Genomic actions of progesterone receptors in human myometrial cells

描述（由申请人提供）：早产是一个主要的公共卫生问题，导致70-80%的新生儿发病率和死亡率。为了解决这一问题，我们的研究重点是了解激素对分娩的控制，特别是类固醇激素黄体酮如何通过核黄体酮受体（nPRs） PR-A和PR-B来控制子宫收缩。我们的中心假设是，在人类妊娠中，黄体酮通过npr介导的基因组途径促进子宫肌静息，分娩涉及到npr介导的黄体酮作用的功能性退出，这使得促收缩途径占上风，导致分娩。我们的研究表明，由于PR-A的表达增加，人类分娩涉及到产前和分娩相关的肌层PR-A/PR-B蛋白比例的增加。我们还发现，相对于PR-B， PR-A水平的升高会抑制PR-B介导的子宫内膜细胞的黄体酮反应性。这些观察结果导致了PR-A/PR-B假说，该假说认为a)在人类怀孕的大部分时间里，黄体酮通过PR-B促进子宫肌层松弛，PR-B调节一组特定松弛基因的表达，b) PR-B的松弛作用在分娩时被PR-A阻断。PR-A对PR-B活性的抑制可能发生在内源性孕激素应答基因的启动子上，特别是那些抑制收缩的基因。另外，PR-A可以控制一组不同基因的表达，这些基因可以增强收缩性，并反对PR-B的松弛作用。为了验证这一假设，我们必须在子宫肌瘤细胞中确定受PR-A和PR-B影响的基因。然而，PR-A和PR-B基因在人类妊娠子宫肌层中的作用靶点尚不清楚。为了解决这一主要的知识差距，拟议的研究将阐明PR-A和PR-B在子宫内膜细胞内源性孕酮应答基因中的基因组作用和相互作用。受PR-A和PR-B影响的基因和启动子元件将在子宫内膜细胞系和子宫内膜组织标本中使用基于微阵列的全基因组表达和DNA定位分析来确定。为了实验控制PR-A和PR-B的水平，我们将开发一种稳定的转基因永生化子宫内膜细胞模型，hTERT-HMA/B细胞，含有独立诱导的PR-A和PR-B转基因。这是一种新颖而有力的方法，可以通过PR-A和PR-B来确定黄体酮在人类子宫肌瘤细胞中单独或联合的特定基因组作用。提出了两个相互关联的具体目标。特异性目的1：鉴定子宫肌瘤细胞中PR-A和PR-B控制的基因。在hTERT-HMA/B细胞中，受PR-A和PR-B单独或联合影响的基因（上调或下调）将使用基于微阵列的全基因组表达谱进行鉴定。将根据每个nPR改变表达的程度以及它们的产物是否会影响基于已知功能的收缩性来选择感兴趣的基因。将在剖宫产获得的子宫肌层中对所选基因进行确证性研究，以确定表达水平是否与PR-A/PR-B蛋白比例、妊娠期和分娩状态相关。特异性目的2：鉴定肌内膜细胞中结合PR-A和PR-B的基因启动子。PR- A和PR-B在hTERT-HMA/B细胞中结合的基因启动子将通过pcr -染色质免疫沉淀（ChIP）鉴定，然后进行基于微阵列的全基因组DNA定位分析（ChIP-on- ChIP）。将对结果进行分析，以确定PR-A和PR-B应答基因启动子内的PR-A和PR-B结合元件。这些基因的启动子将是未来机制研究的主题，以确定PR-A和PR-B如何相互作用，介导人类妊娠肌层中基因组孕酮的作用。这些研究的成功完成将促进对黄体酮如何调节人类出生引擎的理解，并有助于解决早产问题所需的知识基础。公共卫生相关性：早产是一个主要的社会经济问题，影响10-15%的怀孕，造成70-80%的新生儿死亡率和发病率。尽管新生儿护理的进步改善了生存结果，但由于早产引起的混淆问题对早产儿的未来健康产生了深远的负面影响。显然，胎儿发育的最后阶段最好在子宫环境中完成，而不是在新生儿重症监护病房。为此，我们必须预防和/或抑制早产。然而，目前抑制早产的治疗通常是无效的。为了解决这个问题，我们必须填补知识空白，这些空白限制了我们开发有效的早产治疗方法的能力。因此，我们的长期目标是确定控制子宫收缩的激素相互作用。本研究旨在确定孕激素黄体酮在妊娠期促进子宫松弛的机制。清楚地了解这一过程可能会揭示抑制早产和预防早产的新治疗靶点。

DESCRIPTION (provided by applicant): Premature birth is a major public health problem that causes 70-80% of neonatal morbidity and mortality. To address this problem our research focuses on understanding the hormonal control of labor and in particular how the steroid hormone progesterone acting through the nuclear progesterone receptors (nPRs) PR-A and PR-B, controls uterine contractility. Our central hypothesis is that in human pregnancy progesterone promotes myometrial quiescence via nPR-mediated genomic pathways and that parturition involves functional withdrawal of nPR-mediated progesterone actions, which allows the pro-contraction pathways to prevail leading to labor. Our studies show that human parturition involves a pre-partum- and labor-associated increase in the myometrial PR-A/PR-B protein ratio due to increased expression of PR-A. We also found that an increased level of PR-A relative to PR-B represses PR-B-mediated progesterone responsiveness in myometrial cells. Those observations have led to the PR-A/PR-B hypothesis, which posits that a) progesterone promotes myometrial relaxation during most of human pregnancy via PR-B, which modulates the expression of a specific cohort of relaxatory genes, and b) the relaxatory effects of PR-B are blocked at parturition by PR-A. PR-A repression of PR-B activity could occur at the promoters of endogenous progesterone-responsive genes, particularly those that inhibit contraction. Alternatively, PR-A could control the expression of a distinct cohort of genes that augment contractility and oppose the relaxatory effects of PR-B. To test this hypothesis we must identify the genes affected by PR-A and PR-B in myometrial cells. However, the PR-A and PR-B gene targets in the human pregnancy myometrium are not known. To address this major