Estrogen prevents bovine aortic endothelial cells from TNF-alpha-induced apoptosis via opposing effects on p38 and p44/42 CCDPK.
OBJECTIVE
To investigate the effect of 17beta-estradiol (E2) on TNF-alpha-induced apoptosis in cultured bovine aortic endothelial cells (BAEC) and the underlied mechanism.


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 with Hoechst 33258 staining. Cell viability was detected 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) was measured by Western blotting.


RESULTS
TNF-alpha 5000 kU/L elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation, and DNA fragmentation). E2 0.1 pmol/L-100 nmol/L enhanced the expression of phospho-p44/42 CCDPK induced by TNF-alpha, at the same time, inhibited TNF-alpha induced activation of p38 CCDPK. E2 protected BAEC from apoptosis induced by TNF-alpha in a concentration-dependent manner. DNA fragmentation induced by TNF-alpha 5000 kU/L was also reduced by E2 1 nmol/L. Both the E2-induced upregulation of phospho-p44/42 CCDPK and its anti-apoptotic action were prevented by the specific p44/42 CCDPK inhibitor U0126.


CONCLUSIONS
Activation of p44/42 CCDPK signaling together with inhibition of p38 CCDPK signaling by E2 appears to be an important mechanism for its survival effect on endothelial cells.
Liu
Wen-Lan
WL
Laboratory of Molecular Pharmacology, Xiangya College of Medicine, Central South University, Changsha 410078, China.
Guo
Xun
X
Guo
Zhao-Gui
ZG
D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000166:cytology*; D004958:Estradiol / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases
D014409:Tumor Necrosis Factor-alpha; D004958:Estradiol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases