Involvement of TNF receptor-associated factor 6 in IL-25 receptor signaling

IL-25 (IL-17E) induces IL-4, IL-5, and IL-13 production from an unidentified non-T/non-B cell population and subsequently induces Th2-type immune responses such as IgE production and eosinophilic airway inflammation. IL-25R is a single transmembrane protein with homology to IL-17R, but the IL-25R signaling pathways have not been fully understood. In this study, we investigated the signaling pathway under IL-25R, especially the possible involvement of TNFR-associated factor (TRAF)6 in this pathway. We found that IL-25R cross-linking induced NF-kappaB activation as well as ERK, JNK, and p38 activation. We also found that IL-25R-mediated NF-kappaB activation was inhibited by the expression of dominant negative TRAF6 but not of dominant negative TRAF2. Furthermore, IL-25R-mediated NF-kappaB activation, but not MAPK activation, was diminished in TRAF6-deficient murine embryonic fibroblast. In addition, coimmunoprecipitation assay revealed that TRAF6, but not TRAF2, associated with IL-25R even in the absence of ligand binding. Finally, we found that IL-25R-mediated gene expression of IL-6, TGF-beta, G-CSF, and thymus and activation-regulated chemokine was diminished in TRAF6-deficient murine embryonic fibroblast. Taken together, these results indicate that TRAF6 plays a critical role in IL-25R-mediated NF-kappaB activation and gene expression.     

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.     

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.     

IL-1R-associated kinase 4 is required for lipopolysaccharide-induced activation of APC

The bacterial product LPS is a critical stimulus for the host immune system in the response against the corresponding bacterial infection. LPS provides an activation stimulus for macrophages and a maturation signal for dendritic cells to set up innate and adaptive immune responses, respectively. The signaling cascade of myeloid differentiation factor 88-->IL-1R-associated kinase (IRAK)-->TNFR-associated factor 6 has been implicated in mediating LPS signaling. In this report, we studied the function of IRAK-4 in various LPS-induced signals. We found that IRAK-4-deficient cells were severely impaired in producing some IFN-regulated genes as well as inflammatory cytokines in response to LPS. Among the critical downstream signaling pathways induced by LPS, NF-kappaB activation but not IFN regulatory factor 3 or STAT1 activation was defective in cells lacking IRAK-4. IRAK-4 was also required for the proper maturation of dendritic cells by LPS stimulation, particularly in terms of cytokine production and the ability to stimulate Th cell differentiation. Our results demonstrate that IRAK-4 is critical for the LPS-induced activations of APCs.     

Interleukin-7 and transforming growth factor-beta play counter-regulatory roles in protein kinase C-delta-dependent control of fibroblast collagen synthesis in pulmonary fibrosis

Transforming growth factor-beta (TGF-beta) is a potent fibrogenic factor responsible for promoting synthesis of extracellular matrix. Interleukin-7 (IL-7) inhibits TGF-beta signaling by up-regulating Smad7, a major inhibitor of the Smad family. In a variety of cells, TGF-beta-mediated activation of target genes requires active protein kinase C-delta (PKC-delta) in addition to Smads (1). We determined the role of PKC-delta in the regulation of pulmonary fibroblast collagen synthesis in response to TGF-beta and IL-7 stimulation. Here we show that TGF-beta and IL-7 have opposing effects on PKC-delta; TGF-beta stimulates, while IL-7 inhibits, PKC-delta activity. IL-7 inhibits TGF-beta-induced PKC-delta phosphorylation at Ser-645 and Thr-505. Inhibition of PKC-delta with specific small inhibitory RNA restores TGF-beta-mediated induction of Smad7 and in parallel significantly reduces TGF-beta-mediated collagen synthesis. Thus, PKC-delta may play a critical role in the pathogenesis of pulmonary fibrosis and may serve as a molecular target for therapeutic intervention to suppress fibrosis.     

X-linked inhibitor of apoptosis (XIAP) inhibits c-Jun N-terminal kinase 1 (JNK1) activation by transforming growth factor beta1 (TGF-beta1) through ubiquitin-mediated proteosomal degradation of the TGF-beta1-activated kinase 1 (TAK1)

Active NF-kappaB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factorbeta1 (TGF-beta1). NF-kappaB counteracts TGF-beta1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-X(L), which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-kappaB by TGF-beta1 inhibits JNK signaling, thereby attenuating TGF-beta1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-kappaB and JNK pathways during TGF-beta1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-kappaB-mediated suppression of JNK signaling. We show that NF-kappaB plays a role in the up-regulation of XIAP gene expression in response to TGF-beta1 treatment and forms a TGF-beta1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK1 protein expression inhibits TGF-beta1-mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-beta1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-kappaB during TGF-beta1 signaling and raise the possibility that pharmacologic inhibition of the NF-kappaB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-beta1 in advanced hepatocellular carcinomas (HCCs) without affecting the pro-apoptotic effects of TGF-beta1 involved in normal liver homeostasis.