Estradiol Production by Prostaglandin F2alpha

描述(申请人提供)：这项建议的目的是阐明调节大鼠黄体(CL)中P450芳香酶(P450arom)表达的分子和细胞机制。大鼠CL产生孕酮和雌二醇，这两种激素都是生殖所必需的。P450arom催化E2生物合成的最后一步，在大鼠的CL中表达，直到分娩前一天，然后迅速下降。调控大鼠CL中P450arom表达的机制(S)以及导致其在分娩前下降的因素尚不清楚。初步研究表明，前列腺素F2pha(PGF2pha)可降低黄体P450arom基因的转录水平和编码P450arom的cyp19基因的转录速率。这项拟议的研究将解决这样的假设，即PGF2α作用于黄体细胞，调节特定的细胞内信号通路，从而减少cyp19的转录。为了进一步研究PGF2α对cyp19表达的影响，我们拟通过使用PGF2α受体的激动剂和拮抗剂来确定PGF2α效应的特异性，并检测PGF2α是否通过使用基因报告结构来降低cyp19启动子的活性；ii)在体内鉴定哪些转录因子调节CL中P450arom的基础表达，并检测哪些因素介导了PGF2α对cyp19转录的影响。我们发现GATA-4与P450arom近端启动子结合，这种结合被PGF2pha增强。在继续我们的实验来确定GATA-4是否介导了PGF2pha对P450arom表达的抑制之前，我们建议先检查GATA-4是否在体内调节P450arom的表达。我们还建议研究LRH-1、COUP-Tf和/或SF-1是否参与黄体P450arom表达的调节，因为它们调节颗粒细胞和子宫内膜细胞中P450arom的表达。利用RT-PCR、Gel Shift、染色质免疫沉淀和Western印迹等方法，我们将在体内和体外检测这些因子在黄体细胞中的表达和结合活性。考虑到E2参与了乳腺癌和子宫内膜异位症等人类疾病的发展，该项目的结果可能会为未来的肿瘤细胞研究提供信息，特别是如果我们认为相同的cyp19启动子在黄体、子宫内膜异位症斑块和乳腺肿瘤中使用的话。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the molecular and cellular mechanisms that regulate P450 aromatase (P450arom) expression in the rat corpus luteum (CL). The rat CL produces progesterone and estradiol (E2), both of which are essential for reproduction. P450arom, which catalyzes the last step in E2 biosynthesis, is expressed in the rat CL throughout pregnancy until the day before parturition, when it rapidly decreases. The mechanism(s) that control P450arom expression in rat CL and the factors that cause its fall before parturition, are not known. Preliminary studies indicate that prostaglandin F2alpha (PGF2alpha) decreases luteal P450arom mRNA levels and the transcription rate of the cyp19 gene, which encodes P450arom. The proposed study will address the hypothesis that PGF2alpha acts on luteal cells to regulate specific intracellular signaling pathways oriented to decrease cyp19 transcription. We propose to investigate the following specific aims: i) to further characterize the effect of PGF2alpha on cyp19 expression, we intend to determine the specificity of the PGF2alpha effect by using agonists and antagonists of the PGF2alpha receptor and to examine whether PGF2alpha decreases cyp19 promoter activity by using gene reporter constructs, ii) to identify in vivo which transcription factors regulate basal expression of P450arom in the CL and to examine which factors mediate the effect of PGF2alpha on cyp19 transcription. We have found that GATA-4 binds to the P450arom proximal promoter and that such binding is increased by PGF2alpha. Before continuing our experiments to determine whether GATA-4 mediates PGF2alpha-inhibition of P450arom expression, we propose to examine whether GATA-4 regulates P450arom expression in vivo. We also propose to study whether LRH-1, COUP-TF and/or SF-1 are involved in the regulation of luteal P450arom expression as they regulate P450arom expression in granulosa and endometrial cells. Using RT-PCR, Gel Shift, Chromatin Immunoprecipitation, and Western blot, we will examine in vivo and in vitro the expression and binding activity of these factors in luteal cells. Considering that E2 is involved in the development of human pathologies such as breast cancer and endometriosis, the outcome of this project may contribute information that can be the foundation of future studies on tumor cells, especially if we consider that the same cyp19 promoter is used in corpus luteum, endometriotic plaque, and breast tumor.