Activation of Protein Kinase Cα and/or ε Enhances Transcription of the Human Endothelial Nitric Oxide Synthase Gene

Abstract

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.

Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

“…These data suggest that the PLC metabolite DAG activates the PKC, limiting the possible PKC isoforms engaged in the Ca 2+ entry as those that belong to the "classical" (activated by Ca 2+ and DAG) or "novel" family (activated by DAG but nor by Ca 2+ ). On EA.hy926 cells, the PKC␣ and PKC␤, from the "classical" family, as well as the PKC␦, PKC and PKC from the "novel" family are present [50]. Thus we applied inhibitors directed against different isoforms of PKC.…”

Red Wine Polyphenols Enhance Endothelial Nitric Oxide Synthase Expression and Subsequent Nitric Oxide Release From Endothelial Cells

“…11 Protein isolation and western blotting were performed essentially as described by Li et al 11 eNOS protein was detected by a monoclonal mouse anti-eNOS antibody (1:2500, BD Biosciences, Heidelberg, Germany, clone 3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by a monoclonal anti-GAPDH antibody (1:1200, Chemicon, Hofheim, Germany, MAB374) and visualized on a Kodak Image station 440CF using the chemiluminescence reagent Renaissance Plus (both NEN Life Science).…”

“…EA.hy926 cells stably transfected with a plasmid containing 3600 bp of the human eNOS promotor driving a luciferase gene 11 were stimulated as indicated for 18 hours. PMA (2 nmol/L) served as positive control.…”

“…PMA (2 nmol/L) served as positive control. 11 Cells were washed and lysed and the assay performed according to the manufacturer's instructions (Promega) with the use of a luminometer (AutoLumatPlus, Berthold).…”

“…Edgell, University of North Carolina, Chapel Hill, NC) 9 were cultivated as described. 10 EA.hy926 stably transfected with a plasmid containing 3600 bp of the human eNOS promotor controlling a luciferase reporter gene 11 were used for measuring eNOS promotor activity.…”