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


CONCLUSIONS
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.
Liu
W L
WL
Laboratory of Molecular Pharmacology, Hu-nan Medical University, Changsha 410078, China.
Guo
X
X
Chen
Q Q
QQ
Guo
Z G
ZG
D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis; D002070:Butadienes / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases
D002070:Butadienes; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009570:Nitriles; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; C113580:U 0126; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580