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Published on: August 18, 2014
Expression and proliferation profiles of PKC, JNK and p38MAPK in physiologically stretched human bladder smooth
Romel Wazir1, De-Yi Luo, Yi Dai
1Department of Urology, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China.
Objective:
To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro.
Materials And Methods:
HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments.
Results:
Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837±0.026 (control) to 1.462±0.023)%, (P<0.05) and apoptotic cell death rate decreased from 16.4±0.21% (control) to 4.5±0.13% (P<0.05) applied at 0.1Hz. Expression of PKC was upregulated with slight increase in JNK and no change in p38MAPK after application of stretch. Inhibition had effects on proliferation (1.075±0.024, P<0.05 GF109203X); (1.418±0.021, P>0.05 SP600125) and (1.461±0.01, P>0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs.
