Hydrogen peroxide signaling mediates DHEA-induced vascular endothelial cell proliferation

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Dehydroepiandrosterone (DHEA) activates a putative plasma membrane G i-protein coupled receptor to induce vascular endothelial proliferation. We now test the hypothesis that hydrogen peroxide (H 2O 2) signaling mediates this effect. Incubation of EA.hy926 cells, a human vascular endothelial cell line, with DHEA for 5 min produced a significant increase in H 2O 2 production, measured by oxidation of either p-hydroxyphenylacetate or dichlorodihydrofluorescein. The DHEA effect on H 2O 2 production was maximal at 1 nM DHEA, was evident within the first minute of incubation, and remained for 10 min. Similar results were present in primary bovine aortic endothelial cells. The induction of H 2O 2 in EA.hy926 cells was mimicked by a membrane-impermeable albumin-conjugated DHEA and was inhibited by either catalase or pertussis toxin. Incubation of endothelial cells with DHEA for 5 min resulted in a 2-fold increase of cyclin D1 mRNA and protein expression at 4 h. These effects were abolished by co-incubation with catalase. DHEA induced a 50 ± 7% increase in cell proliferation over 24 h, measured as cellular Ki-67 immunoreactivity. This proliferative effect was abolished by either catalase or pertussis toxin co-incubation, indicating an H 2O 2 and G i-protein-dependent effect. We conclude that H 2O 2 is a key signaling molecule mediating the proliferative effects of DHEA in vascular endothelial cells, possibly by up-regulating cell-cycle associated genes, such as cyclin D1.