IFNgamma-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-kappaB 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 beta-catenin accumulation and p53 mutations at 6 months of age. However, interferon (IFN)gamma-/- SOCS1-/- mice and SOCS1-/- Tg mice treated with anti-IFNgamma antibody did not develop such tumors. STAT3 and NF-kappaB activation was evident in SOCS1-/- Tg mice, but these were not sufficient for tumor development because these are also activated in IFNgamma-/- SOCS1-/- mice. However, colons of SOCS1-/- Tg mice, but not IFNgamma-/- 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 IFNgamma/STAT1 pathways.

An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1beta 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.

Peptidoglycan and lipopolysaccharide activate PLCgamma2, 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 Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein.

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-kappaB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-kappaB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

[Regulation of cytokine and toll-like receptor signaling by SOCS family genes].

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through the Toll-like receptor (TLR) 4. Regulation of TLR signaling is a key step for inflammation, septic shock and innate/adaptive immunity. TLR signaling is shown to be regulated by cytokines, such as interferon-gamma (positive) and interleukin-10 and IL-4 (negative). However, molecular mechanisms of the regulation of LPS signaling by cytokines have not been clarified. Cytokine signaling is regulated by CIS/SOCS family proteins. Both SOCS1 and SOCS3 can inhibit JAK tyrosine kinase activity. We demonstrate that SOCS1 and SOCS3 play an important regulatory role in macrophages and dendritic cells (DCs) by modulating TLR signaling. SOCS1 negatively regulates not only the JAK/STAT pathway, but also the TLR-NF-kappaB pathway. SOCS3 protein was strongly induced by both IL-6 and IL-10 in the presence of LPS, but selectively inhibited IL-6 signaling. Therefore lack of SOCS3 gene in macrophages resulted in suppression of TLR signaling by hyperactivation of STAT3.

An RNA-binding protein alphaCP-1 is involved in the STAT3-mediated suppression of NF-kappaB transcriptional activity.

Signal transducer and activator of transcription 3 (STAT3) has been shown to mediate the anti-inflammatory effect of IL-10. Activated STAT3 suppresses LPS-induced IL-6, tumor necrosis factor-alpha and IL-12 gene expression in macrophages and dendritic cells. However, the mechanism of Toll-like receptor (TLR) signal suppression by STAT3 has not been clarified. In this study, we investigated the effect of constitutively activated STAT3 (STAT3C) on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The forced expression of STAT3C in HEK293/TLR4 cells, but neither wild-type STAT3 nor dominant-negative form of STAT3, suppressed LPS-TLR4-mediated NF-kappaB reporter activation. The over-expression of STAT3C did not affect the signal transduction of TLR4, such as the phosphorylation of inhibitory nuclear factor-kappaBalpha and mitogen-activated protein kinases and the DNA-binding activity of NF-kappaB. Thus, STAT3C could suppress the transcriptional and/or translational activity of NF-kappaB. To define the molecular mechanism, we searched STAT3C-binding proteins by using a proteomic approach and found that a novel RNA-binding protein, alphaCP-1, interacted with STAT3C. alphaCP-1 is a K-homology domain-containing RNA-binding protein with specificity for C-rich pyrimidine tracts. Such proteins play pivotal roles in a broad-spectrum of transcriptional and translational events. The over-expression of alphaCP-1 augmented the suppressive effect of STAT3C on NF-kappaB activation in HEK293/TLR4 cells. Furthermore, the forced expression of alphaCP-1 enhanced the antagonistic effect of IL-10 on IL-6 production in RAW264.7 cells, while small interfering RNA against alphaCP-1 reduced it. These data suggest that alphaCP-1 is involved in the STAT3-mediated suppression of NF-kappaB activity.

TGF-beta1 suppresses IFN-gamma-induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation.

TGF-beta1 is a well-known immunosuppressive cytokine; however, little is known of the effect of TGF-beta1 on antigen-presenting cells (APCs). In this report, we investigated the molecular mechanisms of the suppressive effects of TGF-beta1 on APCs including dendritic cells and macrophages. Although TGF-beta1 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-gamma-induced nitric oxide (NO) production from macrophages and dendritic cells. Using murine macrophage-like cell line RAW 264.7, we demonstrated that TGF-beta1 not only reduced the inducible NO synthase (iNOS) protein stability but also suppressed the iNOS gene transcription. We also found that TGF-beta1 directly inhibited IFN-gamma-induced STAT1 activation by reducing STAT1 tyrosine phosphorylation. The IFN-gamma Type I receptor (IFNGR1) was found to be associated with the TGF-beta1 Type I receptor (TGF-betaRI) and was phosphorylated by the TGF-betaRI. Reduced activation of STAT1 by TGF-beta1 was abrogated by the mutation in the IFNGR1 in which the serine residues of potential sites of phosphorylation by TGF-betaRI were replaced by alanine residues. Thus, multiple mechanisms are present for the TGF-beta1-mediated reduction of iNOS production, and we propose a novel mechanism for regulating inflammatory cytokine by an anti-inflammatory cytokine, TGF-beta1; i.e. suppression of IFN-gamma-induced STAT1 activation by an association of the IFNGR1 with the TGF-betaRI.

A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling.

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IkappaB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappaB activation. The N-terminal region (ZCCHC11S) including C2H2-type [corrected] Zn-finger motif was sufficient for suppression of NF-kappaB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappaB.

Deletion of the SOCS3 gene in liver parenchymal cells promotes hepatitis-induced hepatocarcinogenesis.

BACKGROUND & AIMS: A recent study has suggested that the methylation silencing of the suppressor of cytokine signaling-3 (SOCS3), a negative regulator of interleukin-6-related cytokines, could be involved in hepatocellular carcinoma (HCC). However, the roles of SOCS3 in hepatocellular carcinogenesis and hepatitis have not been established. We investigated the effect of deleting the SOCS3 gene on the development of hepatitis and HCC in hepatitis C virus-infected patients and mouse models. METHODS: The expression of SOCS genes in HCC and non-HCC regions of patient samples was determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The conditional knockout approach in mice was used to determine the hepatocyte-specific roles of SOCS3. To generate a liver-specific deletion, floxed SOCS3 (SOCS3(fl/fl)) mice were crossed with albumin-Cre transgenic mice. Hepatitis and HCC were induced by administering concanavalin A and diethylnitrosamine, respectively. RESULTS: SOCS3 expression was reduced in the HCC regions compared with the non-HCC regions. Carcinogen-induced hepatic tumor development was enhanced by deletion of the SOCS3 gene, which was associated with higher levels of the targets of signal transducers and activators of transcription (ie, B-cell lymphoma-XL, B-cell lymphoma-2, C-myelocytomatosis, cyclin D1, and vascular endothelial growth factor). In the concanavalin A-mediated hepatitis model, deletion of the SOCS3 gene in the hepatocytes protected against liver injury through suppression of interferon-gamma signaling and induction of the antiapoptotic protein Bcl-XL. CONCLUSIONS: Deletion of the SOCS3 gene in hepatocytes promotes the activation of STAT3, resistance to apoptosis, and an acceleration of proliferation, resulting in enhanced hepatitis-induced hepatocarcinogenesis.

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-alpha, was down-regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4-mediated activation of IkappaB-alpha, 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.

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-kappaB 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-kappaB 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-kappaB 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.