Alternative NF-κB Signaling Research Articles

TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Abstract 3665Poster Board III-601 IntroductionHodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. MethodsElectromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. ResultsIn this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. ConclusionsNot only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures:No relevant conflicts of interest to declare.

Human T-cell leukemia virus type 1 bZIP factor selectively suppresses the classical pathway of NF-κB

AbstractAdult T-cell leukemia (ATL) is a highly aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). The activation of NF-κB by Tax has been reported to play a crucial role in HTLV-1–induced transformation. The HTLV-1 bZIP factor (HBZ), which is encoded by an mRNA of the opposite polarity of the viral genomic RNA, is involved in both T cell proliferation and suppression of Tax-mediated viral gene transcription, suggesting that HBZ cooperates closely with Tax. In the present study, we observed that HBZ specifically suppressed NF-κB–driven transcription mediated by p65 (the classical pathway) without inhibiting the alternative NF-κB signaling pathway. In an immunoprecipitation assay, HBZ bound to p65 and diminished the DNA binding capacity of p65. In addition, HBZ induced p65 degradation through increasing the expression of the PDLIM2 gene, which encodes a ubiquitin E3 ligase for p65. Finally, HBZ actually repressed the transcription of some classical NF-κB target genes, such as IL-8, IL2RA, IRF4, VCAM-1, and VEGF. Selective suppression of the classical NF-κB pathway by HBZ renders the alternative NF-κB pathway predominant after activation of NF-κB by Tax or other stimuli, which might be critical for oncogenesis.

Networking Modules of Canonical and Alternative NFkB Signaling in Growth and Survival Regulations of Large B Cell Lymphomas

Constitutive NF-kB activation is considered a “hallmark” in B-cell malignancies, especially in large B-cell lymphoma (LBCL), a common but heterogenous Non-Hodgkin's lymphoma type. However, the mechanism(s) of activation and interactions of NF-kB components in the nuclear compartment of B-cell lymphomas are still poorly defined. Our findings demonstrate that both the canonical and the alternative NF-kB pathways are constitutively activated in both LBCL cell lines and primary lymphoma cells. NF-kB DNA binding ELISA analysis showed that the canonical NF-kB components p65 and c-rel as well as the non-canonical NF-kB components p52 and relB are constitutively activated in 14 LBCL cell lines and in 14 primary lymphoma patients' cells (both ABC-and GCB-like subtypes). Based on the ELISA data, we found that the p65/c-rel ratio is relatively higher in ABC-like LBCL subtype when compared to GCB-like LBCL subtype, but the alternative NF-kB members' p52 and rel-B are differentially activated in both GCB- and ABC-like LBCL subtypes. Micro-tissue array (MTA) studies confirmed that both NF-kB pathways are constitutively active in LBCL biopsy-cores. The components of both NF-kB pathways were also found to merge and form functional “hybrid” NF-kB complexes in the nuclear compartment. Besides the common hetero-dimeric complexes occur between p50, p65, and c-rel of the canonical NF-kB pathway and between p52 and relB of the alternative NF-kB pathway, components of both pathways also merged to form “hybrid” dimeric complexes, such as p52-relB, p52-c-rel, relB-p65, and relB-c-rel in LBCL cells. Interestingly, p65 and c-rel binds to relB and p52, respectively, with higher affinity in GCB- than in ABC-like LBCL subtype. Our data also showed that activation of both NF-kB pathways and their cross-interaction are not restricted to only neoplastic B cells but that they are also activated and utilized in highly proliferative activated normal B-lymphocyte. Specific siRNA that target individual NF-kB members p65, c-rel, and rel-B have inhibitory activity in lymphoma cell growth, but similar p52 siRNAs do not have inhibitory activity. These studies indicated that in both LBCL cell lines and patient samples (GCB-and ABC-like subtypes), both the canonical and the alternative NF-kB pathways are not only constitutively activated but also interact to form multimeric NF-kB complexes in the nuclear compartment, giving rise to an NF-kB signaling module that not only controls lymphoma cell growth and survival but likely contributes to the heterogeneity of the disease processes. These findings reveal additional aspects of molecular heterogeneity and complexity in NF-kB signaling mechanisms and interacting transcriptional modules, contributing to the pathophysiology of LBCL. These results also highlight the critical importance of multiple constitutively active NF-kB signaling pathways in LBCL and may also contribute to therapeutic refractoriness, particularly in relapsed/refractory LBCLs.

Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-κB signaling

The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-kappaB (NF-kappaB) signaling in resting cells by targeting NF-kappaB-inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-kappaB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-kappaB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-kappaB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.

Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-κB family member RelB

RelB is the key component of the alternative NF-kappaB (nuclear factor kappaB) signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1 (DNA methyltransferase 1)-Daxx (death domain-associated protein) complex to NF-kappaB target genes. Importantly, the molecular mechanisms which determine the functions of RelB are still largely unknown. In the present study, we aimed to analyse whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively polyubiquitinated in the B-cell lines Namalwa and 70Z/3. Although a PMA+ionomycin-induced augmentation of RelB polyubiquitination was linked to its proteasomal degradation in B-cells, the constitutive RelB polyubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB polyubiquitination in HEK (human embryonic kidney)-293 cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA binding was enhanced by RelB polyubiquitination. Interestingly, basal RelB polyubiquitination depends neither on Lys(48) nor on Lys(63) conjugates, but might involve unconventional ubiquitin conjugates. Mapping of the ubiquitination target sites in RelB revealed the existence of various lysine residues, which serve as ubiquitination acceptors. However, only the substitution of Lys(273/274) and Lys(305/308) significantly decreased the basal RelB activity and the ubiquitin-induced augmentation of the RelB activity. Collectively, these results imply a dual role of RelB polyubiquitination for the stability and activity of this transcription factor.

NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells

BAFF-R-dependent activation of the alternative NF-kappaB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-kappaB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-kappaB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked.

The Role of TRAF2 Binding to the Type I Interferon Receptor in Alternative NFκB Activation and Antiviral Response

Type I interferons (IFNs) play critical roles in the host defense by modulating gene expression through the IFN-dependent activation of STAT and NFkappaB transcription factors. Previous studies established that IFN activates NFkappaB through a classical NFkappaB pathway that results in IkappaBalpha degradation and formation of p50-containing NFkappaB complexes, as well as an alternative pathway that involves NFkappaB-inducing kinase and TRAF2, which results in the formation of p52-containing NFkappaB complexes. In this study, we examined the interaction of TRAF proteins with the type I IFN receptor. We found that TRAF2 was directly coupled to the signal-transducing IFNAR1 subunit of the IFN receptor. By immunoprecipitation, overexpression of epitope-tagged IFNAR1 constructs, and glutathione S-transferase pulldown experiments, we demonstrate that TRAF2 rapidly binds to the IFNAR1 subunit of the IFN receptor upon IFN binding. The membrane proximal half of the IFNAR1 subunit was found to directly bind TRAF2. Moreover, analysis of mouse embryo fibroblasts derived from TRAF2 knock-out mice demonstrated that TRAF2 plays a critical role in the activation of the alternative NFkappaB pathway by IFN, but not the classical NFkappaB pathway, as well as in the antiviral action of IFN. Our results place TRAF2 directly in the signaling pathway transduced through the IFNAR1 subunit of the IFN receptor. These findings provide an important insight into the molecular mechanisms by which IFN generates signals to induce its biological effects.

Constitutive Production of NF-κB2 p52 Is Not Tumorigenic but Predisposes Mice to Inflammatory Autoimmune Disease by Repressing Bim Expression

Normal development of the immune system requires regulated processing of NF-kappaB2 p100 to p52, which activates NF-kappaB2 signaling. Constitutive production of p52 has been suggested as a major mechanism underlying lymphomagenesis induced by NF-kappaB2 mutations, which occur recurrently in a variety of human lymphoid malignancies. To test the hypothesis, we generated transgenic mice with targeted expression of p52 in lymphocytes. In contrast to their counterparts expressing the tumor-derived NF-kappaB2 mutant p80HT, which develop predominantly B cell tumors, p52 transgenic mice are not prone to lymphomagenesis. However, they are predisposed to inflammatory autoimmune disease characterized by multiorgan infiltration of activated lymphocytes, high levels of autoantibodies in the serum, and immune complex glomerulonephritis. p52, but not p80HT, represses Bim expression, leading to defects in apoptotic processes critical for elimination of autoreactive lymphocytes and control of immune response. These findings reveal distinct signaling pathways for actions of NF-kappaB2 mutants and p52 and suggest a causal role for sustained NF-kappaB2 activation in the pathogenesis of autoimmunity.

