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.
