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

AIM: To investigate the pro-apoptotic role of tumor necrosis factor a (TNF- alpha) in cultured bovine aortic endothelial 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 quantifi cation of apoptotic cells were determined under fluorescence microscope aft er TNF-alpha treated BAEC for 24 h with Hoechst 33258 staining. Cell viabil ity was determined with MTT method. DNA fragmentation was visualized by aga rose gel electrophoresis. The expression of phospho-p38 and phospho-p44/42 Ca2+-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) was measured by Western blotting. RESULTS: TNF-alpha elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DN A fragmentation. At 1000-5000 kU/L, incubation with TNF-alpha for 24 h indu ced BAEC apoptosis and both of phospho-p38 and phospho-p44/42 CCDPK express ion in a concentration-dependent manner. Interestingly, TNF-alpha -stimulat ed activation of p44/42 CCDPK was completely blocked, TNF-alpha -induced ap optosis was markedly increased by preincubation with U0126, a specific p44/ 42 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, comp letely blocked TNF-alpha -stimulated activation of p38 CCDPK, and enhanced the expresssion of phospho-p44/42 CCDPK induced by TNF-alpha, substantially inhibited the pro-apoptotic effect of TNF-alpha. CONCLUSION: TNF-alpha sim ultaneously activates p38 CCDPK and P44/42 CCDPK, and these two CCDPK signa ling pathways appeared to play opposing roles in TNF-alpha -induced apoptos is in BAEC.