Yasumasa Minoda

IFNγ-dependent, spontaneous development of colorectal carcinomas in SOCS1-deficient mice

Approximately 20% of human cancers are estimated to develop from chronic inflammation. Recently, the NF-κB pathway was shown to play an essential role in promoting inflammation-associated cancer, but the role of the JAK/STAT pathway, another important signaling pathway of proinflammatory cytokines, remains to be investigated. Suppressor of cytokine signaling-1 (SOCS1) acts as an important physiological regulator of cytokine responses, and silencing of the SOCS1 gene by DNA methylation has been found in several human cancers. Here, we demonstrated that SOCS1-deficient mice (SOCS1−/−Tg mice), in which SOCS1 expression was restored in T and B cells on a SOCS1−/− background, spontaneously developed colorectal carcinomas carrying nuclear β-catenin accumulation and p53 mutations at 6 months of age. However, interferon (IFN)γ−/−SOCS1−/− mice and SOCS1−/−Tg mice treated with anti-IFNγ antibody did not develop such tumors. STAT3 and NF-κB activation was evident in SOCS1−/−Tg mice, but these were not sufficient for tumor development because these are also activated in IFNγ−/−SOCS1−/− mice. However, colons of SOCS1−/−Tg mice, but not IFNγ−/−SOCS1−/− mice, showed hyperactivation of STAT1, which resulted in the induction of carcinogenesis-related enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. These data strongly suggest that SOCS1 is a unique antioncogene which prevents chronic inflammation-mediated carcinogenesis by regulation of the IFNγ/STAT1 pathways.

Peptidoglycan and lipopolysaccharide activate PLCγ2, leading to enhanced cytokine production in macrophages and dendritic cells

In macrophages and monocytes, microbial components trigger the production of pro‐inflammatory cytokine through Toll‐like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram‐positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram‐positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cγ‐2 (PLCγ2), leading to intracellular free Ca2+ mobilization in bone marrow‐derived macrophages (BMMφ) and bone marrow‐derived dendritic cells (BMDC). PGN‐ and LPS‐induced Ca2+ mobilization was not observed in BMDC from PLCγ2 knockout mice. Thus, PLCγ2 is essential for TLR2 and TLR4‐mediated Ca2+ flux. In PLCγ2‐knockdown cells, PGN‐induced IκB‐α phosphorylation and p38 activation were reduced. Moreover, PLCγ2 was necessary for the full production of TNF‐α and IL‐6. These data indicate that the PLCγ2 pathway plays an important role in bacterial ligands‐induced activation of macrophages and dendritic cells.

TGF‐β1 suppresses IFN‐γ‐induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation

TGF‐β1 is a well‐known immunosuppressive cytokine; however, little is known of the effect of TGF‐β1 on antigen‐presenting cells (APCs). In this report, we investigated the molecular mechanisms of the suppressive effects of TGF‐β1 on APCs including dendritic cells and macrophages. Although TGF‐β1 did not greatly affect the activation of APCs, as assessed by the induction of IL‐12 or the upregulation of CD40 in response to LPS, it strongly inhibited IFN‐γ‐induced nitric oxide (NO) production from macrophages and dendritic cells. Using murine macrophage‐like cell line RAW 264.7, we demonstrated that TGF‐β1 not only reduced the inducible NO synthase (iNOS) protein stability but also suppressed the iNOS gene transcription. We also found that TGF‐β1 directly inhibited IFN‐γ‐induced STAT1 activation by reducing STAT1 tyrosine phosphorylation. The IFN‐γ Type I receptor (IFNGR1) was found to be associated with the TGF‐β1 Type I receptor (TGF‐βRI) and was phosphorylated by the TGF‐βRI. Reduced activation of STAT1 by TGF‐β1 was abrogated by the mutation in the IFNGR1 in which the serine residues of potential sites of phosphorylation by TGF‐βRI were replaced by alanine residues. Thus, multiple mechanisms are present for the TGF‐β1‐mediated reduction of iNOS production, and we propose a novel mechanism for regulating inflammatory cytokine by an anti‐inflammatory cytokine, TGF‐β1; i.e. suppression of IFN‐γ‐induced STAT1 activation by an association of the IFNGR1 with the TGF‐βRI.

FLN29, a Novel Interferon- and LPS-inducible Gene Acting as a Negative Regulator of Toll-like Receptor Signaling *

Lipopolysaccharide (LPS) activates macrophages through toll-like receptor (TLR) 4. Although the mechanism of the TLR signaling pathway has been well documented, the mechanism of the negative regulation in response to LPS, particularly LPS tolerance, is still poorly understood. In this study we identified and characterized a novel interferon- and LPS-inducible gene, FLN29, which contains a TRAF6-related zinc finger motif and TRAF family member-associated NF-κB activator-related sequences. The induction of FLN29 was dependent on STAT1. The forced expression of FLN29 in macrophage-like RAW cells resulted in the suppression of TLR-mediated NF-κB and mitogen-activated protein kinase activation, while a reduced expression of FLN29 by small interfering RNA partly cancelled the down-regulation of LPS signaling. Furthermore, we demonstrated that NF-κB activation induced by TRAF6 and TAB2 was impaired by co-expression of FLN29, suggesting FLN29 may regulate the downstream of TRAF6. Taken together, FLN29 is a new negative feedback regulator of TLR signaling.

Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages

In macrophages and monocytes, lipopolysaccharide (LPS) triggers the production of pro‐inflammatory cytokine through Toll‐like receptor (TLR) 4. Although major TLR signalling pathways are mediated by serine or threonine kinases including IKK, TAK1, p38 and JNKs, a number of reports suggested that tyrosine phosphorylation of intracellular proteins is involved in LPS signalling. Here, we identified several tyrosine‐phosphorylated proteins using mass spectrometric analysis in response to LPS stimulation. Among these proteins, we characterized C‐terminal Src kinase (Csk), which negatively regulates Src‐like kinases in RAW 264.7 cells using RNAi knockdown technology. Unexpectedly, LPS‐induced CD40 activation and the secretion of pro‐inflammatory cytokine such as IL‐6 and TNF‐α, was down‐regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4‐mediated activation of IκB‐α, Erk and p38 but not of JNK, were also down‐regulated in Csk knockdown cells. The protein expression levels of a tyrosine kinase, Fgr, were reduced in Csk knockdown cells, suggesting that Csk is a critical regulator of TLR4‐mediated signalling by modifying the levels of Src‐like kinases.

An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1β signaling pathway

TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1beta and TNFalpha signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1beta-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1beta-dependent manner. Overexpression of FBXW5 inhibited IL-1beta-induced activation of JNK/p38 MAPKs and NF-kappaB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1beta stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1beta signaling pathway.