Polymorphisms and promoter overactivity of the p22phox gene in vascular smooth muscle cells from spontaneously hypertensive rats

Abstract

In a previous study, we found that the p22phox subunit of the NADH/NADPH oxidase is overexpressed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) with enhanced vascular production of superoxide anion (O2-). Thus, we have investigated whether changes in the sequence or activity of the promoter region of p22phox gene are present in SHRs. To carry out this analysis, first of all, we characterized the rat gene structure and promoter region for the p22phox subunit. The p22phox gene spans ≈10 kb and contains 6 exons and 5 introns. Primer extension analysis indicated the transcriptional start site 100 bp upstream from the translational start site. The immediate promoter region of the p22phox gene does not contain a TATA box, but there are a CCAC box and putative recognition sites for nuclear factors, such as SP1, γ-interferon, and nuclear factor-κB. Using reporter-gene transfection analysis, we found that this promoter was functional in VSMCs. Furthermore, we observed that p22phox promoter activity was significantly higher in VSMCs from SHRs than from normotensive Wistar-Kyoto rats. In addition, we found that there were 5 polymorphisms in the sequence of p22phox promoter between Wistar-Kyoto rats and SHRs and that they were functional. The results obtained in this study provide a tool to explore the mechanisms that regulate the expression of p22phox gene in rat VSMCs. Furthermore, our findings show that changes in the sequence of p22phox gene promoter and in the degree of activation of VSMCs are responsible for upregulated expression of p22phox in SHRs.