOS theca cells. We will also examine whether dysregulation of signaling components from one pathway result(s) in compensatory changes in signaling through parallel pathway(s). In Aim 2 we will investigate the extent to which alterations in ERK and p38 signaling contribute to increased CYP17 and CYP11A1 gene transcription and mRNA stability in PCOS theca cells. Throughout the literature there are data to support that tumor necrosis factor a (TNF) inhibits thecal CYP17 gene expression and androgen biosynthesis. However, these data are contradictory with the observation that PCOS women have elevated circulating plasma and follicular fluid levels of TNF. In Aim 3, we will examine the extent to which defects in MAPK signaling in PCOS theca cells affects TNF-dependent regulation of androgen biosynthesis. Results of these studies will provide both new insights regarding the cause and molecular basis for increased ovarian androgen production, and lead to the development of new targets for treatment of women with PCOS.