Activation of extracellular signal-regulated kinases potentiates hemin toxicity in astrocyte cultures.
Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30-120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury.
Regan
R F
RF
Division of Emergency Medicine, Department of Surgery, Thomas Jefferson University, Philadelphia 19107, USA. Raymond.F.Regan@mail.tju.edu
Wang
Y
Y
Ma
X
X
Chong
A
A
Guo
Y
Y
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D001798:Blood Proteins / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D005419:Flavonoids / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D006427:Hemin / Q000633:toxicity*; D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010618:Phenanthrolines / Q000494:pharmacology; D011281:Pregnatrienes / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000590:Amino Acid Chloromethyl Ketones; D000975:Antioxidants; D001798:Blood Proteins; D002070:Butadienes; D015853:Cysteine Proteinase Inhibitors; D005419:Flavonoids; D005609:Free Radicals; D009570:Nitriles; D010618:Phenanthrolines; D011281:Pregnatrienes; D011480:Protease Inhibitors; D017382:Reactive Oxygen Species; C113580:U 0126; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D006427:Hemin; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse; C053356:U 74500A; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; C025205:1,10-phenanthroline