Opposing effect of p38 CCDPK and p44/42 CCDPK signaling on TNF-alpha-induced apoptosis in bovine aortic endothelial cells
Abstract
Aim: To investigate the pro-apoptotic role of tumor necrosis factor alpha (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 quantification of apoptotic cells were determined under fluorescence microscope after TNF-alpha 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 kinase (CCDPK, formerly called MAPK) was measured by Western blotting.
Results: TNF-alpha elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 1000-5000 kU/L, incubation with TNF-alpha for 24 h induced BAEC apoptosis and both of phospho-p38 and phospho-p44/42 CCDPK expression in a concentration-dependent manner. Interestingly, TNF-alpha-stimulated activation of p44/42 CCDPK was completely blocked, TNF-alpha-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-alpha-stimulated activation of p38 CCDPK, and enhanced the expression of phospho-p44/42 CCDPK induced by TNF-alpha, substantially inhibited the pro-apoptotic effect of TNF-alpha.
Conclusion: TNF-alpha simultaneously activates p38 CCDPK and p44/42 CCDPK, and these two CCDPK signaling pathways appeared to play opposing roles in TNF-alpha-induced apoptosis in BAEC.
Keywords:
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-alpha 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 kinase (CCDPK, formerly called MAPK) was measured by Western blotting.
Results: TNF-alpha elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 1000-5000 kU/L, incubation with TNF-alpha for 24 h induced BAEC apoptosis and both of phospho-p38 and phospho-p44/42 CCDPK expression in a concentration-dependent manner. Interestingly, TNF-alpha-stimulated activation of p44/42 CCDPK was completely blocked, TNF-alpha-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-alpha-stimulated activation of p38 CCDPK, and enhanced the expression of phospho-p44/42 CCDPK induced by TNF-alpha, substantially inhibited the pro-apoptotic effect of TNF-alpha.
Conclusion: TNF-alpha simultaneously activates p38 CCDPK and p44/42 CCDPK, and these two CCDPK signaling pathways appeared to play opposing roles in TNF-alpha-induced apoptosis in BAEC.