Saved from Destruction

Survival of naïve B lymphocytes depends on the cytokine BAFF (B cell-activating factor of the tumor necrosis factor family). Although BAFF can signal through several receptors, only one of them (BAFF-R) is essential for survival (see Cancro). Noting that BAFF-R binds TNF receptor-associated factor 3 (TRAF3) and that TRAF3 and TRAF2 have been implicated in B cell survival, Gardam et al . examined the effects on lymphocyte survival of lineage-specific inactivation of TRAF2 or TRAF3 or both. Mice lacking B cell TRAF2 ( Traf2ΔB mice) or B cell TRAF3 ( Traf3ΔB mice) showed an indistinguishable phenotype, characterized by increased numbers of lymph node and splenic marginal zone B cells, that resembled that of mice overexpressing BAFF (BAFF-tg mice). B cells from Traf2ΔB and Traf3ΔB mice survived in vitro in the absence of BAFF and, like BAFF-tg B cells, showed increased activation of the alternative (noncanonical) NF-κB (nuclear factor κB) signaling pathway. No further effects on B cell phenotype were seen when both TRAF2 and TRAF3 were absent, and microarray analysis revealed that mature B cells from Traf2ΔB and Traf3ΔB mice showed nearly identical gene expression profiles, which were similar to those of BAFF-tg B cells. Elimination of TRAF2 from the B cells rescued B cell development in BAFF-deficient mice. The abundance of TRAF3 protein (but not Traf3 mRNA) was increased in B cells from mice lacking BAFF, BAFF-R, or TRAF-2. The authors thus propose that BAFF acts as a survival factor by stimulating TRAF2-dependent TRAF3 depletion and reversing the cooperative TRAF2- and TRAF3-dependent inhibition of B cell survival. S. Gardam, F. Sierro, A. Basten, F. Mackay, R. Brink, TRAF2 and TRAF3 signal adapters act cooperatively to control the maturation and survival signals delivered to B cells by the BAFF receptor. Immunity 28 , 391-401 (2008). [PubMed] M. P. Cancro, Living in context with the survival factor BAFF. Immunity 28 , 300-301 (2008). [PubMed]

Inactivation of Tumor Necrosis Factor 3 Gene (TRAF3) Is Frequently Identified in Waldenström's Macroglobulinemia Patients.

Constitutive activation of NF-κB signaling pathways has been identified in a variety of malignancies, however the molecular basis has remained largely unknown. Recently, we have shown mutations in 10 genes encoding positive and negative regulators of NF-κB signaling pathways in 20% of multiple myeloma (MM) patients, with TRAF3 being the most commonly affected gene. TRAF3 is a negative regulator of NF-κB signal and its inactivation results in the constitutive activation of the alternative or noncanonical NF-κB signaling pathway. With the exception of two recent studies in MM, there is no available data regarding the mechanisms that dysregulate NF-κB in other B-cell malignancies. The aim of this study was to analyze the TRAF3 status in a cohort of Waldenström's macroglobulinemia (WM) patients. Forty-nine WM were analyzed by using a combination of array-based comparative genomic hybridization (aCGH) and cIgM-FISH. Genome sequencing from 28 of these patients was performed on all coding exons of TRAF3 and the adjacent intron-exon junctions. A NF-κB index, as a measure of NF-κB transcription activity, was calculated from 21 patients with gene expression data. The index is the mean expression level of four probe sets differentially expressed between human myeloma cell lines with identified mutations compared to those without identified NF-κB pathway mutations (CD74, IL2RG and 2x TNFAIP3). By performing aCGH we identified a small monoallelic deletion (∼460 Kb), including the TRAF3 locus in one of 22 patients. Genome sequencing identified a missense mutation (D483N) in the remaining allele. This substitution affects a highly conserved amino acid throughout vertebrate evolution, suggesting its functional relevance. We used cIgM-FISH to screen 27 patients with no available aCGH data and we found one biallelic TRAF3 deletion and one chromosome 14 monosomy. In the later case, no DNA was available to sequence the remaining allele. Both samples with biallelic inactivation of TRAF3 possess the two highest NFKB index values of our WM cohort, suggesting NF-κB transcription activity. Moreover, the case with biallelic deletion showed the lowest level of TRAF3 gene expression. Additionally, the entire TRAF3 coding region was sequenced in 28 of these 49 patients and one homozygous nonsense mutation (K286X) was identified. No GEP was available in this sample to calculate the NF-κB index. TRAF3 was only recently implicated in a malignant process, when inactivating mutations and biallelic deletions were reported in approximately 13% of MM patients, making it the most frequently inactivated tumor suppressor in MM. Our study has identified biallelic TRAF3 inactivation in at least 3 of 49 WM patients, suggesting that the constitutive activation of NF-κB signaling pathways may be involved in the pathogenesis of WM. Recent findings showing an association between TRAF3 loss of function and good response to bortezomib in MM patients suggest that proteasome inhibitor-based therapy may be an attractive option in the treatment of the WM patients with constitutive activation of NF-κB signaling pathways. Additional mutational screening of TRAF3 and other regulators of these pathways are ongoing to determine the extent of constitutive activation present in WM and other B-cell malignancies.