knowledge gap, the proposed studies will elucidate the genomic actions and interactions of PR-A and PR-B at endogenous progesterone responsive genes in myometrial cells. The genes and promoter elements affected by PR-A and PR-B will be determined using microarray-based genome-wide expression and DNA location analyses in a myometrial cell line and myometrial tissue specimens. To experimentally control the levels of PR-A and PR-B we will develop a stable genetically modified immortalized myometrial cell model, hTERT-HMA/B cells, containing independent inducible PR-A and PR-B transgenes. This is a novel and powerful approach to determine the specific genomic actions of progesterone via PR-A and PR-B, alone and in combination, in a human myometrial cell context. Two inter-related Specific Aims are proposed. SPECIFIC AIM 1: Identify the genes controlled by PR-A and PR-B in myometrial cells. The genes affected (up- or down-regulated) by PR-A and PR-B, alone and in combination, in hTERT-HMA/B cells will be identified using microarray-based genome-wide expression profiling. Genes of interest will be chosen based on the extent to which expression is altered by each nPR and whether their products could influence contractility based on known function. Confirmatory studies of selected genes will be performed in myometrium obtained from caesarean deliveries to determine whether expression levels correlate with the PR-A/PR-B protein ratio, stage of gestation and labor status. SPECIFIC AIM 2: Identify gene promoters that bind PR-A and PR-B in myometrial cells. The gene promoters to which PR- A and PR-B bind in hTERT-HMA/B cells will be identified by nPR-chromatin immunoprecipitation (ChIP) followed by microarray-based genome-wide DNA location analysis (ChIP-on-chip). Outcomes will be analyzed to identify PR-A and PR-B binding elements that lie within the promoters of PR-A and PR-B responsive genes identified in Specific Aim 1. The promoters of those genes will be the subjects of future mechanistic studies to determine how PR-A and PR-B interact to mediate genomic progesterone actions in the human pregnancy myometrium. Successful completion of the proposed studies will advance understanding of how progesterone regulates the engine for human birth, and contribute to the knowledge base needed to address the problem of preterm birth. PUBLIC HEALTH RELEVANCE: Preterm birth is a major socioeconomic problem that affects 10-15% of pregnancies and causes 70-80% of neonatal mortality and morbidity. Despite advances in neonatal care that have improved survival outcomes, the confounding problems due to preterm birth have a profound negative impact on a preterm infant's future health. Clearly, the final phase of fetal development is best achieved in the womb environment rather than in the neonatal intensive care unit. To this end, we must prevent and/or suppress preterm labor. However, current therapies to suppress preterm labor are generally ineffective. To resolve this problem we must fill the knowledge-gaps that limit our capacity to develop effective treatments for preterm labor. Therefore, our long- term goal is to determine the hormonal interactions that control uterine contractility. This proposal seeks to determine the mechanism by which the steroid hormone progesterone promotes uterine relaxation for most of pregnancy. A clear understanding of this process may reveal novel therapeutic targets for the suppression of preterm labor and the prevention of preterm birth.