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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) F2α 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 PGF2α 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 PGF2α-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 PGF2α. 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 PGF2α- 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.