Stretch Regulation and Function of Cyr61 in the Bladder

膀胱出口梗阻是神经源性、前列腺性、先天性和尿道狭窄疾病的结果。其特征是膀胱平滑肌质量增加，伴有肥大/增生和纤维化，膀胱容量改变，残留尿和膀胱不稳定。尽管手术解除了梗阻，但仍有明显的临床后遗症。据推测，异常的机械应变是引发这些变化的主要病因。膀胱壁内正常拉伸模式的破坏改变了关键生长因子基因的表达，这些基因编码的蛋白质在局部起作用，协调平滑肌细胞（SMC）表型特征的变化。本研究的目的是确定机械力影响膀胱SMCs表型特征的程度。利用体外机械系统，我们已经确定了富含半胱氨酸的蛋白61 （Cyr61）作为膀胱SMCs机械力的直接早期靶标。Cyr61是一种即时早期生长因子，具有细胞增殖、粘附和细胞外基质合成的功能。因此，Cyr61在膀胱壁对异常应变反应的整个过程中可能成为药理学操作的潜在靶点。本提案的第一个目的是验证机械拉伸诱导Cyr61基因表达影响膀胱SMCs生长、肥大和/或细胞外基质合成的假设。此外，由于Cyr61的细胞质和细胞核定位，我们将确定Cyr61效应是否涉及与反式作用因子的相互作用。第二个目标将定义机械拉伸改变Cyr61基因表达的机制，并在连接机械拉伸与Cyr61基因表达的信号通路中建立层次化级联。第三个目的是检查Cyr61在正常膀胱壁力学被部分出口阻塞改变的动物模型中的表达谱。我们将确定参与Cyr61机械调节的信号通路是否可以通过药理学操纵来改变膀胱对出口阻塞的反应。利用Northern blot、核糖核酸酶保护实验、免疫印迹、免疫染色技术和酵母双杂交系统评估Cyr61基因表达、蛋白定位和分子相互作用的变化。DNA转染和DNA-蛋白相互作用将用于确定Cyr61基因的转录要求。膀胱的功能改变将通过监测组织收缩性来评估。

Bladder outlet obstruction occurs as a result of neurogenic, prostatic, congenital and urethral stricture diseases. It is characterized by increased bladder smooth muscle mass with hypertrophy/hyperplasia and fibrosis, changes in bladder capacity, residual urine and bladder instability. Significant clinical sequelae remain despite the surgical relief of the obstruction. It is hypothesized that abnormal mechanical strain is a major etiologic factor that triggers these changes. Disruption of the normal pattern of stretch within the bladder wall alters the expression of key growth factor genes whose encoded proteins act locally to orchestrate changes in smooth muscle cell (SMC) phenotypical features. The objectives of this study are to define the extent to which mechanical forces affect the phenotypical features of bladder SMCs. Using an in vitro mechanical system, we have identified the Cysteine-rich protein 61 (Cyr61) as an immediate early target of mechanical forces in bladder SMCs. The Cyr61 is an immediate early growth factor with functions in cell proliferation, adhesion and extracellular matrix synthesis. Therefore, the Cyr61 appears as a potential target for pharmacologic manipulation in the whole process of bladder wall response to abnormal strain. The first aim of this proposal will test the hypothesis that induced-gene expression of Cyr61 by mechanical stretch affects the growth, hypertrophy and/or extracellular matrix synthesis in bladder SMCs. Additionally, because of its cytoplasmic and nuclear localization, we will determine whether Cyr61 effects involve interactions with trans-acting factors. The second aim will define the mechanisms whereby mechanical stretch alters Cyr61 gene expression and establish a hierarchical cascade in the signaling pathways linking mechanical stretch to Cyr61 gene expression. The third aim will examine the expression profile of Cyr61 in an animal model in which normal bladder wall mechanics were altered by partial outlet obstruction. We will determine whether the signaling pathways involved in the mechanical regulation of Cyr61 can be pharmacologically manipulated in ways that alter the bladder response to outlet obstruction. Changes in gene expression, protein localization and molecular interactions of Cyr61 will be evaluated using Northern blot, ribonuclease protection assay, immunoblotting, immunostaining techniques and yeast Two-Hybrid system. DNA transfection, and DNA-protein interactions will be used to define the transcriptional requirements for Cyr61 gene. Functional alterations of the bladder will be assessed by monitoring the tissue contractility.