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

长期目标是证明新的孕酮调节基因在子宫中的病理作用。 子宫肌瘤组织。我们发现孕酮结合的孕激素受体(PR)被招募到多个 在整个基因组范围内，它是肌瘤平滑肌细胞中许多基因的主调节者。在……里面 在体内，黄体酮及其激动剂会导致子宫肌瘤的生长，而治疗 RU486或其他拮抗剂可缩小肿瘤大小并减少相关的子宫出血。这个 孕酮及其拮抗剂在子宫肌瘤中的作用机制尚不清楚 为人所知。我们假设黄体酮调节一些关键基因，这些基因有利于增加 孕激素拮抗剂对子宫肌瘤细胞增殖和凋亡的抑制作用 逆转这些影响。使用两种无偏见的方法，染色质免疫沉淀-PCR克隆和 通过基因芯片分析，我们发现了两个关键基因bcl2和Kruppel-like factor11(KLF11)。 在体外和体内均受孕酮和RU486的高度调控。孕酮诱导的bcl2 表达可抑制子宫肌瘤细胞的凋亡，而抑癌基因KLF11 转录因子和PR的新靶点被黄体酮下调，导致 扩散。RU486抑制BCL2，诱导KLF11表达。为了进一步定义BCL2和BCL2的作用 KLF11作为子宫肌瘤的关键PR靶点，我们提出了以下目标。目标1是确定 PR和bcl2调控子宫肌瘤细胞凋亡的机制 纸巾。我们假设黄体酮及其拮抗剂调节bcl2介导的线粒体 PR介导的细胞凋亡途径。目的2是确定新的PR靶基因KLF11在调节 子宫肌瘤平滑肌细胞和组织的增殖。我们假设，通过公关， 孕酮通过抑制KLF11的表达促进子宫肌瘤细胞增殖，而其拮抗剂 促进KLF11的表达，抑制细胞增殖。目标3是定义PR相关的增强子和抑制因子 占据BCL2和KLF11基因调节区的转录复合体 孕酮或其拮抗剂对子宫肌瘤细胞的作用。我们将检验这一假设 BCL2和KLF11的启动子背景决定了协同调控因子对 用黄体酮或其拮抗剂处理导致转录激活或抑制。 相关性(请参阅说明)： 子宫肌瘤是最常见的良性妇科疾病。然而，潜在的 肌瘤生长和发展的分子和细胞机制还不是很清楚。我们 在此建议对子宫肌瘤的激素反应进行深入的分子和细胞分析。定义 孕激素及其拮抗剂的新分子靶点将导致开发更有效的药物 副作用少的孕激素受体调节剂治疗子宫肌瘤。

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.