Mechanisms of p53 action on primate steroidogenesis

描述(由申请人提供)：最近对非人类灵长类动物的研究表明，在排卵促性腺激素推注后30分钟内，颗粒细胞合成类固醇孕酮显著增加。在增加的同时，几种关键的类固醇生成酶的基因表达也增加，特别是类固醇生成急性调节蛋白(STAR)和3β-羟基类固醇脱氢酶(3bHSD)。虽然STAR和3bHSD的调节部分是由于转录因子如类固醇生成因子-1(SF-1)、GATA 4/6和CAAT增强子结合蛋白β(CEBPB)，但我们最近发现，灵长类颗粒细胞最初类固醇合成的增加依赖于肿瘤抑制因子P53。用药物或病毒癌基因抑制P53可减弱STAR和3bHSD(而不是SF-1)对hCG的刺激，表明P53在这一过程中起关键作用，颗粒细胞提取物中的P53不与3bHSD启动子上的P53结合，因此推测P53是Sf-1、GATA和/或CEBPB的转录辅助因子。具体目标1将确定hCG注射到黄素化的人颗粒细胞是否会导致P53翻译后的变化，以及使用RNA干扰技术识别和阻止这些变化的激酶。目标1的目的是证明P53的翻译后调节是类固醇合成的关键因素。特殊目的2将通过直接与SF-1、GATA和/或CEBPB相互作用来检验核P53对最大STAR启动子活性至关重要的假设。将使用一种非类固醇激素生成、P53缺失的细胞系(H1299)，其中可以操纵P53、SF-1、GATA4/6或CEBPB的表达质粒的各种组合来证明每种蛋白质的必要性。黄素化的颗粒细胞将被导入SF-1、GATA4/6或CEBPB和P53，以检验这些蛋白质直接接触的假设。这项拟议的研究有望证明肿瘤抑制基因P53在灵长类动物和人类类固醇合成中的新作用。

DESCRIPTION (provided by applicant): Recent studies in non-human primates demonstrate that granulosa cell synthesis of the steroid progesterone increases significantly within 30 min of an ovulatory gonadotropin bolus. Concurrent with this increase, gene expression of several key steroidogenic enzymes also increases, notably the steroidogenic acute regulatory protein (STAR) and 3beta-hydroxysteroid dehydrogenase (3bHSD). While the regulation of both StAR and 3bHSD is due in part to transcription factors such as steroidogenic factor-1 (SF-1), GATA 4/6, and CAAT enhancer binding protein beta (CEBPb), we have recently discovered that the initial increase in steroid synthesis by primate granulosa cells depends upon the tumor suppressor p53. Inhibition of p53 with pharmacologic agents or viral oncogenes attenuates hCG stimulation of StAR and 3bHSD (but not SF-1), indicating that p53 plays a key role in this process, p53 in granulosa cell extracts does not bind to putative p53 sites from the 3bHSD promoter, thus it is hypothesized that p53 works as a transcriptional co-factor for SF-1, GATA, and/or CEBPb. Specific aim 1 will determine if a hCG administration to luteinized human granulosa cells results in post-translational changes to p53, and the kinases responsible for these changes identified and blocked using RNA interference technology. The goal of aim 1 is to demonstrate that post-translational regulation of p53 is a key factor in steroidogenesis. Specific aim 2 will test the hypothesis that nuclear p53 is essential for maximal StAR promoter activity by directly interacting with SF-1, GATA, and/or CEBPb. A non-steroidogenic, p53 deficient cell line (H1299) will be used in which various combinations of expression plasmids for p53, SF-1, GATA4/6, or CEBPb can be manipulated to demonstrate the necessity of each protein. Luteinized granulosa cells will be transfected with SF-1, GATA4/6, or CEBPb and p53 to test the hypothesis that these proteins make direct physical contact. The proposed studies are expected to demonstrate a novel role for the tumor suppressor p53 in the onset of primate and human steroidogenesis.