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(Circulation Research. 2001;88:217.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Polymorphisms and Promoter Overactivity of the p22^phox Gene in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

Guillermo Zalba, Gorka San José, Francisco J. Beaumont, María A. Fortuño, Ana Fortuño, Javier Díez

From the Vascular Pathophysiology Unit, School of Medicine, University of Navarra, Pamplona, Spain.

Correspondence to Javier Díez, MD, PhD, Unidad de Fisiopatología Vascular, Facultad de Medicina, C/Irunlarrea s/n, 31080 Pamplona, Spain. E-mail jadimar{at}unav.es

Abstract—In a previous study, we found that the p22^phox 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 p22^phox gene are present in SHRs. To carry out this analysis, first of all, we characterized the rat gene structure and promoter region for the p22^phox subunit. The p22^phox 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 p22^phox gene does not contain a TATA box, but there are a CCAC box and putative recognition sites for nuclear factors, such as SP1, -interferon, and nuclear factor-B. Using reporter-gene transfection analysis, we found that this promoter was functional in VSMCs. Furthermore, we observed that p22^phox 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 p22^phox 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 p22^phox gene in rat VSMCs. Furthermore, our findings show that changes in the sequence of p22^phox gene promoter and in the degree of activation of VSMCs are responsible for upregulated expression of p22^phox in SHRs.

Key Words: NADH/NADPH oxidase • gene promoter • vascular smooth muscle cells • superoxide anion

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