Received for publication August 7, 2006.
Revised November 13, 2006.
Accepted for publication December 12, 2006.

4-Hydroxy-2-nonenal Adduction of Extracellular Signal-Regulated Kinase and the Inhibition of Hepatocyte Erk-Elk-AP-1 Signal Transduction

Abstract

4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation (LPO) product formed during oxidative stress. 4-HNE is highly reactive towards cellular nucleophiles and is implicated in the evolution of numerous pathologies associated with oxidative stress and LPO. Recent evidence suggests that chronic pro-oxidant exposure results in the loss of extracellular signal-regulated kinase 1/2 (Erk-1/2) phosphorylation in vivo, a signaling pathway associated with cellular proliferation, survival, and homeostasis. Immunodetection and molecular analysis were used in this study to evaluate the hypothesis that 4-HNE modification of Erk-1/2 inhibits constitutive Erk-Elk-AP1 signaling. Primary rat hepatocytes treated with sub-cytotoxic, pathologically relevant concentrations of 4-HNE demonstrated a concentration-dependent loss of constitutive Erk-1/2 phosphorylation, activity, and nuclear localization. These findings were consistent with iron-induced intracellular LPO, which also resulted in a concentration-dependent decrease in hepatocyte Erk-1/2 phosphorylation and activity. 4-HNE and iron-induced inhibition of Erk-1/2 were inversely correlated with the accumulation of 4-HNE-Erk-1/2 monomer adducts. 4-HNE treatment of hepatocytes decreased nuclear total and phosphorylated Erk-1/2, Elk-1 and AP-1 phosphorylation, as well as cFos and cJun activities. The cytosolic modification of unphosphorylated Erk-1/2 was evaluated in vitro using molar ratios of inactive Erk-2 to 4-HNE consistent with increasing oxidative stress in vivo. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) confirmed monomer-adduct formation and identified the major adduct species at the histidine 178 residue within the kinase phosphorylation lip. These novel results show that the formation of 4-HNE-Erk-1/2 monomer-adducts results in the inhibition of Erk-Elk-AP-1 signaling in hepatocytes, and implicates the His 178 residue with the mechanism of inhibition.

Key words: MAP Kinase, Mass Spectroscopy, Regulation - transcriptional, Alcohol, Oxidative stress/antioxidants, Reactive intermediates, Oxidative stress

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