title: Transcriptional regulation of the NADPH oxidase isoform, Nox1, in colon epithelial cells: role of GATA-binding factor(s).
abstract: Nonphagocytic NADPH oxidases (Noxs) are major sources of reactive oxygen species (ROS) and exist as a family of isoenzymes with tissue-restricted expression and functions. Nox1, expressed in colon epithelium and vascular smooth muscle, is suggested to be involved in innate immune defense and cell growth or proliferation. The transcriptional regulation of Nox1 appears to be particularly important in the modulation of its activity but the underlying mechanisms are unknown. Here we have identified the functional Nox1 promoter in human colon epithelial Caco-2 cells, and show that a 520-bp genomic fragment encompassing the CAP site is sufficient to direct high levels of expression of a linked reporter gene in these cells. Deletion analyses together with electrophoretic mobility-shift assays (EMSAs) suggest that maximal promoter activity is dependent on a GATA-binding site, conserved between human and mouse, within the proximal promoter region. The ability of mouse GATA factors to transactivate the Nox1 promoter was demonstrated in Cos-7 cells and site-directed mutagenesis of the conserved GATA-binding site further demonstrates that the regulation of Nox1 transcription is mediated by the direct binding of a GATA factor to the Nox1 proximal promoter. We also identified more distal, upstream regions which act to repress significantly expression from the Nox1 promoter.
authors.affiliation: King's College London, Department of Cardiology, GKT School of Medicine and Dentistry, New Medical School Building, Bessemer Road, London SE5 9PJ, UK. alison.brewer@kcl.ac.uk
authors.forename: Alison C
authors.initials: AC
authors.lastname: Brewer
authors.forename: Emma C
authors.initials: EC
authors.lastname: Sparks
authors.forename: Ajay M
authors.initials: AM
authors.lastname: Shah
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018938:Caco-2 Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D050991:GATA4 Transcription Factor / Q000378:metabolism* / Q000494:pharmacology; D050992:GATA5 Transcription Factor / Q000378:metabolism* / Q000494:pharmacology; D050993:GATA6 Transcription Factor / Q000378:metabolism* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D050991:GATA4 Transcription Factor; D050992:GATA5 Transcription Factor; D050993:GATA6 Transcription Factor; C484171:Gata4 protein, mouse; C494756:Gata5 protein, mouse; C494760:Gata6 protein, mouse; D007527:Isoenzymes; D012333:RNA, Messenger; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human