Stretch Regulation and Function of Cyr61 in the Bladder

膀胱中 Cyr61 的伸展调节和功能

• 批准号: 6418450
• 负责人: BRAHIM CHAQOUR
• 金额: $ 22.71万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6418450
• 关键词: DNA binding protein; biomechanics; cell proliferation; cytoskeletal proteins; extracellular matrix; fibrosis; gene expression; genetic transcription; hyperplasia; hypertrophy; intermolecular interaction; laboratory rat; muscle cells; northern blottings; oligonucleotides; phenotype; protein localization; protein structure function; smooth muscle; stretch reflex; transcription factor; transfection /expression vector; urinary bladder; urinary tract obstruction; western blottings; yeast two hybrid system

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.

膀胱出口梗阻是由神经源性、前列腺、先天性和尿道狭窄疾病引起的。 其特征是膀胱平滑肌质量增加并伴有肥大/增生和纤维化、膀胱容量改变、残余尿和膀胱不稳定。尽管通过手术缓解了梗阻，但仍然存在明显的临床后遗症。 据推测，异常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会改变关键生长因子基因的表达，其编码的蛋白质在局部发挥作用，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对膀胱 SMC 表型特征的影响程度。 使用体外机械系统，我们已经确定富含半胱氨酸的蛋白 61 (Cyr61) 是膀胱 SMC 中机械力的直接早期目标。 Cyr61 是一种立即早期生长因子，具有细胞增殖、粘附和细胞外基质合成的功能。 因此，Cyr61 似乎是膀胱壁对异常应变反应整个过程中药物操控的潜在靶点。该提案的第一个目标将检验以下假设：机械拉伸诱导的 Cyr61 基因表达会影响膀胱 SMC 的生长、肥大和/或细胞外基质合成。 此外，由于其细胞质和核定位，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分层级联。 第三个目标是检查动物模型中 Cyr61 的表达谱，在该动物模型中，正常膀胱壁力学因部分出口阻塞而改变。 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方式进行操纵，从而改变膀胱对出口阻塞的反应。 Cyr61 的基因表达、蛋白质定位和分子相互作用的变化将使用 Northern 印迹、核糖核酸酶保护测定、免疫印迹、免疫染色技术和酵母双杂交系统进行评估。 DNA 转染和 DNA-蛋白质相互作用将用于确定 Cyr61 基因的转录要求。通过监测组织收缩性来评估膀胱的功能改变。