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。为了解决这个问题，我们的研究重点是了解分娩的激素控制，特别是类固醇激素孕酮如何通过核孕酮受体（nPR）PR-A和PR-B控制子宫收缩力。我们的中心假设是，在人类妊娠中，孕酮通过nPR介导的基因组途径促进子宫肌层静止，分娩涉及nPR介导的孕酮作用的功能性撤回，这使得促收缩途径占上风，导致分娩。我们的研究表明，人类分娩涉及产前和分娩相关的子宫肌层PR-A/PR-B蛋白比例的增加，由于PR-A的表达增加。我们还发现，PR-A相对于PR-B水平的增加抑制了PR-B介导的子宫肌层细胞孕酮反应。这些观察结果导致了PR-A/PR-B假说，该假说假定a）孕酮在大多数人类妊娠期间通过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，和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酮反应基因的启动子处，特别是那些抑制收缩的基因。或者，PR-A可以控制一组不同的基因的表达，这些基因增强收缩力并对抗PR-B的舒张作用。为了验证这一假设，我们必须确定子宫肌层细胞中受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在子宫肌层细胞中内源性孕酮反应基因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肌层细胞系和子宫肌层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。为了实验性地控制PR-A和PR-B的水平，我们将开发稳定的遗传修饰的永生化子宫肌层细胞模型，hTERT-HMA/B细胞，其含有独立的诱导型PR-A和PR-B转基因。这是一种新的和强大的方法，以确定特定的基因组行动的孕酮通过PR-A和PR-B，单独和组合，在人类子宫肌层细胞的情况下。提出了两个相互关联的具体目标。具体目的1：鉴定子宫肌层细胞中受PR-A和PR-B调控的基因。在hTERT-HMA/B细胞中受PR-A和PR-B单独和组合影响（上调或下调）的基因将使用基于微阵列的全基因组表达谱进行鉴定。将基于每个nPR改变表达的程度以及其产物是否可以基于已知功能影响收缩性来选择感兴趣的基因。将在从剖腹产获得的子宫肌层中进行所选基因的验证性研究，以确定表达水平是否与PR-A/PR-B蛋白比率、妊娠阶段和分娩状态相关。特定目的2：鉴定子宫肌层细胞中结合PR-A和PR-B的基因启动子。将通过nPR-染色质免疫沉淀（ChIP），随后进行基于微阵列的全基因组DNA定位分析（ChIP-on-chip），鉴定hTERT-HMA/B细胞中PR-A和PR-B结合的基因启动子。将对结果进行分析，以鉴定特异性目的1中鉴定的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，以介导人类妊娠子宫肌层中的基因组孕酮作用。成功完成拟议的研究将促进对孕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，并造成70-80%的新生儿死亡和发病率。尽管新生儿护理的进步改善了生存结果，但早产造成的混淆问题对早产儿未来的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室。为此，我们必须预防和/或抑制早产。然而，目前抑制早产的疗法通常无效。为了解决这个问题，我们必须填补知识空白，限制我们的能力，以开发有效的治疗早产。因此，我们的长期目标是确定控制子宫收缩力的激素相互作用。这项建议旨在确定类固醇激素孕酮在怀孕期间促进子宫舒张的机制。对这一过程的清楚理解可能会揭示抑制早产和预防早产的新的治疗靶点。

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.