Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4).

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4-GFP (T4-GFP) was clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259-470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259-470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259-470) recruited full-length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259-470). Thus, it seems that individual T4(259-470) mutant molecules are sufficient to direct the respective TRAF4-T4(259-470) heteromeric complexes into the nucleus. In cells forming cell-cell contacts, TRAF4 was recruited to the sites of contact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-kappaB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-kappaB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidly up-regulated. In Jurkat T-cells deficient for I-kappaB kinase gamma (IKKgamma, also known as NEMO), an essential component of the NF-kappaB-inducing-IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-kappaB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-kappaB pathway is involved in up-regulation of TRAF4 in T-cells.     

NF-kappaB signaling pathway governs TRAIL gene expression and human T-cell leukemia virus-I Tax-induced T-cell death.

The Tax oncoprotein encoded by human T-cell leukemia virus induces both T-cell activation and apoptosis. The mechanism by which Tax induces apoptosis has remained unclear. Using genetically manipulated T-cell lines, we demonstrate that Tax-induced T-cell death is dependent on NF-kappaB signaling. Tax fails to induce apoptosis in T cells lacking IkappaB kinase gamma (IKKgamma), an essential component of the NF-kappaB signaling pathway. This defect was rescued when the mutant cells were reconstituted with exogenous IKKgamma. We further demonstrate that the Tax-induced T-cell death is mediated by TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), because this event can be effectively inhibited by a TRAIL-blocking antibody. Consistent with this functional aspect, Tax stimulates the expression of TRAIL mRNA. Finally, we provide genetic evidence demonstrating that the NF-kappaB signaling pathway is essential for TRAIL gene induction by both Tax and T-cell activation signals. These studies reveal a novel function of the NF-kappaB signaling pathway and suggest a key mechanism by which Tax induces T-cell death.     

Regulation of Ikappa B kinase (IKK)gamma /NEMO function by IKKbeta -mediated phosphorylation

The IkappaB kinase (IKK) complex includes the catalytic components IKKalpha and IKKbeta in addition to the scaffold protein IKKgamma/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IkappaB and the activation of the NF-kappaB pathway. Recent data indicate that the constitutive activation of the NF-kappaB pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKKgamma/NEMO by IKKbeta. To address further the significance of IKKbeta-mediated phosphorylation of IKKgamma/NEMO, we determined the sites in IKKgamma/NEMO that were phosphorylated by IKKbeta, and we assayed whether IKKgamma/NEMO phosphorylation was involved in modulating IKKbeta activity. IKKgamma/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-alpha and interleukin-1 that activate the NF-kappaB pathway. By using both in vitro and in vivo assays, IKKbeta was found to phosphorylate IKKgamma/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKKgamma/NEMO to stimulate IKKbeta kinase activity. These results indicate that the differential phosphorylation of IKKgamma/NEMO by IKKbeta and perhaps other kinases may be important in regulating IKK activity.     

The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.     

Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.     

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.     

A role for NF-kappaB essential modifier/IkappaB kinase-gamma (NEMO/IKKgamma) ubiquitination in the activation of the IkappaB kinase complex by tumor necrosis factor-alpha

NEMO (NF-kappaB essential modifier)/IKKgamma (IkappaB kinase-gamma) is required for the activation of the IkappaB kinase complex (IKK) by inflammatory stimuli such as tumor necrosis factor (TNF-alpha). Here we show that TNF-alpha stimulates the ubiquitination of NEMO in a manner that does not appear to target it for degradation and that is impaired by mutations in the NEMO zinc finger. Mutations of the zinc finger are found in patients with hypohidrotic ectodermal dysplasia with immunodeficiency (HED-ID) and lead to the impairment of TNF-alpha-stimulated IKK phosphorylation and activation. In addition, the ubiquitination of NEMO is mediated by c-IAP1, an inhibitor of apoptosis protein that is a component of the TNF receptor signaling complex. Thus, the ubiquitination of NEMO mediated by c-IAP1 likely plays an important role in the activation of IKK by TNF-alpha. Also, defective NEMO ubiquitination may be responsible for the impaired cellular NF-kappaB signaling found in patients with HED-ID.     

Differential regulation of the expression of CD95 ligand, receptor activator of nuclear factor-kappa B ligand (RANKL), TNF-related apoptosis-inducing ligand (TRAIL), and TNF-alpha during T cell activation

