title: Buthionine sulfoximine, an inhibitor of glutathione biosynthesis, induces expression of soluble epoxide hydrolase and markers of cellular hypertrophy in a rat cardiomyoblast cell line: roles of the NF-κB and MAPK signaling pathways.
abstract: Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy.
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.
authors.forename: Ghada
authors.initials: G
authors.lastname: Abdelhamid
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Electronic address: aelkadi@pharmacy.ualberta.ca.
authors.forename: Ayman O S
authors.initials: AO
authors.lastname: El-Kadi
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis; D002070:Butadienes / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D006332:Cardiomegaly / Q000473:pathology*; D002460:Cell Line; D002470:Cell Survival; D004789:Enzyme Activation; D004851:Epoxide Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D005978:Glutathione / Q000096:biosynthesis; D006333:Heart Failure / Q000473:pathology; D020672:I-kappa B Proteins / Q000096:biosynthesis / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D032386:Myoblasts, Cardiac / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D052002:NF-kappa B p50 Subunit / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D020097:Natriuretic Peptide, Brain / Q000096:biosynthesis; D009570:Nitriles / Q000494:pharmacology; D018384:Oxidative Stress; D010766:Phosphorylation; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D013859:Thiocarbamates / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000378:metabolism; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000975:Antioxidants; D002070:Butadienes; D005419:Flavonoids; D020672:I-kappa B Proteins; D052002:NF-kappa B p50 Subunit; C000606206:Nfkbia protein, rat; D009570:Nitriles; D012333:RNA, Messenger; C498145:Rela protein, rat; D013859:Thiocarbamates; D051996:Transcription Factor RelA; C113580:U 0126; C495963:natriuretic peptide precursor type B, rat; D020097:Natriuretic Peptide, Brain; C066229:prolinedithiocarbamate; D000072000:NF-KappaB Inhibitor alpha; D019328:Buthionine Sulfoximine; D009320:Atrial Natriuretic Factor; D011392:Proline; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D004851:Epoxide Hydrolases; C529022:EPHX2 protein, rat; D005978:Glutathione; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
keywords: MAPKs; NF-κB; sEH