Positive regulation of the NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F.
The generation of reactive oxygen species (ROS) by the NOX family of NADPH oxidases is known to be involved in the regulation of many physiological cellular functions. Unlike other members of this family, NOX4 generates ROS constitutively without the need for a stimulus. The activity of NOX4 is known to be regulated, at least in part, at the level of mRNA expression. However, nothing is known of the molecular mechanisms which underlie its transcriptional regulation. We have therefore determined the transcriptional initiation site of NOX4 in vascular smooth muscle cells (VSMC) and identified NOX4 genomic sequences necessary to effect high levels of expression of a linked luciferase reporter gene in both rat and mouse VSMCs. A potential binding site for members of the E2F family of transcription factors was identified, and electrophoretic mobility-shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays confirmed that this site binds E2F1 both in vitro and in vivo. siRNA against E2F1 decreased NOX4 promoter activity, while site-specific mutation of the core-binding site both downregulated the NOX4 promoter and abolished transregulation by E2F1. These data therefore demonstrate that E2F factor(s) are positive regulators of NOX4 transcription in VSMCs.
Cardiovascular Division, King's College London British Heart Foundation Centre, James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK.
Li
L
Zhang
Olivia R
OR
Sheppard
Ajay M
AM
Shah
Alison C
AC
Brewer
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D050684:E2F Transcription Factors / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000235:genetics
D050684:E2F Transcription Factors; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; C498911:Nox4 protein, rat