Members of TNF superfamily are characterized by their ability to inflict apoptosis upon binding to their cognate receptors in a homotrimeric manner. These proteins are expressed on different cell types under various conditions. However, the mechanisms governing the expression of these molecules remain elusive. We have found that the TCR signal can elicit the expression of receptor activator of NF-kappaB ligand (RANKL), TNF-alpha, CD95L, and TNF-related apoptosis inducing ligand (TRAIL) in T cell hybridoma A1.1 cells, thus allowing us to examine the expression pattern of these molecules under precisely the same conditions. We have previously reported that CD95L expression requires both protein kinase C (PKC) translocation and Ca2+ mobilization and is inhibited by cyclosporin A, and dexamethasone. We demonstrate now that activation-induced expression of RANKL is mediated by Ca2+ mobilization. PKC activation does not induce RANKL expression nor does it synergize with the Ca2+ signal. Activation-induced RANKL expression is blocked by cyclosporin A, but not by dexamethasone. The expression of TNF, in contrast, is mediated by PKC, but not by Ca2+. TNF-alpha expression is not inhibited by cyclosporin A, but is sensitive to dexamethasone. A1.1 cells constitutively express TRAIL at low levels. Stimulation with anti-CD3 leads to an initial reduction and subsequent increase in TRAIL expression. TRAIL induction is not inhibited by cyclosporin A, but highly sensitive to dexamethasone. Therefore, expression of the TNF superfamily genes is regulated by distinct signals. Detailed understanding of the regulatory mechanisms could provide crucial information concerning the role of these molecules in the modulation of the immune system.     

ABIN-1 binds to NEMO/IKKgamma and co-operates with A20 in inhibiting NF-kappaB

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-kappaB is dependent on the phosphorylation of the inhibitory subunit IkappaBalpha that is mediated by a multimeric, high molecular weight complex, called IkappaB kinase (IKK) complex. This is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, NEMO/IKKgamma. The latter protein is essential for the activation of IKKs and NF-kappaB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-kappaB) as a NEMO/IKKgamma-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-kappaB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-kappaB at the level of the IKK complex and that A20 inhibits activation of NF-kappaB by de-ubiquitination of NEMO/IKKgamma. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKgamma and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-kappaB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKgamma and facilitates A20-mediated de-ubiquitination of NEMO/IKKgamma, thus resulting in inhibition of NF-kappaB.     

IKKgamma /NEMO facilitates the recruitment of the IkappaB proteins into the IkappaB kinase complex

IKKgamma/NEMO is an essential regulatory component of the IkappaB kinase complex that is required for NF-kappaB activation in response to various stimuli including tumor necrosis factor-alpha and interleukin-1beta. To investigate the mechanism by which IKKgamma/NEMO regulates the IKK complex, we examined the ability of IKKgamma/NEMO to recruit the IkappaB proteins into this complex. IKKgamma/NEMO binding to wild-type, but not to a kinase-deficient IKKbeta protein, facilitated the association of IkappaBalpha and IkappaBbeta with the high molecular weight IKK complex. Following tumor necrosis factor-alpha treatment of HeLa cells, the majority of the phosphorylated form of endogenous IkappaBalpha was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKKgamma/NEMO-deficient cells. Finally, we demonstrate that IKKgamma/NEMO facilitates the association of the IkappaB proteins and IKKbeta and leads to increases in IKKbeta kinase activity. These results suggest that an important function of IKKgamma/NEMO is to facilitate the association of both IKKbeta and IkappaB in the high molecular weight IKK complex to increase IkappaB phosphorylation.     

Failure of Bcl-2 to block cytochrome c redistribution during TRAIL-induced apoptosis

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis and NF-kappaB activation. Here we show that recombinant hu-TRAIL initiates the activation of multiple caspases, the loss of mitochondrial transmembrane potential, the cleavage of BID and the redistribution of mitochondrial cytochrome c. However, whereas Bcl-2 efficiently blocked UV radiation-induced cytochrome c release and consequent apoptosis of CEM cells, it failed to do either in the context of TRAIL treatment. Thus, TRAIL engages a death pathway that is at least partially routed via the mitochondria, but in contrast with other stimuli that engage this pathway, TRAIL-induced cytochrome c release is not regulated by Bcl-2.     

Tumor necrosis factor alpha up-regulates non-lymphoid Fas-ligand following superantigen-induced peripheral lymphocyte activation

Members of the tumor necrosis factor (TNF) and TNF receptor families play important roles in inducing apoptosis and mediating the inflammatory response. Activated T lymphocytes can trigger the expression of Fas-ligand on non-lymphoid tissue, such as intestinal epithelial cells (IEC), and this, in turn, can induce apoptosis in the T cells. Here, we examine the role of TNFalpha in this feedback regulation. Injection of TNFalpha into mice caused a rapid up-regulation of Fas-ligand mRNA in IEC. TNFalpha-induced activation of the Fas-ligand promoter in IEC requires NF-kappaB as this was blocked by an I-kappaBalphaM super-repressor and by mutation of an NF-kappaB site in the Fas-ligand promoter. Activation of T cells by antigen induced Fas-ligand expression in IEC in vivo in wild type, but not in TNFalpha-/- or TNFR1-/- mice. These results define a novel pathway wherein TNFalpha, produced by activated T cells in the intestine, induce Fas-ligand expression in IEC. This is the first observation that one member of the TNF superfamily mediates the regulation of another family member and represents a potential feedback mechanism controlling lymphocyte infiltration and inflammation in the small intestine.