Role of E2F transcriptional factor family in hydrogen peroxide regulation of dihydrofolate reductase in endothelial cells (855.5)

Abstract

Endothelial nitric oxide synthase (eNOS) uncoupling consequent to a deficiency in eNOS cofactor tetrahydrobiopterin (H4B) is an important mechanism of vascular dysfunction. Our previous work has shown that endothelial hydrogen peroxide (H2O2) overproduction specifically downregulates H4B salvage enzyme dihydrofolate reductase (DHFR), which mediates persistent H4B deficiency, eNOS uncoupling and elevated blood pressure in Angiotensin II‐infused mice. However, detailed molecular mechanisms underlying H2O2 regulation of DHFR in endothelial cells remain unclear. In this study, we found that hydrogen peroxide dose and time dependently downregulated DHFR protein expression and its enzyme activity in bovine aortic endothelial cells (BAECs). The DHFR mRNA levels were also decreased by H2O2, implicating transcriptional regulation. Based on the fact that DHFR promoter contains cis‐acting elements for Sp1 and E2F, we constructed wildtype DHFR promoter reporter construct (WT‐DHFR), and mutant DHFR promoter constructs that were deleted of either Sp1 or E2F binding sites. The dual‐luciferase reporter assay showed that while the promoter activities of both WT‐DHFR and Sp1 deletion mutant remained inhibited by H2O2, deletion of the E2F binding sites completely abolished H2O2 attenuation of DHFR promoter activity. RT‐PCR analyses indicated that H2O2 treatment resulted in marked downregulation of E2F1, E2F7 and E2F8 by 38%, 43% and 82% respectively. Intriguingly, overexpression of E2F1, E2F7 or E2F8 restored DHFR expression in response to H2O2, and cotransfection with E2F1 also significantly increased DHFR promoter activity. In conclusion, these results suggest that DHFR is downregulated by H2O2 in endothelial cells transcriptionally via a E2F‐dependent manner, and that restoration of E2F family‐transcription factor activity is effective in preserving DHFR function under oxidative stress.Grant Funding Source: Supported by NHLBI awards HL077440 (HC), HL088975 (HC) and an AHA EI award 12EIA8990025 (HC)