MECHANICAL REGULATION OF THE IGF SYSTEM IN THE BLADDER

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Bladder outlet obstruction is a common complication that occurs as a result of neurogenic, prostatic, congenital and/or urethral stricture disease. It is characterized by increased bladder smooth muscle mass, bladder capacity and size and urinary retention. The cellular force generation decreases and bladder function is often irreversibly impaired despite the relief of the obstruction. Mechanical strain is the most likely type of stimulus that triggers changes in smooth muscle cell (SMC) growth and synthetic phenotype in the bladder. A lack of either an adequate level of mechanical exercise or volume work, or a disruption of the normal pattern of stretch within the bladder wall, alters the expression of key growth factor genes like the insulin-like growth factor-I (IGF-I) and potentially the protein involved in the regulation of its bioactivity including receptor and binding proteins. The objectives of this study are to define the extent to which a mechanical stimulus affects the expression of the IGF system components in vitro and in vivo and to provide new information regarding the molecular mechanisms responsible for these changes in the bladder. These goals will be achieved by addressing three specific aims. In the first aim, the effect of strain levels on IGF-I, IGF-I receptor and IGF-I binding protein (IGFBP) gene expression will be examined in cultured bladder SMCs to determine whether there is merely a threshold above which these genes respond. The functional significance of these changes on cell growth and matrix protein synthesis will also be studied. The second aim will examine the potential regulation of IGF-I gene at transcriptional or post-transcriptional levels in response to a mechanical input. Transcriptional regulation will be studied by functional mapping of the 5' flanking region with emphasis on the identification of regions that are crucial in the strain- response. The third aim will examine the expression and secretory profile of the IGF system components in a fetal animal model in which normal bladder wall mechanics were altered by partial outlet obstruction and after bladder obstruction reversal. Changes in gene expression will be evaluated at the mRNA and protein levels using Northern blots, ribonuclease protection assays, western ligand blotting and radioimmunoassays. DNA transfection experiments and DNA-protein interaction analyses will be used to define stretch-regulatory sequence(s) and trans- acting factor(s) in the promoter region. Immunohistochemical analysis will determine the tissue and cellular localization of these proteins.