Interactions between p38 mitogen-activated protein kinase and caspase-3 in cerebral endothelial cell death after hypoxia-reoxygenation.
OBJECTIVE
The emerging concept of the neurovascular unit in stroke reemphasizes the need to focus on endothelial responses in brain. In this study we examined the role of mitogen-activated protein (MAP) kinase signaling in the regulation of hypoxic cell death in cerebral endothelial cells.


METHODS
Human cerebral microvascular endothelial cells were exposed to 4 to 12 hours of hypoxia followed by 12 to 24 hours of reoxygenation. Cytotoxicity was measured by quantifying lactate dehydrogenase release. DNA laddering and caspase-3 activity were assessed to document a role for caspase-dependent cell death. zVAD-fmk and zDEVD-fmk were used to inhibit caspases. Activation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. U0126, SB203580, and SP600125 were used to interrupt the ERK, p38, and JNK pathways, respectively.


RESULTS
Endothelial cell death occurred primarily during reoxygenation. DNA laddering and caspase activation were observed, and cytotoxicity was ameliorated by caspase inhibitors (20 micromol/L of zVAD-fmk or zDEVD-fmk). Among the 3 major MAP kinases, only p38 was transiently activated during reoxygenation, and inhibition with 10 micromol/L of SB203580 significantly reduced cytotoxicity. No effects were observed with other MAP kinase inhibitors. Cytoprotection with SB203580 was not accompanied by caspase downregulation. In contrast, cytoprotection with zVAD-fmk was associated with a decrease in p38 activation. Furthermore, cleavage of MEKK1 (an upstream kinase of p38) was significantly reduced by zVAD-fmk.


CONCLUSIONS
Cerebral endothelial cell death after hypoxia-reoxygenation is mediated by interactions between caspases and p38 MAP kinase. Surprisingly, p38 pathways lie downstream of caspase mechanisms in this model system.
Lee
Sun-Ryung
SR
Neuroprotection Research Laboratory, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass 02129, USA.
Lo
Eng H
EH
D001921:Brain / Q000098:blood supply* / Q000166:cytology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases