17 alpha-Hydroxylase Expression in Human Ovarian Cells

描述（由申请人提供）：本实验旨在阐明正常经期女性和多囊卵巢综合征（PCOS）女性卵巢内膜细胞雄激素生物合成的调控机制。在过去的资助期间，我们获得了令人兴奋的数据，证明PCOS膜细胞中雄激素产生增加和P450 17 α -羟化酶（CYP17）基因表达增强是由于MEK1/ERK途径激活状态的降低和MKK3途径激活状态的刺激。我们观察到，表达显性阴性MEK1和组成型活性MKK3的腺病毒感染正常的卵泡膜细胞，再现了PCOS的卵泡膜细胞表型。我们现在有工具来充分探索MAPK信号通路的其他组成部分的贡献。在本提案中，我们将验证卵巢雄激素产生增加是PCOS卵泡细胞中丝裂原活化蛋白激酶（MAPK）信号传导改变的结果。Aim 1研究旨在探讨ERK和p38信号通路上游信号组分（即Raf-1、MEKK1、MEKK3）的失调是否直接影响各同源通路的下游信号组分，从而导致PCOS膜细胞中CYP17和CYP11A1基因表达和雄激素生物合成上调。我们还将研究一个信号通路中信号成分的失调是否会导致平行通路中信号的代偿性变化。在Aim 2中，我们将研究ERK和p38信号的改变在多大程度上促进PCOS膜细胞中CYP17和CYP11A1基因转录和mRNA稳定性的增加。纵观文献，有数据支持肿瘤坏死因子a （tumor necrosis factor a， TNF）抑制鞘内CYP17基因表达和雄激素生物合成。然而，这些数据与PCOS妇女循环血浆和卵泡液TNF水平升高的观察结果相矛盾。在Aim 3中，我们将研究PCOS膜细胞中MAPK信号缺陷在多大程度上影响tnf依赖的雄激素生物合成调节。这些研究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PCOS女性治疗的新靶点的开发。

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.