Mechanical and Developmental Gene Regulation in Bladder

描述（由申请人提供）：机械力在膀胱发育和几种膀胱相关的病理生理过程中发挥重要作用。特别是，在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，异常/过度应变被认为是病理性重塑的主要引发者，病理性重塑是由于神经源性、前列腺、先天性和/或尿道狭窄疾病后的解剖或功能性膀胱出口梗阻而发生的。我们最近已经积累了几条线的证据表明，称为Cyr 61（富含半胱氨酸的蛋白质61）或CCN 1的立即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着重要的作用。Cyr 61基因的表达是通过机械转导途径介导的，机械转导途径是肥大信号在细胞中转导的主要手段。Cyr 61蛋白活性似乎影响几种肌肉和非肌肉特异性基因的表达。目前的建议包括一个战略，梳理出适当的影响，机械力对培养的膀胱平滑肌细胞的基因表达，并确定在膀胱发育过程中这些分子变化的体内相关性。这些目标将通过实现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来研究在mRNA转录水平上，机械刺激和Cyr 61过表达之间在培养的膀胱平滑肌细胞中的重叠。首先，我们将使用机械拉伸装置鉴定与急性（0.5和6小时）和慢性（24小时）机械刺激的应用相关的常见和/或独特的基因簇。第二，我们将确定一组基因与Cyr 61的过度表达，使用腺病毒基因转移和微阵列筛选。因此，将鉴定其表达依赖于Cyr 61的机械敏感基因。这种全球基因表达谱将使我们能够确定是否共调节基因的功能相关，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Cyr 61和Cyr 61靶向基因在发育中的膀胱中的时空表达，并确定这些机械敏感基因是否在组织特异性中发挥作用，特别强调逼尿肌平滑肌分化。这些研究将使我们能够定义将机械信号转化为生物反应的关键分子中间体，并产生新的有生物学意义的假设，即潜在的分子靶点在膀胱发育和阻塞性疾病中的作用。

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.