Hydrogen Peroxide Regulates Osteopontin Expression through Activation of Transcriptional and Translational Pathways

• Type of Material: Article
• Authors: Alicia Lyle, Emory University; Ebony W. Remus, Emory University; Aaron Fan, Emory University; Bernard Lassegue, Emory University; Grant A. Walter, Emory University; Arihiro Kiyosue, University of Tokyo Hospital; Kathy Griendling, Emory University; W Robert Taylor, Emory University
• Language: English
• Date: 2014-01-03
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M113.489641
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 289
• Issue: 1
• Start Page: 275
• End Page: 285
• Grant/Funding Information: This work was supported, in whole or in part, by National Institutes of Health Grants P01 HL095070, R01 HL090584, and R01HL062820 (to W. R. T.).; This work was also supported by American Heart Association Post-doctoral Grant 11POST7570026 (to A. N. L.).
• Keywords: Transcription; DEFICIENT MICE; RECEPTOR; ESSENTIAL-HYPERTENSION; PROTEIN-SYNTHESIS; Translation; Osteopontin; Biochemistry & Molecular Biology; Science & Technology; INITIATION; Life Sciences & Biomedicine; ATHEROSCLEROSIS; SMOOTH-MUSCLE-CELLS; Reactive Oxygen Species (ROS); DEPENDENT TRANSLATION; PHOSPHORYLATION; Hydrogen Peroxide
• Research Categories: Biology, Physiology; Chemistry, Biochemistry

Abstract

Recent in vivo studies establish that osteopontin (OPN) expression is hydrogen peroxide (H2O2)-dependent. However, the mechanisms by which H2O2 increases OPN expression remain poorly defined. OPN protein expression increased in an unusual biphasic pattern in response to H2O2. To investigate whether these increases were mediated through transcriptional and/or translational regulation of OPN, smooth muscle cells stimulated with 50 μm H2O2 were used as an in vitro cell system. Early protein increases at 6 h were not preceded by increased mRNA, whereas later increases (18 h) were, suggesting multiple mechanisms of regulation by H2O2. Polyribosomal fractionation assays established that early increases (6 h) in OPN expression were due to increased translation. This increase in translation occurred through phosphorylation of 4E-BP1 at the reactive oxygen species-sensitive Ser-65, which allowed for release and activation of eukaryotic initiation factor eIF4E and subsequent OPN translation. This early increase (6 h) in OPN was blunted in cells expressing a phospho-deficient 4E-BP1 mutant. H2O2 stimulation increased rat OPN promoter activity at 8 and 18 h, and promoter truncation studies established that promoter region −2284 to −795 is crucial for H2O2-dependent OPN transcription. ChIP studies determined that H2O2-dependent transcription is mediated by the reactive oxygen species-sensitive transcription factors NF-κB and AP-1. In conclusion, H2O2 stimulates OPN expression in a unique biphasic pattern, where early increases are translational and late increases are transcriptional.