The goal of this proposal is to determine the molecular mechanism by which bladder smooth muscle and lamina propria fibroblast regulate fibrillar collagen gene expression. The hypothesis is that mechanical stimulation alone can modulate the expression of transcription factor genes whose encoded proteins regulate and trigger the expression of other genes that are responsible for substantial phenotypic changes of bladder cells. By identifying these genes and characterizing their activation in bladder smooth muscle and fibroblast cells, the investigators expect to gain valuable information on the mechanisms by which the structural and functional properties of the bladder are regulated. They have developed an apparatus that permits the application of precise mechanical load, i.e., mechanical stretch, to cells in vitro. They will use such a device to apply an equiaxial stretch to bladder wall cells and study the regulation process of mechano-sensitive genes. They propose: 1) to study the effects of strain and strain rate on cell growth and matrix protein synthesis, 2) to characterize the mechano-response element in the type I and III collagen genes, 3) to identify and characterize genes whose expression is modulated as a result of mechanical stimulation, and 4) to use targeted primers to screen the family of transcription factors whose expression is modulated by the application of mechanical forces and to characterize the promoter activity of their gene targets. Changes in gene expression will be evaluated at the mRNA and protein levels using molecular biological and biochemical techniques, i.e., Northern blot, Western blot and differential display RT-PCR. Transfection experiments and DNA-protein interaction analyses will be used to define stretch-regulatory sequence(s) and trans acting factor(s) in the promoter of mechano-sensitive genes. With these approaches, the investigators expect to correlate changes in collagen expression to changes in expression and/or activity of trans acting factors, and to define the role of physical forces at the molecular level in normal functioning of the bladder.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG4-UROL (01))
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Mullins, Christopher V
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University of Pennsylvania
Anatomy/Cell Biology
Schools of Dentistry
United States
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