title: Molecular mechanisms in H2O2-induced increase in AT1 receptor gene expression in cardiac fibroblasts: A role for endogenously generated Angiotensin II.
abstract: The AT1 receptor (AT1R) mediates the manifold actions of angiotensin II in the cardiovascular system. This study probed the molecular mechanisms that link altered redox status to AT1R expression in cardiac fibroblasts. Real-time PCR and western blot analysis showed that H2O2 enhances AT1R mRNA and protein expression via NADPH oxidase-dependent reactive oxygen species induction. Activation of NF-κB and AP-1, demonstrated by electrophoretic mobility shift assay, abolition of AT1R expression by their inhibitors, Bay-11-7085 and SR11302, respectively, and luciferase and chromatin immunoprecipitation assays confirmed transcriptional control of AT1R by NF-κB and AP-1 in H2O2-treated cells. Further, inhibition of ERK1/2, p38 MAPK and c-Jun N-terminal kinase (JNK) using chemical inhibitors or by RNA interference attenuated AT1R expression. Inhibition of the MAPKs showed that while ERK1/2 and p38 MAPK suffice for NF-κB activation, all three kinases are required for AP-1 activation. H2O2 also increased collagen type I mRNA and protein expression. Interestingly, the AT1R antagonist, candesartan, attenuated H2O2-stimulated AT1R and collagen mRNA and protein expression, suggesting that H2O2 up-regulates AT1R and collagen expression via local Angiotensin II generation, which was confirmed by real-time PCR and ELISA. To conclude, oxidative stress enhances AT1R gene expression in cardiac fibroblasts by a complex mechanism involving the redox-sensitive transcription factors NF-κB and AP-1 that are activated by the co-ordinated action of ERK1/2, p38 MAPK and JNK. Importantly, by causally linking oxidative stress to Angiotensin II and AT1R up-regulation in cardiac fibroblasts, this study offers a novel perspective on the pathogenesis of cardiovascular diseases associated with oxidative stress.
authors.affiliation: Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum 695011, Kerala, India.
authors.forename: V
authors.initials: V
authors.lastname: Anupama
authors.affiliation: Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum 695011, Kerala, India.
authors.forename: Mereena
authors.initials: M
authors.lastname: George
authors.affiliation: Neuro Stem Cell Biology, Neurobiology Division, Rajiv Gandhi Center for Biotechnology, Trivandrum 695014, Kerala, India.
authors.forename: Sivadasan Bindu
authors.initials: SB
authors.lastname: Dhanesh
authors.affiliation: Bacterial and Parasite Disease Biology, Tropical Disease Biology, Rajiv Gandhi Center for Biotechnology, Trivandrum 695014, Kerala, India.
authors.forename: Aneesh
authors.initials: A
authors.lastname: Chandran
authors.affiliation: Neuro Stem Cell Biology, Neurobiology Division, Rajiv Gandhi Center for Biotechnology, Trivandrum 695014, Kerala, India.
authors.forename: Jackson
authors.initials: J
authors.lastname: James
authors.affiliation: Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum 695011, Kerala, India. Electronic address: shivak@sctimst.ac.in.
authors.forename: K
authors.initials: K
authors.lastname: Shivakumar
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D005786:Gene Expression Regulation / Q000187:drug effects*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D008954:Models, Biological; D058628:Myofibroblasts / Q000187:drug effects* / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D017382:Reactive Oxygen Species; D044140:Receptor, Angiotensin, Type 1; D018808:Transcription Factor AP-1; D000804:Angiotensin II; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: AT1R; Angiotensin II; Cardiac fibroblasts; Collagen; Oxidative stress