PT  - JOURNAL ARTICLE
AU  - Huige Li
AU  - Silke A. Oehrlein
AU  - Thomas Wallerath
AU  - Irmgard Ihrig-Biedert
AU  - Paulus Wohlfart
AU  - Thomas Ulshöfer
AU  - Timm Jessen
AU  - Thomas Herget
AU  - Ulrich Förstermann
AU  - Hartmut Kleinert
TI  - Activation of Protein Kinase Cα and/or ε Enhances Transcription of the Human Endothelial Nitric Oxide Synthase Gene
AID  - 10.1124/mol.53.4.630
DP  - 1998 Apr 01
TA  - Molecular Pharmacology
PG  - 630--637
VI  - 53
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/53/4/630.short
4100  - http://molpharm.aspetjournals.org/content/53/4/630.full
SO  - Mol Pharmacol1998 Apr 01; 53
AB  - 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 μm), Gö 6976 [12-(2cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3,4-c]carbazole] (1 μm), Ro-31–8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H-indoyl-3-yl) maleimide methane sulfonate] (1 μm), and chelerythrine (3 μm) 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 (α, βI, δ, ε, η, ζ, λ, and μ), only PKCα and PKCε 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 PKCα and PKCε 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 PKCα, PKCε, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.