Myocyte enhancer factor 2B is involved in the inducible expression of NOX1/NADPH oxidase, a vascular superoxide-producing enzyme

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F-2 alpha and platelet-derived growth factor (PDGF). To clarify the molecular basis of this transcriptional activation, we delineated the promoter region of the NOX1 gene. RT-PCR and 5 '-rapid amplification of cDNA ends-based analyses revealed a novel 5 '-terminal exon of the rat NOX1 gene located approximately 28 kb upstream of the exon containing the start codon. Both PGF(2 alpha) and PDGF enhanced the transcriptional activity of the - 3.6 kb 5 '-flanking region of the NOX1 gene in A7r5 cells, a rat vascular smooth muscle cell line. A PGF(2 alpha)-response element was located between -146 and -125 in the 5 '-flanking region containing a consensus binding site for myocyte enhancer factor 2 (MEF2), to which binding of MEF2 was augmented by PGF(2 alpha). Gene silencing of MEF2B by RNA interference significantly suppressed the expression of NOX1, while silencing of activating transcription factor (ATF)-1, previously implicated in up-regulation of NOX1, abolished the PGF(2 alpha)- or PDGF-induced expression of MEF2B. These results indicate that superoxide production in vascular smooth muscle cells is regulated by the ATF-1-MEF2B cascade by induction of the expression of the NOX1 gene.