Abstract 699: Biphasic Regulation of Vascular NADPH Oxidase Subunit Nox4 by Protein Kinase C in Human Endothelial Cells
NADPH oxidases (Nox) are a major source of superoxide in the vascular wall. By phosphorylation of the cytosolic regulatory subunit p47phox, protein kinase C (PKC) increases the activity of the NADPH oxidase complex. Previous animal studies have demonstrated that increased PKC activity is also associated with an enhanced expression of NADPH oxidase subunits. Here we show that the regulation of NADPH oxidase gene expression by PKC is a complex event with a biphasic kinetics. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with the PKC activator phorbol-12-myristate-13-acetate (PMA, 10 and 100 nmol/L) for 6h led to a down-regulation of the gp91phox homolog Nox4 (to about 40% in response to 10 nmol/L PMA). This effect was reproducible with a second PKC activator, phorbol-12,13-dibutyrate (PDBu), but not with the inactive phorbol ester analog 4alpha-phorbol-12,13-didecanoate. These results indicate that the effects of PMA and PDBu were PKC-dependent. Accordingly, the reduction of Nox4 mRNA expression after a 6h-treatment with PMA was preventable with PKC inhibitors. Interestingly, the mRNA expression of Nox4 returned to normal levels after a 24h-incubation with 10 nmol/L PMA, and turned into an upregulation (about 4-fold) when the incubation time was extended to 48h. Human Nox4 mRNA had a half-life of about 6h in endothelial cells and PMA treatment did not change Nox4 mRNA stability. The Nox4 upregulation at 48h was associated with an enhanced, NADPH oxidase-dependent superoxide production. PMA activated the PKC isoforms epsilon and alpha in EA.hy 926 cells. Knockdown of PKC epsilon by siRNA prevented the initial Nox4 down-regulation at 6h, whereas knockdown of PKC alpha by siRNA abolished the late Nox4 up-regulation at 48h. Thus, the biphasic expression pattern of Nox4 in vascular endothelium in response to PKC activation seems to involve two PKC isoforms with PKC epsilon being responsible for the initial down-regulation and PKC alpha for the late up-regulation. Thus, a selective inhibition/knockdown of PKC alpha may represent a novel therapeutic strategy for the prevention of oxidative stress in the vasculature.