Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene.

In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.