In vitro selection of optimal RelB/p52 DNA-binding motifs

Rel/NF-kB dimers of different subunit composition can activate distinct subsets of target genes in vivo, however, the role of DNA recognition in this specificity is not well understood. We set out to study the DNA-binding ability of RelB/p52, the least studied of all NF-kB proteins and the main transcriptionally active product of the alternative NF-kB signaling pathway. We searched for optimal binding sites for RelB/p52 by random site selection method, using full-length proteins expressed in eukaryotic cells. The subset of RelB/p52-binding sequences defines a consensus which is very similar to the classical RelA/p50 consensus. Importantly, each of these binding sites is also recognized by RelA/p50 heterodimer with comparable affinity, questioning the existence of RelB/p52-specific kappa B sites.

Constitutive alternative NF-κB signaling promotes marginal zone B-cell development but disrupts the marginal sinus and induces HEV-like structures in the spleen

Nuclear factor-kappaB (NF-kappaB) plays a crucial role in B-cell and lymphoid organ development. Here, we studied the consequences of constitutive, signal-independent activation of the alternative NF-kappaB pathway for the splenic marginal zone (MZ). In contrast to nfkb2(-/-) mice, which lack both p100 and p52, mice that lack only the inhibitory p100 precursor but still express the p52 subunit of NF-kappaB2 (p100(-/-)) had markedly elevated MZ B-cell numbers. Both cell-intrinsic mechanisms and increased stromal expression of vascular cell adhesion molecule-1 (VCAM-1) contributed to the accumulation of MZ B cells in p100(-/-) spleens. While migration of p100(-/-) MZ B cells toward the lysophospholipid sphingosine-1 phosphate (S1P) was not affected, CXCL13-stimulated chemotaxis was impaired, correlating with reduced migration of MZ B cells into follicles in response to lipopolysaccharide (LPS). Strikingly, p100 deficiency resulted in the absence of a normal marginal sinus, strongly induced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and glycosylated cell adhesion molecule-1 (GlyCAM-1), and the formation of nonfunctional ectopic high endothelial venule (HEV)-like structures in the red pulp. Thus, constitutive activation of the alternative NF-kappaB pathway favors MZ B-cell development and accumulation but leads to a disorganized spleen microarchitecture.

Thymus Medulla Formation and Central Tolerance Are Restored in IKKα−/− Mice That Express an IKKα Transgene in Keratin 5+ Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-kappaB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IkappaB kinase (IKK)alpha, a required intermediate in the noncanonical NF-kappaB signaling pathway. Introduction of an IKKalpha transgene driven by a K5 promoter restores the K5(+)K14(+) mTEC subset in IKKalpha(-/-) mice. Unexpectedly, the K5-IKKalpha transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKalpha transgene ameliorates autoimmune symptoms in IKKalpha(-/-) mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-kappaB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.

Alternative and Classical NF-κB Signaling Retain Autoreactive B Cells in the Splenic Marginal Zone and Result in Lupus-like Disease

Expression of B cell-activating factor (BAFF), a critical B cell survival factor, is elevated in autoimmune and lymphoproliferative disorders. Mice overproducing BAFF develop systemic lupus erythematosus (SLE)-like disease and exhibit B cell activation of classical and alternative NF-kappaB-signaling pathways. We used a genetic approach and found that both NF-kappaB-signaling pathways contributed to disease development but act through distinct mechanisms. Whereas BAFF enhanced long-term B cell survival primarily through the alternative, but not the classical, NF-kappaB pathway, it promoted immunoglobulin class switching and generation of pathogenic antibodies through the classical pathway. Activation of the alternative NF-kappaB pathway resulted in integrin upregulation, thereby retaining autoreactive B cells in the splenic marginal zone, a compartment that contributes to their survival. Thus, both classical and alternative NF-kappaB signaling are important for development of lupus-like disease associated with BAFF overproduction. The same mechanisms may be involved in the pathogenesis of human SLE.

RelB/p52 NF-κB Complexes Rescue an Early Delay in Mammary Gland Development in Transgenic Mice with Targeted Superrepressor IκB-α Expression and Promote Carcinogenesis of the Mammary Gland

Classical NF-kappaB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-kappaB factors in breast development, we generated a transgenic mouse expressing the IkappaB-alpha S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IkappaB-alpha mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-kappaB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IkappaB-alpha-glutathione S-transferase, indicating that their weak interaction with IkappaB-alpha can account for the observed recovery of mammary gland development. Activation of IKKalpha and NF-kappaB-inducing kinase was detected by d5.5, implicating the alternative NF-kappaB signaling pathway in RelB/p52 induction. Constitutively active IKKalpha induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.