Gene expression of rat uterine endometrial stromal cells treated with ethanol (vehicle) or 0.1 nM progestin R5020

Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene, and Usf1 knockdown abrogated Cdc2 progestin-dependent transcriptional regulation providing a mechanism for direct regulation of its expression. Finally, Cdc2 knockdown blocked R5020 induced UIII cell proliferation. We conclude that progestin induced proliferation of endometrial stromal cells requires ERK1-2 and AKT mediated early regulation of USF1, that induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets.尽管非基因组类固醇受体通路在过去十年间已得到广泛研究，但针对其激活所介导的直接基因表达变化，目前仍鲜有深入认知。孕酮可在妊娠围着床期调控大鼠子宫内膜基质细胞的增殖。我们的研究证实，皮摩尔浓度的孕激素R5020可通过孕酮受体（Progesterone Receptor, PR）与雌激素受体β（Estrogen Receptor beta, ERβ）相互作用介导的ERK1-2与AKT激活，在UIII子宫内膜基质细胞中模拟这一调控效应，且该过程不依赖内源性PR与ER的转录活性。本研究旨在鉴定胞质PR信号通路的早期下游靶基因，及其在子宫内膜基质细胞增殖中可能发挥的作用。对经孕激素处理45分钟的UIII细胞进行全基因表达变化的基因芯片（Microarray）分析，共鉴定出97个上调基因与341个下调基因。富集度最高的分子功能类别为与细胞增殖及细胞周期相关的转录因子与调控因子，其中大部分为激素下调的阻遏因子。进一步分析验证：在无基因组PR结合的情况下，孕激素可通过配体非依赖型ER、ERK1-2或AKT的介导，调控Ccnd1、JunD、Usf1、Gfi1、Cyr61及Cdkn1b的表达。染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验显示，孕激素可促进USF1与Cdc2基因近端启动子的结合，而Usf1基因敲除则会废除Cdc2的孕激素依赖型转录调控，这为其表达的直接调控机制提供了依据。此外，Cdc2基因敲除可阻断R5020诱导的UIII细胞增殖。我们由此得出结论：孕激素诱导子宫内膜基质细胞增殖，需要ERK1-2与AKT介导的USF1早期调控，而USF1可进一步诱导Cdc2的表达。据我们所知，这是首次通过与蛋白激酶的交叉对话、且不依赖激素受体与基因组靶位点结合的方式，描述孕激素激活经典PR的早期靶基因。