Estradiol Production by Prostaglandin F2alpha

描述（由申请人提供）：本提案的目的是阐明调节大鼠黄体（CL）中P450芳香化酶（P450 arom）表达的分子和细胞机制。大鼠CL产生孕酮和雌二醇（E2），这两者都是生殖所必需的。P450 arom催化E2生物合成的最后一步，在大鼠整个妊娠期CL中表达，直到分娩前一天，当它迅速下降。控制大鼠CL中P450 arom表达的机制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F2 α（PGF 2 α）降低黄体P450 arom mRNA水平和编码P450 arom的cyp 19基因的转录速率。这项研究将阐明PGF 2 α作用于黄体细胞以调节特定的细胞内信号通路从而降低cyp 19转录的假设。我们建议研究以下具体目标：i）为了进一步表征PGF 2 α对cyp 19表达的作用，我们打算通过使用PGF 2 α受体的激动剂和拮抗剂来确定PGF 2 α作用的特异性，并通过使用基因报道构建体来检查PGF 2 α是否降低cyp 19启动子活性，ii）在体内鉴定哪些转录因子调节CL中P450 arom的基础表达，并检查哪些因子介导PGF 2 α对cyp 19转录的影响。我们已经发现加塔-4与P450 arom近端启动子结合，并且这种结合被PGF 2 α增强。在继续我们的实验以确定加塔-4是否介导PGF 2 α抑制P450 arom表达之前，我们建议检查加塔-4是否在体内调节P450 arom表达。我们还建议研究LRH-1，COUP-TF和/或SF-1是否参与调节黄体P450 arom表达，因为它们调节颗粒细胞和子宫内膜细胞中P450 arom的表达。我们将利用RT-PCR、凝胶迁移、染色质免疫沉淀和Western印迹技术，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E2参与了人类病理学的发展，如乳腺癌和子宫内膜异位症，该项目的结果可能会提供信息，这些信息可以成为未来肿瘤细胞研究的基础，特别是如果我们考虑到相同的cyp 19启动子用于黄体，子宫内膜斑块和乳腺肿瘤。

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.