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确定与膀胱平滑肌发育、功能和发病相关的分子通路的特征。在美国，膀胱疾病每年影响数百万人，对生活质量和发病率有毁灭性的影响(1，2)。尽管有大量的人受到影响，这些疾病对我们的社区造成了巨大的经济影响，但控制膀胱发育、功能和发病机制的分子途径(S)尚不清楚。我们实验室的研究已经确定了一种研究膀胱畸形发生的独特的小鼠模型(3)。这些动物被命名为MGB的巨型膀胱，由于缺乏逼尿肌平滑肌发育，在子宫内形成下尿路梗阻(3)。互补分析、表达研究和定量聚合酶链式反应表明，肌钙蛋白(Myocardin，MYOCD)与MGB小鼠的膀胱平滑肌缺陷有关。根据这些研究的观察结果，我们假设膀胱平滑肌的发育高度依赖于MYOCD表达水平的改变，并对其易感性。为了解决这一假设，我们建议：1)在野生型和MGB-/-膀胱中，研究Myocardin在典型的平滑肌特异性启动子上形成的转录复合体。我们将使用EMSA分析比较正常(WT)和突变(MGB)膀胱核蛋白与典型的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MYOCD在我们的MGB动物中的缺失如何影响膀胱平滑肌特异性基因转录蛋白复合体的形成。质谱学将被用来确定EMSA确定的膀胱平滑肌基因表达所需的复合体的精确蛋白质组成。2)通过改变Myocardin的表达，在体外重现和挽救MGB的表型。我们将使用体外全器官外植体培养来表征和操纵膀胱平滑肌，以评估改变MYOCD的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.