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.

STAT1 represses hypoxia-inducible factor-1-mediated transcription.

Tumor hypoxia is associated with tumor promotion, malignant progression, and resistance to cancer therapy. The hypoxia-induced phenotypic changes in tumors result, at least partially, from the induction of hypoxia-responsive genes, such as chemokine receptor CXCR4. Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor involved in the transcriptional regulation of these genes. Although interferon-gamma (IFNgamma) exerts anti-tumor responses against various tumors, the effect of IFNgamma on HIF-1-dependent transcription remains to be determined. In this study, we examined the inhibitory effect of IFNgamma on hypoxia-induced CXCR4 gene expression in human glioblastoma cell lines and explored the mechanism of inhibition. Hypoxia (1% O(2)) and the iron chelator deferoxamine (DFX), a hypoxia mimetic, increased the levels of CXCR4 mRNA in A172 and T98G cells, and treatment with IFNgamma inhibited the expression of CXCR4 mRNA. Although hypoxia and DFX induced the expression of HIF-1alpha protein and its hypoxia response element (HRE) DNA-binding activity, IFNgamma failed to inhibit its expression or DNA-binding activity. The results of a luciferase reporter assay using a heterologous promoter construct containing the HRE sequence revealed that IFNgamma suppressed the hypoxia- and DFX-induced reporter activities. Lentivirus-mediated RNAi of signal transducer and activator of transcription 1 (STAT1) expression abolished the inhibitory effect of IFNgamma on hypoxia-induced reporter gene activity. Furthermore, this activity was not detected in a stable cell line expressing dominant-negative STAT1. These results indicate that IFNgamma-activated STAT1 functions as a negative regulator of HIF-1-dependent transcription in tumor cells.

Mechanisms of resistance to interferon-gamma-mediated cell growth arrest in human oral squamous carcinoma cells.

Interferon-gamma (IFNgamma) has an antiproliferative effect on a variety of tumor cells. However, many tumor cells resist treatment with IFNs. Here, we show that IFNgamma fails to inhibit the growth of some types of oral squamous cell carcinoma (OSCC) cells that possess a fully functional IFNgamma/STAT1 (signal transducer and activator of transcription-1) signaling pathway. IFNgamma inhibited the growth of the HSC-2, HSC-3, and HSC-4 OSCC cell lines. However, Ca9-22 cells were resistant to IFNgamma despite having intact STAT1-dependent signaling, such as normal tyrosine phosphorylation, DNA binding activity, and transcriptional activity of STAT1. The growth inhibition of HSC-2 cells resulted from S-phase arrest of the cell cycle. IFNgamma inhibited cyclin A2 (CcnA2)-associated kinase activity, which correlated with the IFNgamma-mediated down-regulation of CcnA2 and Cdk2 expression at both the transcriptional and post-transcriptional level in HSC-2 cells but not in Ca9-22 cells. RNAi-mediated knockdown of CcnA2 and Cdk2 resulted in growth inhibition in both cell lines. These results indicate that the resistance of OSCC to IFNgamma is not due simply to the deficiency in STAT1-dependent signaling but results from a defect in the signaling component that mediates this IFNgamma-induced down-regulation of CcnA2 and Cdk2 expression at the transcriptional and post-transcriptional levels.

Sulindac, a nonsteroidal anti-inflammatory drug, selectively inhibits interferon-gamma-induced expression of the chemokine CXCL9 gene in mouse macrophages.

Sulindac, a non-steroidal anti-inflammatory drug, has been shown to exert an anti-tumor effect on several types of cancer. To determine the effect of sulindac on intracellular signaling pathways in host immune cells such as macrophages, we investigated the effect of the drug on interferon gamma (IFNgamma)-induced expression of signal transducer and activator of transcription 1 (STAT1) and other genes in mouse macrophage-like cell line RAW264.7 cells. Sulindac, but not aspirin or sodium salicylate, inhibited IFNgamma-induced expression of the CXC ligand 9 (CXCL9) mRNA, a chemokine for activated T cells, whereas the interferon-induced expression of CXCL10 or IFN regulatory factor-1 was not affected by sulindac. Luciferase reporter assay demonstrated that sulindac inhibited IFNgamma-induced promoter activity of the CXCL9 gene. Surprisingly, sulindac had no inhibitory effect on IFNgamma-induced STAT1 activation; however, constitutive nuclear factor kappaB activity was suppressed by the drug. These results indicate that sulindac selectively inhibited IFNgamma-inducible gene expression without inhibiting STAT1 activation.