Role of protein kinase C beta2 activation in TNF-alpha-induced human vascular endothelial cell apoptosis.
The circulatory inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is increased in pathologic conditions that initiate or exacerbate vascular endothelial injury, such as diabetes. Protein kinase C (PKC) has been shown to play a critical role in TNF-alpha-induced human endothelial cell apoptosis. However, the relative roles played by specific isoforms of PKC in TNF-alpha-induced human endothelial cell apoptosis have not been addressed. We investigated the effects of a selective PKCbeta(2) inhibitor (CGP53353) on TNF-alpha-induced apoptosis in human vascular endothelial cells (cell line ECV304) and on the production of reactive oxygen species and nitric oxide, and compared its effects with rottlerin, a reagent that has been shown to reduce PKCdelta protein levels. Cultured human vascular endothelial cells (ECV304) were treated for 24 h with one of 4 regimes: 40 ng/mL TNF-alpha alone (TNF-alpha), TNF-alpha with 10 micromol/L rottlerin (T+rottlerin), TNF-alpha with 1 micromol/L CGP53353 (T+CGP), or untreated (control). Cell viability was measured by MTT assay, and cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with dramatic increases in production of intracellular hydrogen peroxide (approximately 20 times greater than control) and superoxide (approximately 16 times greater than control), as measured by dichlorofluorescein and dihydroethidium fluorescent staining, respectively. This increase was accompanied by reduced activity of superoxide dismutase and glutathione peroxidase and, subsequently, an increase in the lipid peroxidation product malondialdehyde. CGP53353, but not rottlerin, abolished or attenuated all these changes. We conclude that PKCbeta(2) plays a major role in TNF-alpha-induced human vascular endothelial cell apoptosis.
Wang
Fang
F
Anesthesiology Research Laboratory, Renmin Hospital, Wuhan University, Wuhan, Hubei, China.
Liu
Hui-Min
HM
Irwin
Michael G
MG
Xia
Zhong-Yuan
ZY
Huang
Zhiyong
Z
Ouyang
Jingping
J
Xia
Zhengyuan
Z
D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D004789:Enzyme Activation; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008315:Malondialdehyde / Q000032:analysis; D010797:Phthalimides / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D064546:Protein Kinase C beta; D013482:Superoxide Dismutase / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D010797:Phthalimides; D014409:Tumor Necrosis Factor-alpha; D008315:Malondialdehyde; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; C579054:PRKCB protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; C098874:4,5-dianilinophthalimide