Opposing effect of p38 CCDPK and p44/42 CCDPK signaling on TNF-α-induced apoptosis in bovine aortic endothelial cells  

p38和p44/42 CCDPK信号在TNF-α诱导牛主动脉内皮细胞凋亡中的相反作用(英文)

作　　者：刘文兰[1] 郭迅[1] 陈清泉 郭兆贵[1] 

机构地区：[1]湖南医科大学分子药理研究室,长沙中国410078

出　　处：《Acta Pharmacologica Sinica》2001年第5期25-30,共6页中国药理学报（英文版）

摘　　要：AIM: To investigate the pro-apoptotic role of tumor necrosis factor α (TNF-α) in cultured bovine aortic en-dothelial cells (BAEC) and its underlied apoptotic signaling pathways. METHODS: BAEC were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after TNF-a treated BAEC for 24 h with Hoechst 33258 staining. Cell viability was determined with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p44/42 Ca2 + -calmodulin dependent protein ki-nase (CCDPK, formerly called MAPK) was measured by Western blotting. RESULTS: TNF-α elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 1000- 5000 kU/L, incubation with TNF-α for 24 h induced BAEC apoptosis and both of phospho-p38 and phospho-p44/42 CCDPK expression in a concentration-dependent manner. Interestingly, TNF-α-stimulated activation of p44/42 CCDPK was completely blocked, TNF-α-induced apoptosis was markedly increased by preincubation with U0126, a specific p44/42 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, completely blocked TNF-α-stimulated activation of p38 CCDPK, and enhanced the expresssion of phos-pho-p44/42 CCDPK induced by TNF-α, substantially inhibited the pro-apoptotic effect of TNF-α. CONCLUSION: TNF-α simultaneously activates p38 CCDPK and p44/42 CCDPK, and these two CCDPK signaling path-ways appeared to play opposing roles in TNF-α-induced apoptosis in BAEC.目的:研究肿瘤坏死因子(TNF-α)诱导牛主动脉内皮细胞(BAEC)凋亡及其信号途径。方法:BAEC培养并传代于DMEM。经TNF-α处理24h后,Hoechst33258染色,荧光显微镜观察形态学变化及凋亡细胞计数。MIT法测定细胞活性,琼脂糖凝胶电泳分析DNA降解,Western blot法检测磷酸化p38和p44/42CCDPK表达。结果:TNF-α诱导BAEC产生典型的凋亡细胞形态学变化(核浓染,核碎裂)和DNA断片。TNF-α(100-5000kU/L)浓度依赖性诱导BAEC凋亡,并同时刺激磷酸化p44/42和p38CCDPK的表达.p44/42CCDPK抑制剂U0126可完全阻断TNF-α诱导的p44/42CCDPK的活化,显著增强TNF-α致凋亡作用;而p38 CCDPK抑制剂SB203580可完全阻断TNF-α诱导的p38CCDPK的活化,还可增强TNF-α诱导的磷酸化p44/42CCDPK的表达,明显抑制TNF-α促凋亡作用。结论:TNF-α同时激活p38和p44/42CCDPK,这两种CCDPK信号通路在TNF-α诱导BAEC凋亡中起相反作用。

关 键 词：apoptosis DNA fragmentation Ca2+- calmodulin dependent protein kinase tumor necrosis fac- tor Western blotting vascular endothelium cultured cells 

分 类 号：R363[医药卫生—病理学]