[Identification of PKC isozymes and effect of knockdown of PKC alpha by antisense oligodeoxynucleotide on iNOS expression via interleukin-1 receptor in vascular smooth muscle cells].
Protein kinase C (PKC) family, is now classified into three groups; conventional (cPKC), novel (nPKC) and atypical (aPKC), and to date, 10 members of isozymes have been identified. We have suggested that PKC is essential to interleukin-1 (IL-1)-triggered expression of inducible NO synthase (iNOS), and that by pharmacological analysis, cPKC is not involved in iNOS induction in rat vascular smooth muscle cells (VSMC). In the present study, we identified some PKC isozymes and investigated the effect of PKC alpha knockdown by antisense oligodeoxynucleotide (AS-ODN) strategy on iNOS expression and nuclear translocation of NF-kappa B in RASMC. Western blot analysis revealed the presence of cPKC (alpha), nPKCs (delta and epsilon) and aPKCs (tau and lambda). Short-time (10-20 min) treatment with phorbol 12-myristate 13-acetate (PMA) induced translocation of PKC alpha from cytosolic to particulate fraction. PKC alpha was completely downregulated by treatment with 100 nM PMA for 24 hours. Treatment with AS-ODN against PKC alpha mRNA depleted PKC alpha specifically, and had no detectable effect on the other PKCs. The production of iNOS mRNA, but not nuclear translocation of NF-kappa B, stimulated by IL-1 beta was decreased by PKC alpha knockdown. These results suggest that there are 5 PKC isozymes in RASMC, and that PKC alpha is involved in iNOS expression triggered by IL-1 beta, supporting our previous pharmacological conclusion.
Hisayama
T
T
Dept. of Pharmacology, Fac. of Pharmaceutical Sciences, Univ. of Tokushima.
Inomoto
M
M
Hioki
Y
Y
Fukui
H
H
D000818:Animals; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000302:isolation & purification; D004790:Enzyme Induction; D007375:Interleukin-1 / Q000502:physiology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000502:physiology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D016328:NF-kappa B / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000032:analysis*; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011493:Protein Kinase C / Q000302:isolation & purification* / Q000502:physiology*; D051571:Protein Kinase C-alpha; D051381:Rats; D017208:Rats, Wistar; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007375:Interleukin-1; D007527:Isoenzymes; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; C061570:protein kinase N; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate