The regulation of inducible nitric oxide synthase gene expression induced by lipopolysaccharide and tumor necrosis factor-alpha in C6 cells: involvement of AP-1 and NFkappaB.
The roles of AP-1 and NFkappaB in the regulation of inducible nitric oxide synthase (iNOS) mRNA expression induced by the combination of lipopolysaccharide and tumor necrosis factor-alpha (LT) in C6 cells were examined in the present study. The iNOS mRNA level and NO release were increased by several cytokines alone or combination treatments at 24 hr. LT-induced iNOS mRNA level was maximally increased at 6 hr and maintained at higher level at least up to 24 hr. At 6 hr, iNOS protein level and NO release were also increased by LT. By western blot analysis, AP-1, such as Fra-1, Jun B, and phospho-CREB protein levels were increased by LT and translocation of NFkappaB p52 from the cytoplasm to the nucleus was increased. In addition, phosphorylations of MAPKs (ERK 1/2, p38, JNK 1/2) were increased by LT. LT-induced iNOS mRNA level was inhibited by PD98059 (MEK 1/2 inhibitor), SB203580 (p38 inhibitor), and cycloheximide (a protein synthesis blocker), indicating that the phosphorylation of ERK 1/2 and p38, and on-going protein synthesis are necessary for LT-induced iNOS expression. Electrophoretic mobility shift assay (EMSA) showed that AP-1 and NFkappaB DNA binding activities were increased at 6 hr and these AP-1 and NFkappaB DNA bands increased by LT were super-shifted when Fra-1, Jun B, or NFkappaB p50 antibody was coincubated. These findings strongly suggest that, in C6 cells, Fra-1, Jun B, NFkappaB p50, and NFkappaB p52 appear to be involved in the regulation of iNOS mRNA induced by LT.
Lee
Jin-Koo
JK
Department of Pharmacology, College of Medicine and Institute of Natural Medicine, Hallym University, 1 Okchun-Dong, Chunchon, Kangwon-Do 200-702, South Korea.
Choi
Seong-Soo
SS
Won
Je-Seong
JS
Suh
Hong-Won
HW
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007093:Imidazoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D020928:Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one