Silencing of the interferon-inducible gene Ifi204/p204 induces resistance to interferon-γ-mediated cell growth arrest of tumor cells.

Many tumor cells escape from cancer immunosurveillance and resist treatment with interferons (IFNs). Although the mechanism underlying IFN resistance is mostly attributed to a deficiency of components of the IFN-signaling pathway, some types of tumor cells resist IFN-mediated cell growth arrest despite the presence of an intact JAK/STAT signaling pathway. However, the molecular mechanisms underlying the unresponsiveness to IFNs independent of the defective JAK/STAT pathway remain to be clarified. To elucidate the mechanisms underlying IFNγ resistance, we examined the anti-proliferative effect of IFNγ on mouse tumor cell lines. Mouse squamous cell carcinoma (SCCVII) cells were resistant to IFNγ-mediated cell growth arrest despite the presence of the IFNγ-induced STAT1-dependent signaling pathway, whereas IFNγ inhibited cell growth of B16/F1 cells, a well-known IFNγ-sensitive mouse melanoma cell line, at the G1 phase of the cell cycle. Treatment of SCCVII cells with IFNγ neither downregulated the expression of cyclin D1, cyclin A2, and cyclin E1 nor induced a hypo-phosphorylated, active form of retinoblastoma protein (pRb). Interestingly, the hyper-phosphorylated, inactive form of pRb was exclusively localized in the cytoplasm in SCCVII cells. The IFN-inducible 204 gene (Ifi204), whose gene product, p204, binds to pRb and exerts an anti-proliferative effect, was repressed in SCCVII cells. p204 overexpression in SCCVII significantly inhibited cell growth, and mutation of a pRb-binding LXCXE motif decreased the anti-proliferative effect. These results suggest that silencing of Ifi204/p204 induces resistance to IFNγ-mediated cell growth arrest in SCCVII cells.

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.

Irsogladine maleate ameliorates inflammation and fibrosis in mice with chronic colitis induced by dextran sulfate sodium.

Intestinal fibrosis is a common and severe complication of inflammatory bowel disease (IBD), especially Crohn's disease (CD). To investigate the therapeutic approach to intestinal fibrosis, we have developed a mouse model of intestinal fibrosis by administering dextran sulfate sodium (DSS) and examining the effects of irsogladine maleate (IM) [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], which has been widely used as an antiulcer drug for gastric mucosa in Japan, on DDS-induced chronic colitis. In this experimental colitis lesion, several pathognomonic changes were found: increased deposition of collagen, increased number of profibrogenic mesenchymal cells such as fibroblasts (vimentin(+), α-SMA(-)) and myofibroblasts (vimentin(+), α-SMA(+)) in both mucosa and submucosa of the colon with infiltrating inflammatory cells, and increased mRNA expressions of collagen type I, transforming growth factor (TGF)-ß, matrix metalloproteinase (MMP)-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1. When IM was administered intrarectally to this colitis, all these pathological changes were significantly decreased or suppressed, suggesting a potential adjunctive therapy for intestinal fibrosis. IM could consequently reduce fibrosis in DSS colitis by direct or indirect effect on profibrogenic factors or fibroblasts. Therefore, the precise effect of IM on intestinal fibrosis should be investigated further.

Analysis of intestinal fibrosis in chronic colitis in mice induced by dextran sulfate sodium.

Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in intestinal fibrosis, however, their precise role is largely unknown. To investigate their role in intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5 days developed acute colitis that progressed to severe chronic inflammation with dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of proinflammatory cytokines are correlated with extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-ß, and matrix metalloproteinases were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin(+), α-smooth muscle actin (α-SMA)(-)) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin(+), α-SMA(+)) were increased in mucosal but not in submucosal layers. Primary mouse subcutaneous fibroblast cultures experiments revealed that exogenously added TGF-ß 1 substantially augmented the expressions of both vimentin and α-SMA proteins with increased production of collagen. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in this chronic DSS-induced colitis model.

Simultaneous Expression of Th1- and Treg-Associated Chemokine Genes and CD4+, CD8+, and Foxp3+ Cells in the Premalignant Lesions of 4NQO-Induced Mouse Tongue Tumorigenesis.

Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks. Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.