Activation of Mitogen-activated Protein Kinase by H2O2

Abstract

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress sig- nals. In this study we investigated the factors control- ling MAPK activation by H2O2 and explored the impact of altering the pathways to kinase activation on cell survival following H2O2 exposure. Potent activation (10–20-fold) of extracellular signal-regulated protein ki- nase (ERK2) occurred within 10 min of H2O2 treatment, whereupon rapid inactivation ensued. H2O2 activated ERK2in several cell types and also moderately activated (3–5-fold) both c-Jun N-terminal kinase and p38/RK/ CSBP. Additionally, H2O2 increased the mRNA expres- sion of MAPK-dependent genes c-jun,c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhib- ited H2O2 stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by H2O2 was blocked by pretreatment with eitherN-acetyl-cysteine, o-phenanthroline, or man- nitol, indicating that metal-catalyzed free radical forma- tion mediates the initiation of signal transduction by H2O2.H2O2-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to H2O2 toxicity. Moreover, NIH 3T3 cells expressing constitutively activeMAPKkinase (MEK, the immediate upstream regulator of ERK) were more re- sistant to H2O2 toxicity, while those expressing kinase- defective MEK were more sensitive, than cells express- ing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by H2O2 and suggest that ERK plays a critical role in cell survival following oxidant injury