CARD6 is a modulator of NF-kappa B activation by Nod1- and Cardiak-mediated pathways

We cloned a novel cDNA derived from the CARD6 gene locus on chromosome 5p12 of 311 amino acids in length. By immunoprecipitation we detected specific binding of this CARD6-encoding protein to Nod1 (CARD4), Cardiak (Rip2/Rick), NAC (NALP1/DEFCAP/CARD7), and TUCAN (CARD8/Cardinal/NDPP/Dakar), caspase recruitment domain (CARD)-containing proteins implicated in NF-kappa B and caspase-1 activation but not to other CARD family proteins. Cardiak and Nod1 (but not other CARD proteins) also exhibited opposing effects on CARD6 protein phosphorylation and expression, providing further evidence of functional interactions among these proteins in cells. In transfection experiments, the CARD6 protein suppressed NF-kappa B induction by Nod1 or Cardiak but did not interfere with NF-kappa B activation by the CARD-containing adapter protein Bcl10 or the cytokine tumor necrosis factor-alpha, demonstrating specificity of CARD6 for Nod-1 and Cardiak-dependent pathways. In contrast to its effects on Nod1- and Cardiak-dependent NF-kappa B activation, CARD6 did not interfere with caspase-1-dependent interleukin-1 beta secretion induced by Cardiak or Nod1. CARD6 also did not affect caspase activation and apoptosis induced by overexpression of Fas, Bax, or other pro-apoptotic stimuli. Thus, CARD6 represents a selective modulator of NF-kappa B activation by Cardiak and Nod1, adding to the repertoire of CARD-family proteins implicated in inflammatory responses and innate immunity.     

TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta

In many physiological and disease processes, TGF-beta usurps branches of MAP kinase pathways in conjunction with Smads to induce apoptosis and epithelial-to-mesenchymal transition, but the detailed mechanism of how a MAP kinase cascade is activated by TGF-beta receptors is not clear. We report here that TRAF6 is specifically required for the Smad-independent activation of JNK and p38, and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6 and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway.     

Membrane localization of TRAF 3 enables JNK activation

Members of the tumor necrosis factor receptor family as well as other receptors achieve their diverse biological effects through the activation of intracellular signals including the c-Jun N-terminal kinase (JNK) pathway. Such signals are believed to be delivered through mediators known as TNF receptor-associated factors (TRAFs). Although the N-terminal zinc finger region of TRAFs has been shown to be essential for downstream signaling, there is no indication yet as to the nature of its role or of the factors that distinguish the N terminus of TRAF 3, which does not activate JNK in the systems examined thus far, from those of other TRAFs, which do activate this pathway. In the present study, it is shown that, among the known TRAFs, localization to the insoluble cell pellet fraction consistently correlates with JNK activation and that both characteristics map to the TRAF N terminus. Furthermore, it is demonstrated that forced localization of TRAF 3 to the cell membrane is sufficient to convert this molecule into an activator of JNK. This suggests that one of the roles of the TRAF N terminus may be to participate in interactions that promote the recruitment of TRAFs to the membrane and that this localization effect plays an important role in TRAF-mediated JNK activation.     

The ectodermal dysplasia receptor activates the nuclear factor-kappaB, JNK, and cell death pathways and binds to ectodysplasin A

The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-kappaB, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-kappaB or JNK activation. Collectively, the above results suggest that EDAR utilizes a novel signal transduction pathway. Finally, ectodysplasin A can physically interact with the extracellular domain of EDAR and thus represents its biological ligand.     

Induction of apoptosis by X-linked ectodermal dysplasia receptor via a caspase 8-dependent mechanism

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that is highly expressed during embryonic development and binds to ectodysplasin-A2 (EDA-A2). In this report, we demonstrate that although XEDAR lacks a death domain, it nevertheless induces apoptosis in an EDA-A2-dependent fashion. The apoptosis-inducing ability of XEDAR is dependent on the activation of caspase 8 and can be blocked by its genetic and pharmacological inhibitors. Although XEDAR-induced apoptosis can be blocked by dominant-negative Fas-associated death domain (FADD) protein and FADD small interfering RNA, XEDAR does not directly bind to FADD, tumor necrosis factor receptor-associated death domain (TRADD) protein, or RIP1. Instead, XEDAR signaling leads to the formation of a secondary complex containing FADD, caspase 8, and caspase 10, which results in caspase activation. Thus, XEDAR belongs to a novel class of death receptors that lack a discernible death domain but are capable of activating apoptosis in a caspase 8- and FADD-dependent fashion. XEDAR may represent an early stage in the evolution of death receptors prior to the emergence of the death domain and may play a role in the induction of apoptosis during embryonic development and adult life.     

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.     

Mutation identification in a canine model of X-linked ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-κB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue, a cryptic acceptor site within exon 9 is used, leading to a frame shift and use of a premature stop codon that truncates the translation of both isoforms, EDA-A1 and EDA-A2, resulting in the absence of the TNF-like homology domain, the receptor-binding site of ectodysplasin.The sequence data described in this article have been submitted to GenBank under accession numbers AY924407–AY924414.     

Lymphotoxin beta receptor induces sequential activation of distinct NF-kappa B factors via separate signaling pathways

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.     

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.     

IL-6 induces NF-kappa B activation in the intestinal epithelia

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.     

IRAK-2 participates in multiple toll-like receptor signaling pathways to NFkappaB via activation of TRAF6 ubiquitination

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFkappaB signaling axis, recent data have shown that it is dispensable for NFkappaB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFkappaB activation pathway. The poxviral TLR antagonist A52 inhibited NFkappaB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFkappaB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFkappaB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFkappaB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFkappaB.     

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.     

A genetic study of anodontia in X-linked hypohidrotic ectodermal dysplasia.

Dental examinations and tooth measurements were conducted on 16 mothers, 10 fathers, and 23 affected males in 15 families with X-linked hypohidrotic ectodermal dysplasia. Small teeth and congenital missing teeth were sufficiently consistent findings in obligate heterozygotes to suggest that carriers can usually be recognized by clinical criteria.     

High-resolution mapping of the X-linked hypohidrotic ectodermal dysplasia (EDA) locus

The X-linked hypohidrotic ectodermal dysplasia (EDA) locus has been previously localized to the subchromosomal region Xq11-q21.1. We have extended our previous linkage studies and analyzed linkage between the EDA locus and 10 marker loci, including five new loci, in 41 families. Four of the marker loci showed no recombination with the EDA locus, and six other loci were also linked to the EDA locus with recombination fractions of .009-.075. Multipoint analyses gave support to the placement of the PGK1P1 locus proximal to the EDA locus and the DXS453 and PGK1 loci distal to EDA. Further ordering of the loci could be inferred from a human/rodent somatic cell hybrid derived from an affected female with EDA and an X;9 translocation and from studies of an affected male with EDA and a submicroscopic deletion. Three of the proximal marker loci, which showed no recombination with the EDA locus, when used in combination, were informative in 92% of females. The closely linked flanking polymorphic loci DXS339 and DXS453 had heterozygosities of 72% and 76%, respectively, and when used jointly, they were doubly informative in 52% of females. The human DXS732 locus was defined by a conserved mouse probe pcos169E/4 (DXCrc169 locus) that cosegregates with the mouse tabby (Ta) locus, a potential homologue to the EDA locus. The absence of recombination between EDA and the DXS732 locus lends support to the hypothesis that the DXCrc169 locus in the mouse and the DXS732 locus in humans may contain candidate sequences for the Ta and EDA genes, respectively.