CHANGES IN MYOSIN HEAVY CHAIN ISOFORMS AND PROTEINS INVOLVED IN CA 2+-SENSITIZATION PATHWAY IN THE DETRUSOR SMOOTH MUSCLE FROM MEN WITH OUTLET OBSTRUCTION INDUCED BY BENIGN PROSTATIC HYPERPLASIA

Abstract

INTRODUCTION AND OBJECTIVES: Partial Bladder Outlet Obstruction (PBOO) induced remodeling of the detrusor smooth muscle (DSM) is associated with modulation of cell signaling that regulates smooth muscle contraction. The RhoA, ROCK and CPI-17 have been linked to Ca2+-sensitization which maintains smooth muscle tone by keeping the myosin regulatory light chain in the phosphorylated state due to diminished mysoin phosphatase activity. In this study, we show that the transcription factor (TF) NFB and proteins involved in the Ca2+-sensitization (RhoA, ROCK and CPI-17) are overexpressed in DSM from mice with PBOO. Furthermore, we show a direct relationship between NFB and expression of ROCK and CPI-17 by (1) upregulation of RhoA, ROCK and CPI-17 in murine bladder smooth muscle cells transfected with a vector containing NFB cDNA, and (2) showing the down-regulation of RhoA, ROCK and CPI-17 in the DSM of NF B/p65 knout out mouse model with ablation of NFB. METHODS: Male C57bl/6 mice were surgically obstructed and kept for 2-weeks. Unoperated normal mice serve as a control. DSM removed from PBOO and normal control animals were used for PCR, western blotting, and protein/DNA array. The TFs that showed enhanced binding in PBOO were transiently transfected in murine bladder primary smooth muscle cells. The RhoA, ROCK and CPI-17 expressions from transiently transfected murine bladder muscles were analyzed using immunoblotting and RT-PCR. RESULTS: RhoA, ROCK and CPI-17 are upregulated in PBOO as shown by immunoblotting and RT-PCR analyses. Analysis of TFs profiling using protein/DNA array revealed enhanced binding of NFB to their cognate DNA sequences with the nuclear extract isolated from DSM from PBOO. The transient trnasfection of NFB cDNA in murine bladder primary smooth muscle cells increased the RhoA, ROCK and CPI-17 mRNA and protein expression. The RT-PCR and immunoblot analysis of RhoA, ROCK and CPI-17 from NF B/p65 KO mouse showed reduced amount of mRNA and the proteins compared to control. CONCLUSIONS: The increased expression of RhoA, ROCK and CPI-17 observed in PBOO is mediated through NFB. The upregulation of these proteins could alter the signaling pathways that regulates DSM contractility in PBOO.