Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2)

Tumor necrosis factor (TNF)-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and germinal center kinases (GCKs) and the subsequent activation of stress-activated protein kinases (SAPKs and c-Jun NH(2)-terminal kinases) requires TNF receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain binds ASK1, GCK, and the highly related kinase GCKR, the RING finger domain is needed for their activation. Here, we report that TNF activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a minimal effect on ASK1 activation. TNF signaling leads to TRAF2 polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to the disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A is required for TNF-mediated GCKR and SAPK activation, but may not be required for ASK1 activation.     

TANK Potentiates Tumor Necrosis Factor Receptor-Associated Factor-Mediated c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase Activation through the Germinal Center Kinase Pathway

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.     

Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin

The stress-activated protein kinases (SAPKs, also called c-Jun NH(2)-terminal kinases) and the p38s, two mitogen-activated protein kinase (MAPK) subgroups activated by cytokines of the tumor necrosis factor (TNF) family, are pivotal to the de novo gene expression elicited as part of the inflammatory response. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAP3K) that activates both the SAPKs and p38s in vivo. Here we show that TNF receptor (TNFR) associated factor 2 (TRAF2), an adapter protein that couples TNFRs to the SAPKs and p38s, can activate ASK1 in vivo and can interact in vivo with the amino- and carboxyl-terminal noncatalytic domains of the ASK1 polypeptide. Expression of the amino-terminal noncatalytic domain of ASK1 can inhibit TNF and TRAF2 activation of SAPK. TNF can stimulate the production of reactive oxygen species (ROS), and the redox-sensing enzyme thioredoxin (Trx) is an endogenous inhibitor of ASK1. We also show that expression of TRAF2 fosters the production of ROS in transfected cells. We demonstrate that Trx significantly inhibits TRAF2 activation of SAPK and blocks the ASK1-TRAF2 interaction in a reaction reversed by oxidants. Finally, the mechanism of ASK1 activation involves, in part, homo-oligomerization. We show that expression of ASK1 with TRAF2 enhances in vivo ASK1 homo-oligomerization in a manner dependent, in part, upon the TRAF2 RING effector domain and the generation of ROS. Thus, activation of ASK1 by TNF requires the ROS-mediated dissociation of Trx possibly followed by the binding of TRAF2 and consequent ASK1 homo-oligomerization.     

Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.     

Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen-activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1β (IL-1β) stimulation rapidly activate MLK3 kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1β stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1β.     

Direct Activation of Mitogen-Activated Protein Kinase Kinase Kinase MEKK1 by the Ste20p Homologue GCK and the Adapter Protein TRAF2

Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.     

Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2.

Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII we fused the extracellular domain of the mouse CD4 antigen to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. Our findings show that: (i) TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK); (ii) TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression; (iii) dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and (iv) TRAF2 signals the activation of JNK and NF-kappaB through different pathways. Our findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway and that TRAF2 occupies a key role in TNF receptor signaling to JNK.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.     

The tyrosine kinase Syk regulates TPL2 activation signals

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.     

NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain.

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.     

1Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a phosphoprotein possessing various functions. We have previously reported that the HCV NS5A protein interacts with tumor necrosis factor (TNF) receptor-associated factor (TRAF) domain of TRAF2 (Park, K.-J., Choi, S.-H., Lee, S. Y., Hwang, S. B., and Lai, M. M. C. (2002) J. Biol. Chem. 277, 13122-13128). Both TNF-alpha- and TRAF2-mediated nuclear factor-kappaB (NF-kappaB) activations were inhibited by NS5A-TRAF2 interaction. Because TRAF2 is required for the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK), we investigated HCV NS5A protein for its potential capacity to modulate TRAF2-mediated JNK activity. Using in vitro kinase assay, we have found that NS5A protein synergistically activated both TNF-alpha- and TRAF2-mediated JNK in human embryonic kidney 293T cells. Furthermore, synergism of NS5A-mediated JNK activation was inhibited by dominant-negative form of MEK kinase 1. Our in vivo binding data show that NS5A does not inhibit interaction between TNF receptor-associated death domain and TRAF2 protein, indicating that NS5A and TRAF2 may form a ternary complex with TNF receptor-associated death domain. These results indicate that HCV NS5A protein modulates TNF signaling of the host cells and may play a role in HCV pathogenesis.     

The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.     

Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-alpha signaling.

Tumor necrosis factor-alpha (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-kappa B, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with sphingosine kinase (SphK), a lipid kinase that is responsible for the production of sphingosine 1-phosphate. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-kappa B but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-kappa B and antiapoptosis.     

Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaB-inducing kinase. Identification of a novel TRAF6 interaction motif

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappaB (NF-kappaB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappaB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-kappaB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-kappaB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565-568 and 606-611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-kappaB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kappaB activation. However, RANK-mediated JNK activation required a distal portion (427-603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-kappaB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-kappaB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.     

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.     

An 11-amino acid sequence in the cytoplasmic domain of CD40 is sufficient for activation of c-Jun N-terminal kinase, activation of MAPKAP kinase-2, phosphorylation of I kappa B alpha, and protection of WEHI-231 cells from anti-IgM-induced growth arrest

We have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35-45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of I kappa B alpha phosphorylation and degradation. Amino acids 35-45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-kappa B, JNK, and p38 MAPK.     

Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis

Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events.     

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.