Characterizing the Molecular Pathways of Bladder Smooth Muscle Development

描述（由申请人提供）：该项目的长期目标是表征负责膀胱平滑肌发育、功能和发病机制的分子途径。膀胱疾病每年影响数百万人，对生活质量和发病率产生毁灭性影响 (1, 2)。尽管受影响的人数很多，并且这些疾病对我们的社区产生了巨大的经济影响，但控制膀胱发育、功能和发病机制的分子途径尚不清楚。我们实验室的研究已经确定了一种用于研究膀胱畸形发生的独特小鼠模型 (3)。这些动物被称为“mgb”（大膀胱），由于逼尿肌平滑肌发育不足，在子宫内出现下尿路梗阻 (3)。互补分析、表达研究和 qPCR 表明心肌素 (Myocd) 是 mgb 小鼠膀胱平滑肌缺陷的原因。根据这些研究的观察结果，我们假设膀胱平滑肌发育高度依赖且容易受到 Myocd 表达水平改变的影响。为了解决这一假设，我们建议：1）检查野生型和 mgb-/- 膀胱中心肌素在原型平滑肌特异性启动子上形成的转录复合物。我们将使用 EMSA 分析来比较正常 (WT) 和突变 (mgb) 膀胱核蛋白与原型平滑肌特异性启动子元件在平滑肌分化的三个关键发育时间点的相互作用，以表征我们的 mgb 动物中 Myocd 的缺失如何影响膀胱平滑肌特异性基因上转录蛋白复合物的形成。质谱法将用于确定 EMSA 鉴定的膀胱平滑肌基因表达所需的复合物的精确蛋白质组成。 2) 通过改变心肌素表达来概括和挽救体外 mgb 表型。我们将使用体外全器官外植体培养物来表征和操纵膀胱平​​滑肌，以评估改变 Myocd 表达是否足以影响膀胱平滑肌的发育。

DESCRIPTION (provided by applicant): The long-term objective of this project is to characterize the molecular pathways responsible for bladder smooth muscle development, function, and pathogenesis. Disorders of the urinary bladder affect millions of people in the United States annually and have a devastating impact on quality of life and morbidity (1, 2). In spite of the large number of people affected and the substantial financial impact these disorders have on our community, the molecular pathway(s) controlling bladder development, function, and pathogenesis are not well understood. Studies in our lab have identified a unique murine model for studying bladder dysmorphogenesis (3). These animals, designated mgb for megabladder, develop a lower urinary tract obstruction in utero due to a lack of detrusor smooth muscle development (3). Complementation analysis, expression studies, and qPCR have indicated that myocardin (Myocd) is responsible for the bladder smooth muscle defect in the mgb mouse. Based on observations from these studies we hypothesize that bladder smooth muscle development is highly dependent and susceptible to altered levels of Myocd expression. To address this hypothesis, we propose to: 1) Examine the transcriptional complex formed by Myocardin on a prototypic smooth muscle specific promoter in both wild type and mgb-/- bladders. We will use EMSA analysis to compare the interactions of normal (WT) and mutant (mgb) bladder nuclear proteins with a prototypic smooth muscle-specific promoter element at three key developmental time points in smooth muscle differentiation in order to characterize how the loss of Myocd in our mgb animals impacts transcriptional protein complex formation on bladder smooth muscle specific genes. Mass Spectrometry will be used to determine the precise protein composition of the complex(es) identified by EMSA necessary for bladder smooth muscle gene expression. 2) Recapitulate and rescue the mgb phenotype in vitro by altering Myocardin expression. We will use in vitro whole organ explant cultures to characterize and manipulate bladder smooth muscle in order to assess if altering Myocd expression is sufficient to influence bladder smooth muscle development.