Specificity of TRAF3 in its negative regulation of the noncanonical NF-kappa B pathway

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are critical signaling adaptors downstream of many receptors in the TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Whereas TRAF2, 5, and 6 are activators of the canonical NF-kappaB signaling pathway, TRAF3 is an inhibitor of the noncanonical NF-kappaB pathway. The contribution of the different domains in TRAFs to their respective functions remains unclear. To elucidate the structural and functional specificities of TRAF3, we reconstituted TRAF3-deficient cells with a series of TRAF3 mutants and assessed their abilities to restore TRAF3-mediated inhibition of the noncanonical NF-kappaB pathway as measured by NF-kappaB-inducing kinase (NIK) protein levels and processing of p100 to p52. We found that a structurally intact RING finger domain of TRAF3 is required for inhibition of the noncanonical NF-kappaB pathway. In addition, the three N-terminal domains, but not the C-terminal TRAF domain, of the highly homologous TRAF5 can functionally replace the corresponding domains of TRAF3 in suppression of the noncanonical NF-kappaB pathway. This functional specificity correlates with the specific binding of TRAF3, but not TRAF5, to the previously reported TRAF3 binding motif in NIK. Our studies suggest that both the RING finger domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels and the processing of p100 to p52.     

Regulation of FcepsilonRI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells

Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI mediated signaling cascades, which activate nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, and these are critical elements for degranulation in mast cells. We cloned a novel adaptor molecule, signal transducing adaptor protein (STAP)-2 containing PH and SH2-like domains as a c-fms interacting protein. STAP-2 was identical to a recently cloned adaptor molecule, BKS, a substrate of BRK (breast tumor kinase) tyrosine kinase, although its function is still unknown. To examine a novel function of STAP-2/BSK, we expressed STAP-2/BSK or its mutants in rat basophilic leukemia RBL-2H3 cells. Overexpression of STAP-2/BSK resulted in a suppression of FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma.     

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.     

Genetic and biochemical evidence for a critical role of Janus kinase (JAK)-3 in mast cell-mediated type I hypersensitivity reactions

We investigated the role of JAK3 in IgE receptor/FcepsilonRI-mediated mast cell responses. IgE/antigen induced degranulation and mediator release were substantially reduced with Jak3-/- mast cells from JAK3-null mice that were generated by targeted disruption of Jak3 gene in embryonic stem cells. Further, treatment of mast cells with 3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154), a potent inhibitor of JAK3, inhibited degranulation and proinflammatory mediator release after IgE receptor/ FcepsilonRI crosslinking. Thus, JAK3 plays a pivotal role in IgE receptor/ FcepsilonRI-mediated mast cell responses and targeting JAK3 may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.     

NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase

The activation of NF-kappaB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin-1 (IL-1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IkappaB. TANK is a TRAF-binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF-kappaB activation by these receptors. However, TANK also displays the ability to stimulate TRAF-mediated NF-kappaB activation. In this report, we investigate the mechanism of the stimulatory activity of TANK. We find that TANK interacts with TBK1 (TANK-binding kinase 1), a novel IKK-related kinase that can activate NF-kappaB in a kinase-dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase-inactive TBK1 inhibits TANK-mediated NF-kappaB activation but does not block the activation mediated by TNF-alpha, IL-1 or CD40. The TBK1-TANK-TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF-mediated activation of NF-kappaB.     

Critical roles of TRAF2 and TRAF5 in tumor necrosis factor-induced NF-kappa B activation and protection from cell death

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the TNF receptor superfamily. However, the exact roles of TRAF2 and TRAF5 in TNF-induced NF-kappaB activation still remain controversial. To address this issue, we generated TRAF2 and TRAF5 double knockout (DKO) mice. TNF- but not interleukin-1-induced nuclear translocation of NF-kappaB was severely impaired in murine embryonic fibroblasts (MEFs) derived from DKO mice. Moreover, DKO MEFs were more susceptible to TNF-induced cytotoxicity than TRAF2 knockout MEFs. Collectively, these results indicate that both TRAF2 and TRAF5 are involved in TNF-induced NF-kappaB activation and protection from cell death.     

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.     

TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.     

Developmental stage-dependent collaboration between the TNF receptor-associated factor 6 and lymphotoxin pathways for B cell follicle organization in secondary lymphoid organs

Signal transduction pathways regulating NF-kappaB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-kappaB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) beta receptor (LtbetaR) signal induction of nonclassical NF-kappaB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtbetaR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.     

