Project 3: Mechanisms that Regulate Growth of Leiomyoma Smooth Muscle Cells

项目3：调节平滑肌瘤平滑肌细胞生长的机制

• 批准号: 8308001
• 负责人: Romana A. Nowak
• 金额: $ 44.99万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8308001
• 关键词: Adverse effects; Affect; Agonist; Anti-Progestin; Apoptosis; BCL2 gene; Benign; Binding; Cell Nucleus; Cell Proliferation; Cells; Cloning; Complex; Development; Disease; Enhancers; Gene Targeting; Genes; Goals; Growth; Growth and Development function; Gynecologic; Hormonal; In Vitro; Instruction; Lead; Leiomyoma; Mediating; Messenger RNA; Mitochondria; Molecular; Molecular Profiling; Molecular Target; Muscle; Nucleic Acid Regulatory Sequences; Pathologic; Pathology; Patients; Progesterone; Progesterone Receptors; Progestins; RU-486; RU-5020; Recruitment Activity; Regulation; Research; Role; Site; Smooth Muscle; Smooth Muscle Myocytes; Testing; Therapeutic; Tissues; Transcriptional Activation; Tumor Suppressor Proteins; Uterine Fibroids; Uterine hemorrhage; Woman; asoprisnil; chromatin immunoprecipitation; gene repression; genome-wide; in vivo; novel; programs; promoter; response; steroid hormone; transcription factor; tumor

The long-range goal is to demonstrate the pathologic roles of novel progesterone-regulated genes in uterine leiomyoma tissue. We found that progesterone-bound progesterone receptor (PR) is recruited to multiple sites genome-wide and acts as a master-regulator of many genes in leiomyoma smooth muscle cells. In vivo, progesterone and its agonists cause growth of uterine leiomyomata, whereas treatment of patients with RU486 or other antagonists reduces the tumor size and decreases associated uterine bleeding. The mechanisms responsible for these actions of progesterone and its antagonists in uterine leiomyoma are not known. We hypothesize that progesterone regulates a number of critical genes that favors increased proliferation and decreased apoptosis of leiomyoma smooth muscle cells, whereas progesterone antagonists reverse these effects. Using two unbiased approaches, chromatin immunoprecipitation-PCR cloning and mRNA profiling by microarray, we identified two key genes, BCL2 and kruppel-like factor-11 (KLF11), which are highly regulated by progesterone and RU486 in vitro and in vivo. Progesterone-induced BCL2 expression led to an inhibition of leiomyoma cell apoptosis, whereas KLF11, a tumor-suppressor transcription factor and a novel target of PR, was downregulated by progesterone resulting in enhanced proliferation. RU486 inhibited BCL2 and induced KLF11 expression. To further define the roles of BCL2 and KLF11 as critical PR targets in uterine leiomyoma, we propose the following aims. Aim 1 is to determine the mechanisms responsible for regulation of apoptosis via PR and BCL2 in leiomyoma smooth muscle cells and tissues. We hypothesize that progesterone and its antagonists regulate BCL2-mediated mitochondrial pathway of apoptosis via PR. Aim 2 is to define the role of the novel PR-target gene KLF11 in regulating proliferation of uterine leiomyoma smooth muscle cells and tissues. We hypothesize that, via PR, progesterone enhances leiomyoma cell proliferation by inhibiting KLF11 expression, whereas its antagonists favor KLF11 expression and inhibit proliferation. Aim 3 is to define PR-associated enhancer and inhibitory transcriptional complexes that occupy regulatory regions of BCL2 and KLF11 genes as a function of treatment of leiomyoma cells with progesterone or its antagonist. We will test the hypothesis that the promoter contexts of BCL2 and KLF11 determine differential recruitment of coregulators in response to treatment with progesterone or its antagonist resulting in transcriptional activation or repression. RELEVANCE (See instructions): Uterine leiomyomata represent the most prevalent benign gynecologic disorder. However, the underlying molecular and cellular mechanisms of leiomyoma growth and development are not well understood. We propose here in-depth molecular and cellular analysis of hormonal responsiveness of leiomyoma. Defining novel molecular targets of progestins and its antagonists will lead to development of more effective progesterone receptor modulators with fewer side effects for treating uterine leiomyoma.

长期目标是证明新的孕酮调节基因在子宫内膜异位症中的病理作用。 平滑肌瘤组织我们发现孕酮结合的孕激素受体（PR）被募集到多个 位点全基因组，并作为平滑肌瘤平滑肌细胞中许多基因的主调节因子。在 在体内，孕酮及其激动剂引起子宫平滑肌瘤的生长，而用孕酮及其激动剂治疗的患者 RU 486或其它拮抗剂减小肿瘤大小并减少相关子宫出血。的 孕激素及其拮抗剂在子宫平滑肌瘤中的这些作用机制并不清楚， 知道的我们假设孕酮调节了一些关键基因，这些基因有利于增加 增殖和减少平滑肌细胞凋亡，而孕酮拮抗剂 扭转这些影响。采用两种无偏倚的方法，染色质免疫沉淀-PCR克隆和 通过基因芯片的mRNA分析，我们确定了两个关键基因，BCL 2和Kruppel样因子-11（KLF 11）， 在体外和体内都受到孕酮和RU 486的高度调节。孕酮诱导的BCL 2 表达导致平滑肌瘤细胞凋亡的抑制，而肿瘤抑制因子KLF 11， 转录因子和PR的一个新靶点，被孕酮下调，导致增强 增殖RU 486抑制BCL 2和诱导KLF 11表达。为了进一步确定BCL 2的作用， KLF 11作为子宫平滑肌瘤的关键PR靶点，我们提出以下目标。目标1是确定 通过PR和BCL 2调节平滑肌瘤平滑肌细胞凋亡的机制， 组织中我们假设孕酮及其拮抗剂调节BCL 2介导的线粒体 目的2是确定新的PR靶基因KLF 11在调节细胞凋亡中的作用。 子宫平滑肌瘤平滑肌细胞和组织增生。我们假设，通过公关， 孕酮通过抑制KLF 11的表达促进平滑肌瘤细胞增殖，而其拮抗剂 有利于KLF 11的表达，抑制细胞增殖。目的3是确定PR相关的增强子和抑制子 转录复合物占据BCL 2和KLF 11基因的调控区，作为 用孕酮或其拮抗剂治疗平滑肌瘤细胞。我们将测试假设， BCL 2和KLF 11的启动子环境决定了辅助调节因子响应于 用孕酮或其拮抗剂处理导致转录激活或抑制。 相关性（参见说明）： 子宫平滑肌瘤是最常见的良性妇科疾病。但是，底层 平滑肌瘤生长和发育的分子和细胞机制还不清楚。我们 在这里提出深入的分子和细胞分析激素反应的平滑肌瘤。限定 孕激素及其拮抗剂的新的分子靶点将导致开发更有效的 孕酮受体调节剂，用于治疗子宫平滑肌瘤，副作用少。