ACTIVATION OF PROTEIN-KINASE-C-ALPHA AND OR PROTEIN-KINASE-C-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 bradyki nin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,1 3-dibutyrate stimulated endothelial n itric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) wa s observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisi ndolylmaleimide I (1 mu M), Go 6976 l-5-oxo-5H-indolo-[2,3-a]pyrrolo-[ 3,4-c]carbazole] (1 mu M), Ro-31-8220 o)propyl-1H-inoyl-3-yl]3-(1-meth yl-1H-indoyl-3-yl) maleimide methane sulfonate] (1 mu M), and cheleryt hrine (3 mu M) did not change NOS III expression when applied alone, b ut they all prevented the up-regulation of NOS III mRNA produced by PM A. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, de lta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsil on showed changes in protein expression after PMA treatment. Incubatio n of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation o f PKC alpha and PKC epsilon from the cytosol to the cell membrane, ind icating activation of these isoforms. After 24 hr of PMA incubation, b oth isoforms were down-regulated. The time course of activation and do wn-regulation of these two PKC isoforms correlated well with the PMA-s timulated 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 rep orter gene, PMA stimulated promoter activity up to 2.5-fold. On the ot her 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 a ctive phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.