Impaired FcepsilonRI-dependent gene expression and defective eicosanoid and cytokine production as a consequence of Fyn deficiency in mast cells

Fyn kinase is a key contributor in coupling FcepsilonRI to mast cell degranulation. A limited macroarray analysis of FcepsilonRI-induced gene expression suggested potential defects in lipid metabolism, eicosanoid and glutathione metabolism, and cytokine production. Biochemical analysis of these responses revealed that Fyn-deficient mast cells failed to secrete the inflammatory eicosanoid products leukotrienes B4 and C4, the cytokines IL-6 and TNF, and chemokines CCL2 (MCP-1) and CCL4 (MIP-1beta). FcepsilonRI-induced generation of arachidonic acid and normal induction of cytokine mRNA were defective. Defects in JNK and p38 MAPK activation were observed, whereas ERK1/2 and cytosolic phospholipase A2 (S505) phosphorylation was normal. Pharmacological studies revealed that JNK activity was associated with generation of arachidonic acid. FcepsilonRI-mediated activation of IkappaB kinase beta and IkappaBalpha phosphorylation and degradation was defective resulting in a marked decrease of the nuclear NF-kappaB DNA binding activity that drives IL-6 and TNF production in mast cells. However, not all cytokine were affected, as IL-13 production and secretion was enhanced. These studies reveal a major positive role for Fyn kinase in multiple mast cell inflammatory responses and demonstrate a selective negative regulatory role for certain cytokines.     

Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis

Death receptors are a subfamily of the tumor necrosis factor (TNF) receptor subfamily. They are characterized by a death domain (DD) motif within their intracellular domain, which is required for the induction of apoptosis. Fas-associated death domain protein (FADD) is reported to be the universal adaptor used by death receptors to recruit and activate the initiator caspase-8. CD95, TNF-related apoptosis-inducing ligand (TRAIL-R1), and TRAIL-R2 bind FADD directly, whereas recruitment to TNF-R1 is indirect through another adaptor TNF receptor-associated death domain protein (TRADD). TRADD also binds two other adaptors receptor-interacting protein (RIP) and TNF-receptor-associated factor 2 (TRAF2), which are required for TNF-induced NF-kappaB and c-Jun N-terminal kinase activation, respectively. Analysis of the native TNF signaling complex revealed the recruitment of RIP, TRADD, and TRAF2 but not FADD or caspase-8. TNF failed to induce apoptosis in FADD- and caspase-8-deficient Jurkat cells, indicating that these apoptotic mediators were required for TNF-induced apoptosis. In an in vitro binding assay, the intracellular domain of TNF-R1 bound TRADD, RIP, and TRAF2 but did not bind FADD or caspase-8. Under the same conditions, the intracellular domain of both CD95 and TRAIL-R2 bound both FADD and caspase-8. Taken together these results suggest that apoptosis signaling by TNF is distinct from that induced by CD95 and TRAIL. Although caspase-8 and FADD are obligatory for TNF-mediated apoptosis, they are not recruited to a TNF-induced membrane-bound receptor signaling complex as occurs during CD95 or TRAIL signaling, but instead must be activated elsewhere within the cell.     

Role of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) in distinct and overlapping CD40 and TNF receptor 2/CD120b-mediated B lymphocyte activation

Members of the tumor necrosis factor receptor (TNFR) family play a variety of roles in the regulation of lymphocyte activation. An important TNFR family member for B cell activation is CD40. CD40 signals stimulate B cell TNF-alpha secretion, which subsequently signals via TNFR2 (CD120b) to enhance B cell activation. Although the function of the pro-apoptotic and pro-inflammatory receptor TNFR1 (CD120a) has been the subject of much research, less is understood about the distinct contributions of CD120b to cell activation and how it stimulates downstream events. Members of the tumor necrosis factor receptor family bind various members of the cytoplasmic adapter protein family, the tumor necrosis factor receptor-associated factors (TRAFs), during signaling. Both CD40 and CD120b bind TNF receptor-associated factor 2 (TRAF2) upon ligand stimulation. Wild type and TRAF2-deficient B cells expressing CD40 or the hybrid molecule (human) CD40 (mouse)-CD120b were examined. CD40- and CD120b-mediated IgM secretion were partly TRAF2-dependent, but only CD40 required TRAF2 for c-Jun N-terminal kinase activation. CD40 and CD120b used primarily divergent mechanisms to activate NF-kappaB, exemplifying how TNFR family members can use diverse mechanisms to mediate similar downstream events.     

Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, β, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D(2) and leukotriene C(4) in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.