title: A far-upstream AP-1/Smad binding box regulates human NOX4 promoter activation by transforming growth factor-β.
abstract: NADPH oxidase 4 (NOX4) is a member of the NADPH oxidase gene family that regulates cellular differentiation, innate immunity and tissue fibrosis. Transforming growth factor-β (TGF-β1) is known to induce expression of NOX4 mRNA in mesenchymal cells. However, the mechanisms of transcriptional regulation of NOX4 are not well understood. In this study, we examined the transcriptional regulation of NOX4 in human lung fibroblasts by TGF-β1. Five promoter-reporter constructs containing DNA fragments of 0.74kb, 1.35kb, 1.84kb, 3.97kb and 4.76kb upstream from the transcriptional start site (TSS) of the human NOX4 gene were generated and their relative responsiveness to TGF-β1 analyzed. TGF-β1-induced NOX4 gene promoter activation requires a region between -3.97kb and -4.76kb. Bioinformatics analysis revealed a 15bp AP-1/Smad binding element in this region. Mutation or deletion of either the AP-1 or the Smad element attenuated TGF-β1 responsiveness of the -4.76kb NOX4 promoter. Furthermore, insertion of this AP-1/Smad box conferred TGF-β1 inducibility to the non-responsive -3.97kb NOX4 promoter construct. Chromatin immunoprecipitation analysis indicated that phospho-Smad3 and cJun associate with this element in a TGF-β1-inducible manner. These results demonstrate that the AP-1/Smad box located between 3.97kb and 4.76kb upstream of the TSS site of the NOX4 promoter is essential for NOX4 gene transcription induced by TGF-β1 in human lung fibroblasts. Our study provides insights into the molecular mechanisms of NOX4 gene expression, informing novel therapeutic approaches to interfere with upregulation of NOX4 in diseases characterized by activation of the TGF-β1/NOX4 pathway.
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Guangxing
authors.initials: G
authors.lastname: Bai
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hock
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Logsdon
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: vjthan@uab.edu.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Thannickal
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D020871:RNA Stability; D051900:Smad3 Protein / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D015533:Transcriptional Activation; D053773:Transforming Growth Factor beta1 / Q000502:physiology*
chemical_list: D016755:Proto-Oncogene Proteins c-jun; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D018808:Transcription Factor AP-1; D053773:Transforming Growth Factor beta1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human
keywords: NADPH Oxidase-4; Pulmonary fibrosis; TGF-β1