Characterizing the Molecular Pathways of Bladder Smooth Muscle Development

膀胱平滑肌发育的分子途径特征

• 批准号: 8203119
• 负责人: Kristin R DeSouza
• 金额: $ 5.3万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8203119
• 关键词: Address; Affect; Age; Animals; Binding; Bladder; Bladder Control; Bladder Diseases; Bladder Dysfunction; Communities; Complex; DNA Binding; Defect; Detection; Development; Disease; EMSA; Elements; Embryo; Gene Expression; Genes; In Vitro; Knockout Mice; Lower urinary tract; Mass Spectrum Analysis; Methods; Microarray Analysis; Molecular; Morbidity - disease rate; Mus; Muscle Development; Nuclear Proteins; Organ; Organ Culture Techniques; Pathogenesis; Pathway interactions; Phenotype; Proteins; Quality of life; Resources; Role; Smooth Muscle; Structure; Study models; Therapeutic; Time; Tissue Engineering; United States; Up-Regulation; base; design; in utero; knock-down; mouse model; mutant; myocardin; outcome forecast; overexpression; promoter; protein complex; sex; stoichiometry; transcription factor; urinary tract obstruction

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. PUBLIC HEALTH RELEVANCE: Disorders of the urinary bladder and its associated structures affect both sexes of all ages and ethnic backgrounds. The mgb mouse model provides us with an unparalleled resource to specifically study bladder smooth muscle development and bladder dysmorphogenesis. The studies proposed here with the mgb mouse model will provide a basis for the development of new methods of detection, prognosis, and therapeutic strategies, including tissue engineering, to treat a variety of bladder diseases and dysfunction.

描述（由申请人提供）：该项目的长期目标是表征膀胱平滑肌发育、功能和发病机制的分子途径。在美国，膀胱疾病每年影响数百万人，并对生活质量和发病率造成毁灭性影响（1,2）。尽管有大量的人受到影响，并且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尚不清楚。我们实验室的研究已经确定了一种独特的小鼠模型来研究膀胱畸形(3)。这些动物，mgb代表巨型阶梯，由于缺乏逼尿肌平滑肌发育，在子宫内发生下尿路阻塞(3)。互补分析、表达研究和qPCR表明，心肌素（myocardial in，心肌素）在mgb小鼠膀胱平滑肌缺损中起作用。基于这些研究的观察，我们假设膀胱平滑肌的发育高度依赖且易受心肌蛋白表达水平改变的影响。为了解决这一假设，我们建议：1)在野生型和mgb-/-膀胱中检测由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）膀胱核蛋白在平滑肌分化的三个关键发育时间点与原型平滑肌特异性启动子元件的相互作用，以表征mgb动物中心肌的缺失如何影响膀胱平滑肌特异性基因的转录蛋白复合物形成。质谱法将用于确定EMSA鉴定的膀胱平滑肌基因表达所必需的复合体的精确蛋白质组成。2)通过改变心肌素的表达，在体外重现并挽救mgb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，以评估改变心肌表达是否足以影响膀胱平滑肌的发育。