Mechanical and Developmental Gene Regulation in Bladder

描述（由申请人提供）：机械力在膀胱发育和一些与膀胱相关的病理生理过程中起重要作用。特别是，膀胱发育过程中发生的正常循环或体积功对于驱动细胞生长和分化所需的过程是必要的。然而，神经源性、前列腺、先天性和/或尿道狭窄疾病导致解剖性或功能性膀胱出口梗阻，异常/过度的张力被认为是病理性重构的主要发起者。我们最近积累了几条证据，表明被称为Cyr61（富含半胱氨酸的蛋白61）或CCN1的即时早期生长因子在膀胱平滑肌细胞（SMCs）对机械刺激的反应中起重要作用。Cyr61基因的表达是通过机械转导途径介导的，这是细胞中肥厚信号转导的主要途径。Cyr61蛋白活性似乎影响几种肌肉和非肌肉特异性基因的表达。目前的建议包括梳理机械力对培养膀胱SMCs基因表达的适当影响，并确定膀胱发育过程中这些分子变化的体内相关性。这些目标将通过解决以下具体目标来实现。在第一个目标中，我们将使用微阵列技术在mRNA转录水平上检查机械刺激和Cyr61在培养膀胱SMCs中的过表达之间的重叠。首先，我们将确定与使用机械拉伸装置进行急性（0.5和6小时）和慢性（24小时）机械刺激相关的常见和/或独特的基因簇。其次，我们将使用腺病毒基因转移和微阵列筛选来鉴定与Cyr61过表达相关的基因组。因此，将鉴定出表达依赖Cyr61的机械敏感基因。这种全球基因表达谱将使我们能够确定共调控基因是否在功能上相关，并确定共同和/或独特的基因调控网络。在第二个目标中，我们将定义Cyr61和Cyr61靶向基因在发育膀胱中的时空表达，并确定这些机械敏感基因是否在组织规范中发挥作用，特别是在逼尿肌平滑肌分化中。这些研究将使我们能够确定将机械信号转化为生物反应的关键分子中间体，并就潜在分子靶点在膀胱发育和阻塞性疾病中的作用产生新的生物学意义假设。

DESCRIPTION (provided by applicant): Mechanical forces play an important role both during bladder development and in several bladder-related pathophysiological processes. In particular, the normal cycling or volume work that occurs during bladder development is necessary to drive processes required for cell growth and differentiation. However, abnormal/excessive strain is suggested as the prime initiator of pathological remodeling that occurs as a result of anatomic or functional bladder outlet obstruction subsequent to neurogenic, prostatic, congenital and/or urethtral stricture diseases. We have recently accumulated several lines of evidence indicating that the immediate early growth factor referred to as Cyr61 (cysteine-rich protein 61) or CCN1 plays an important role in the response of bladder smooth muscle cells (SMCs) to mechanical stimulation. The expression of the Cyr61 gene is mediated via mechano-transduction pathways that are the prime means by which hypertrophic signals are transduced in the cells. Cyr61 protein activity seems to affect the expression of several muscle and non-muscle-specific genes. The current proposal consists of a strategy to tease out the proper effects of mechanical forces on gene expression in cultured bladder SMCs and determine the in vivo relevance of those molecular changes during bladder development. These goals will be achieved by addressing the following Specific Aims. In the first Aim, we will use microarray technology to examine the overlap, at the level of mRNA transcription, between mechanical stimulation and Cyr61 overexpression in cultured bladder SMCs. We will, first, identify common and/or unique clusters of genes associated with the application of acute (0.5 and 6 hours) and chronic (24 hours) mechanical stimulation using a mechanical stretch device. Second, we will identify the group of genes associated with the over-expression of Cyr61 using adenoviral gene transfer and microarray screening. Therefore, mechano-sensive genes whose expression is Cyr61- dependent will be identified. This global gene expression profiling will allow us to determine whether coregulated genes are related functionally and identify common and/or unique gene regulatory networks. In the second Aim, we will define the temporal and spatial expression of Cyr61 and Cyr61-targeted genes in the developing bladder and determine whether these mechano-sensitive genes play a role during tissue specification with particular emphasis on detrusor smooth muscle differentiation. These studies will allow us to define the key molecular intermediates that convert the mechanical signals into biological responses and generate new biologically meaningful hypotheses as to the role of potential molecular targets in bladder development and obstructive diseases.