Department of Urology, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China.
Biochem Biophys Res Commun. 2013 Aug 30;438(3):479-82. doi: 10.1016/j.bbrc.2013.07.115. Epub 2013 Aug 2.
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.
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.
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.
确定蛋白激酶 C（PKC）、c-Jun NH2-末端激酶（JNK）和 P38 丝裂原活化蛋白激酶（p38MAPK）的表达水平，并研究其各自抑制剂对体外生理拉伸下人膀胱平滑肌细胞（HBSMC）增殖的影响。
HBSMC 在硅橡胶膜上生长，在不同条件下施加拉伸：（等张伸长：2.5%、5%、10%、15%、20%、25%），（频率：0.05、0.1、0.2、0.5、1Hz）。通过 BrdU 检测和流式细胞术评估增殖情况，确定最佳生理拉伸条件。通过 Western blot 分析 PKC、JNK 和 p38 的表达水平。在某些实验中，使用特定抑制剂（PKC 的 GF109203X、JNK 的 SP600125 和 p38MAPK 的 SB203580）来保持特异性。
在 5%等张拉伸时观察到最佳增殖（BrdU：0.837±0.026（对照）至 1.462±0.023）%，（P<0.05），应用 0.1Hz 时细胞凋亡率从 16.4±0.21%（对照）降至 4.5±0.13%（P<0.05）。施加拉伸后，PKC 表达上调，JNK 略有增加，p38MAPK 无变化。抑制作用对增殖有影响（1.075±0.024，P<0.05 GF109203X）；（1.418±0.021，P>0.05 SP600125）和（1.461±0.01，P>0.05 SB203580）。这些发现表明，机械拉伸可以通过 PKC 并可能通过 JNK 但不是通过 p38MAPK 促进与人 HBSMC 增殖相关的幅度依赖性调节。
