Anti-inflammatory cytokine interleukin-4 inhibits inducible nitric oxide synthase gene expression in the mouse macrophage cell line RAW264.7 through the repression of octamer-dependent transcription.
Inducible nitric oxide synthase (iNOS) is a signature molecule involved in the classical activation of M1 macrophages and is induced by the Nos2 gene upon stimulation with Th1-cell derived interferon-gamma (IFNγ) and bacterial lipopolysaccharide (LPS). Although the anti-inflammatory cytokine IL-4 is known to inhibit Nos2 gene expression, the molecular mechanism involved in the negative regulation of Nos2 by IL-4 remains to be fully elucidated. In the present study, we investigated the mechanism of IL-4-mediated Nos2 transcriptional repression in the mouse macrophage-like cell line RAW264.7. Signal transducer and activator of transcription 6 (Stat6) knockdown by siRNA abolished the IL-4-mediated inhibition of Nos2 induced by IFNγ/LPS. Transient transfection of a luciferase reporter gene containing the 5'-flanking region of the Nos2 gene demonstrated that an octamer transcription factor (OCT) binding site in the promoter region is required for both positive regulation by IFNγ/LPS and negative regulation by IL-4. Although IL-4 had no inhibitory effect on the DNA-binding activity of constitutively expressed Oct-1, IL-4-induced Nos2-reporter transcriptional repression was partially attenuated by overexpression of the coactivator CREB-binding protein (CBP). These results suggest that a coactivator/cofactor that functionally interacts with Oct-1 is a molecular target for the IL-4-mediated inhibition of Nos2 and that IL-4-activated Stat6 represses Oct-1-dependent transcription by competing with this coactivator/cofactor.
Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado 350-0283, Saitama, Japan.
Miki
M
Hiroi
Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado 350-0283, Saitama, Japan.
Yoshiichi
Y
Sakaeda
Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado 350-0283, Saitama, Japan.
Hana
H
Yamaguchi
Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado 350-0283, Saitama, Japan.
Yoshihiro
Y
Ohmori
D000818:Animals; D002478:Cells, Cultured; D007371:Interferon-gamma / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics*; D050811:Octamer Transcription Factor-1 / Q000502:physiology*; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D010750:Phosphoproteins / Q000502:physiology; D011401:Promoter Regions, Genetic; D050800:STAT6 Transcription Factor / Q000502:physiology
D008070:Lipopolysaccharides; D008565:Membrane Proteins; D050811:Octamer Transcription Factor-1; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C498709:Pag1 protein, mouse; D010750:Phosphoproteins; C494166:Pou2f1 protein, mouse; D050800:STAT6 Transcription Factor; C491012:Slc22a2 protein, mouse; C494100:Stat6 protein, mouse; D015847:Interleukin-4; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse