Staphylococcus Aureus Induces Osteoclastogenesis via the NF-κB Signaling Pathway.

BACKGROUND Osteomyelitis is one of the refractory diseases encountered in orthopedics, while Staphylococcus aureus (S. aureus) is the most common causative organism in osteomyelitis. However, the precise mechanisms underlying the bone loss caused by S. aureus infection have not been well defined. Here, we investigated the effect of S. aureus on osteoclast differentiation and the probable molecular mechanism. MATERIAL AND METHODS RAW 264.7 cells were treated for 5 days with live S. aureus, inactivated S. aureus, and S. aureus filtrate. Then, the formation of osteoclast-like cells and resorption pits was observed, and the expression of osteoclast-specific genes (TRAP, MMP-9, cathepsin K, CTR and Atp6v0d2) was detected by real-time PCR. Moreover, key proteins in the signaling pathway associated with osteoclast differentiation were detected with Western blot. RESULTS The data showed that live S. aureus, inactivated S. aureus, and S. aureus filtrate induced osteoclast formation, promoted bone resorption, and increased the expression of osteoclast-specific genes in a dose-dependent manner in the absence RANKL. In addition, we found that the S. aureus-induced osteoclastogenesis was related to the degradation of IκB-a, phosphorylation of NF-κB p65, and increased expression of NFATc1. Thus, we used JSH-23 to inhibit NF-κB transcriptional activity. The effect of the S. aureus-induced osteoclastogenesis and the expression of osteoclast-specific genes and NFATc1 were inhibited, which indicated that the NF-κB signaling pathway plays a role in S. aureus-induced osteoclastogenesis. CONCLUSIONS This study demonstrated that S. aureus induces osteoclastogenesis through its cell wall compound and secretion of small soluble molecules, and the NF-κB signaling pathway plays a role in this process.

Tumor-suppressor NFκB2 p100 interacts with ERK2 and stabilizes PTEN mRNA via inhibition of miR-494.

Emerging evidence from The Cancer Genome Atlas has revealed that nuclear factor κB2 (nfκb2) gene encoding p100 is genetically deleted or mutated in human cancers, implicating NFκB2 as a potential tumor suppressor. However, the molecular mechanism underlying the antitumorigenic action of p100 remains poorly understood. Here we report that p100 inhibits cancer cell anchorage-independent growth, a hallmark of cellular malignancy, by stabilizing the tumor-suppressor phosphatase and tensin homolog (PTEN) mRNA via a mechanism that is independent of p100's inhibitory role in NFκB activation. We further demonstrate that the regulatory effect of p100 on PTEN expression is mediated by its downregulation of miR-494 as a result of the inactivation of extracellular signal-regulated kinase 2 (ERK2), in turn leading to inhibition of c-Jun/activator protein-1-dependent transcriptional activity. Furthermore, we identify that p100 specifically interacts with non-phosphorylated ERK2 and prevents ERK2 phosphorylation and nuclear translocation. Moreover, the death domain at C-terminal of p100 is identified as being crucial and sufficient for its interaction with ERK2. Taken together, our findings provide novel mechanistic insights into the understanding of the tumor-suppressive role for NFκB2 p100.

Autophagy-induced RelB/p52 activation mediates tumour-associated macrophage repolarisation and suppression of hepatocellular carcinoma by natural compound baicalin.

The plasticity of tumour-associated macrophages (TAMs) has implicated an influential role in hepatocellular carcinoma (HCC). Repolarisation of TAM towards M1 phenotype characterises an immune-competent microenvironment that favours tumour regression. To investigate the role and mechanism of TAM repolarisation in suppression of HCC by a natural compound baicalin, Orthotopic HCC implantation model was used to investigate the effect of baicalin on HCC; liposome-clodronate was introduced to suppress macrophage populations in mice; bone marrow-derived monocytes (BMDMs) were induced to unpolarised, M1-like, M2-like macrophages and TAM using different conditioned medium. We observed that oral administration of baicalin (50 mg/kg) completely blocked orthotopic growth of implanted HCC. Suppression of HCC by baicalin was diminished when mice macrophage was removed by clodronate treatment. Baicalin induced repolarisation of TAM to M1-like phenotype without specific toxicity to either phenotype of macrophages. Baicalin initiated TAM reprogramming to M1-like macrophage, and promoted pro-inflammatory cytokines production. Co-culturing of HCC cells with baicalin-treated TAMs resulted in reduced proliferation and motility in HCC. Baicalin had minimal effect on derivation of macrophage polarisation factors by HCC cells, while directly induced repolarisation of TAM and M2-like macrophage. This effect was associated with elevated autophagy, and transcriptional activation of RelB/p52 pathway. Suppression of autophagy or RelB abolished skewing of baicalin-treated TAM. Autophagic degradation of TRAF2 in baicalin-treated TAM might be responsible for RelB/p52 activation. Our findings unveil the essential role of TAM repolarisation in suppressive effect of baicalin on HCC, which requires autophagy-associated activation of RelB/p52.

The NFKB Inducing Kinase Modulates Hematopoiesis During Stress.

The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress.

p100, a precursor of NF-κB2, inhibits c-Rel and reduces the expression of IL-23 in dendritic cells.

Nuclear factor κB regulates various genes involved in the immune response, inflammation, cell survival, and development. NF-κB activation is controlled by proteins possessing ankyrin repeats, such as IκBs. A precursor of the NF-κB2 (p52) subunit, p100, contains ankyrin repeats in its C-terminal portion and has been found to act as a cytoplasmic inhibitor of RelA in the canonical pathway of NF-κB activation. Here, we demonstrate that p100 also suppresses c-Rel function in dendritic cells. Expression of the p19 and p40 subunits of IL-23, a c-Rel-dependent cytokine, was enhanced in p100-deficient cells, although expression of a RelA-dependent cytokine, TNF-α, was reduced. Nuclear translocation of c-Rel was enhanced in p100-deficient cells. p100, and not the processed p52 form, associated with c-Rel in the steady state and dissociated immediately after lipopolysaccharide stimulation in wild-type dendritic cells. Four hours after the stimulation, p100 was newly synthesized and associated with c-Rel again. In cells expressing both c-Rel and RelA, c-Rel is preferentially suppressed by p100.

NF-κB inducing kinase, a central signaling component of the non-canonical pathway of NF-κB, contributes to ovarian cancer progression.

Ovarian cancer is one of the leading causes of female death and the development of novel therapeutic approaches is urgently required. Nuclear factor-κB (NF-κB) is constitutively activated in several types of cancer including ovarian cancer and is known to support the survival of cancer cells. However, molecular mechanisms of persistent activation of NF-κB in ovarian cancer remain largely unknown. We report here that, in addition to the previously reported canonical activation, NF-κB is activated through the noncanonical pathway in ovarian cancer cells. RNA interference-mediated silencing of NF-κB inducing kinase (NIK), a central regulator of the noncanonical pathway, reduced the NF-κB2/p52 DNA binding activity and NF-κB-dependent reporter gene expression as well as NF-κB target gene expression. Notably, anchorage-dependent and -independent cell growth was impaired in NIK-depleted cells. Depletion of NIK also suppressed tumor formation in the nude mouse xenograft assay. These results indicate that NIK plays a key role in constitutive NF-κB activation and the progression of ovarian cancer cells and suggest that NIK represents an attractive therapeutic target for ovarian cancer.

NF-κB p65/p52 plays a role in GDNF up-regulating Bcl-2 and Bcl-w expression in 6-OHDA-induced apoptosis of MN9D cell.

Glial-cell-line-derived neurotrophic factor (GDNF) has been shown to protect dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA) toxicity. The mechanism underlying the antiapoptosis role of GDNF still needs further studies. We previously observed that nuclear factor-kappaB (NF-κB) signaling pathway, i.e. p65/p52, mediated the antiapoptosis role of GDNF in MN9D cells. Here, the DA cell line MN9D was used to explore the mechanisms underlying NF-κB p65/p52-mediated protection role of GDNF in DA neurons. The results showed that GDNF pretreatment blocked the apoptotic effects induced by 6-OHDA, with the upregulation of the antiapoptotic protein, Bcl-2 and Bcl-w, as well as the downregulation of the proapoptotic proteins, Bax and Bad. Furthermore, when sip100 plasmids were transfected into MN9D cells to inhibit the expression of p100, which was the precursor of p52, the effects of GDNF on upregulating Bcl-2 and Bcl-w were attenuated. These results indicated that GDNF could protect MN9D cells from apoptosis induced by 6-OHDA via upregulating Bcl-2 and Bcl-w expressions and downregulating Bax and Bad expressions. Moreover, NF-κB p65/p52 signaling mediated the effects of GDNF on Bcl-2 and Bcl-w expressions.

Expression of Id proteins is regulated by the Bcl-3 proto-oncogene in prostate cancer.

B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. As high levels of Bcl-3 expression and activation have been detected in different types of human cancer, Bcl-3 has been labeled a proto-oncogene. Our study uncovered a markedly upregulated Bcl-3 expression in human prostate cancer (PCa), where inflammatory cell infiltration was observed. Elevated Bcl-3 expression in PCa was dependent on the proinflammatory cytokine interleukin-6-mediated STAT3 activation. Microarray analyses, using Bcl-3 knockdown in PCa cells, identified the inhibitor of DNA-binding (Id) family of helix-loop-helix proteins as potential Bcl-3-regulated genes. Bcl-3 knockdown reduced the abundance of Id-1 and Id-2 proteins and boosted PCa cells to be more receptive to undergoing apoptosis following treatment with anticancer drug. Our data imply that inactivation of Bcl-3 may lead to sensitization of cancer cells to chemotherapeutic drug-induced apoptosis, thus suggesting a potential therapeutic strategy in PCa treatment.

RelB/NF-κB2 regulates corticotropin-releasing hormone in the human placenta.

Placental CRH may be part of a clock that governs the length of human gestation. The mechanism underlying differential regulation of CRH in the human placenta is poorly understood. We report here that constitutively activated RelB/nuclear factor-κB2 (NF-κB)-2 (p100/p52) acts as an endogenous stimulatory signal to regulate CRH by binding to an NF-κB enhancer of CRH gene promoter in the human placenta. Nuclear staining of NF-κB2 and RelB in villous syncytiotrophoblasts and cytotrophoblasts was coupled with cytoplasmic CRH in syncytial knots of cytotrophoblasts. Chromatin immunoprecipitation identified that CRH gene associated with both RelB and NF-κB2 (p52). Dexamethasone increased synthesis and nuclear translocation of RelB and NF-κB2 (p52) and their association with the CRH gene. In contrast, progesterone, a down-regulator of placental CRH, repressed NF-κB2 (p100) processing, nuclear translocation of RelB and NF-κB2 (p52), and their association with the CRH gene. Luciferase reporter assay determined that the NF-κB enhancer of CRH was sufficient to regulate transcriptional activity of a heterologous promoter in primary cytotrophoblasts. RNA interference-mediated repression of RelB or NF-κB2 resulted in significant inhibition of CRH at both transcriptional and translational levels and prevented the dexamethasone-mediated up-regulation of CRH transcription and translation. These results suggest that the noncanonical NF-κB pathway regulates CRH production in the human placenta and is responsible for the positive regulation of CRH by glucocorticoids.

The IKKα-dependent NF-κB p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1.

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)ß- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKß-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.

Nuclear factor-κB2 represses Sp1-mediated transcription at the CD99 promoter.

Downregulation of the CD99 antigen on the surface of Hodgkin's lymphoma (HL) cells via EBV LMP1-mediated NF-κB suppression of Sp1 transcriptional activity is known to be associated with the appearance of pathogenic Reed-Sternberg cells. Here, we show that in addition, EBV LMP1 heterologous NF-κB activators such as CD30 and CD40 repress the CD99 promoter, which contains multiple Sp1-binding sites but no NF-κB binding sites. In addition, NF-κB-inducing kinase (NIK) repressed the CD99 promoter while NIK kinase mutants and JNK inhibitory protein failed to do so. Of the NF-κB subunits, NF-κB2 (p52) alone or in combination with other Rel subunits consistently inhibited the CD99, while NF-κB1 (p50) showed a marginal repressive effect. Furthermore, while transfection of LMP1 repressed the CD99 promoter in wild-type or NF-κB1 deficient MEFs, the same repression was not observed in NF-κB2 (p52)-deficient MEFs, indicating that NF-κB2 (p52) is required for LMP1-mediated repression of the CD99 promoter. Consistently, basal activity of the CD99 promoter was significantly higher in IKKα(-/-) and IKKß(-/-) MEFs, but not in IKKΓ(-/-) MEFs compared to the wild-type control MEFs. Sp1-binding sites were directly used in the repression, because a synthetic Sp1 reporter with 10 Sp1-binding sites from the CD99 promoter was repressed by LMP1 or p52 transfection. These data indicate that LMP1-mediated NF-κB2 exhibits the major inhibitory role in the transcription at the CD99 promoter.

The Nfkb1 and Nfkb2 proteins p105 and p100 function as the core of high-molecular-weight heterogeneous complexes.

Nfkb1 and Nfkb2 proteins p105 and p100 serve both as NF-kappaB precursors and inhibitors of NF-kappaB dimers. In a biochemical characterization of endogenous cytoplasmic and purified recombinant proteins, we found that p105 and p100 assemble into high-molecular-weight complexes that contribute to the regulation of all NF-kappaB isoforms. Unlike the classical inhibitors IkappaBalpha, -beta, and -epsilon, high-molecular-weight complexes of p105 and p100 proteins bind NF-kappaB subunits in two modes: through direct dimerization of Rel homology domain-containing NF-kappaB polypeptides and through interactions of the p105 and p100 ankyrin repeats with preformed NF-kappaB dimers, thereby mediating the bona fide IkappaB activities, IkappaBgamma and IkappaBdelta. Our biochemical evidence suggests an assembly pathway in which kinetic mechanisms control NF-kappaB dimer formation via processing and assembly of large complexes that contain IkappaB activities.

Cell-autonomous role for NF-kappa B in immature bone marrow B cells.

The NF-kappaB transcription factors have many essential functions in B cells, such as during differentiation and proliferation of Ag-challenged mature B cells, but also during final maturation of developing B cells in the spleen. Among the various specific functions NF-kappaB factors carry out in these biologic contexts, their ability to assure the survival of mature and maturing B cells in the periphery stands out. Less clear is what if any roles NF-kappaB factors play during earlier stages of B cell development in the bone marrow. Using mice deficient in both NF-kappaB1 and NF-kappaB2, which are thus partially compromised in both the classical and alternative activation pathways, we demonstrate a B cell-autonomous contribution of NF-kappaB to the survival of immature B cells in the bone marrow. NF-kappaB1 and NF-kappaB2 also play a role during the earlier transition from proB to late preB cells; however, in this context these factors do not act in a B cell-autonomous fashion. Although NF-kappaB1 and NF-kappaB2 are not absolutely required for survival and progression of immature B cells in the bone marrow, they nevertheless make a significant contribution that marks the beginning of the profound cell-autonomous control these factors exert during all subsequent stages of B cell development. Therefore, the lifelong dependency of B cells on NF-kappaB-mediated survival functions is set in motion at the time of first expression of a full BCR.

Reduced thymic Aire expression and abnormal NF-kappa B2 signaling in a model of systemic autoimmunity.

The thymic stromal niche normally directs the production and export of a self-tolerant T cell repertoire. Many models of spontaneous autoimmunity, however, develop thymic architectural abnormalities before disease onset. Although this is suspected to affect central tolerance induction, creating an autoimmune predisposition, in-depth analysis of the microenvironment within these thymi is lacking, such that the mechanisms and likely direct effects on the T cell repertoire are unknown or speculative. Here we show that NZB mice, the first described model for systemic autoimmunity, demonstrate a complex thymic phenotype, including a lack of the autoimmune regulator (Aire), early defects in thymic epithelial cell (TEC) expansion, and evidence for altered NF-kappaB2 signaling. Analysis of medullary TEC revealed a numerical loss of the Aire-expressing MHC class II(high) (mTEC-high) subset as well reduced Aire protein and mRNA per cell. RelB expression was also reduced, while chemokines CCL19 and CCL21 were increased. Unexpectedly, the proportion of cortex and medulla in the NZB mice was normal from 36 wk, despite worsening architectural abnormalities. These data show that the NZB defect is more complex than previously appreciated, segregating into early numerical TEC deficiencies that correct with age, late degeneration of the niche architecture that does not affect TEC number, and a persistent reduction in Aire and RelB expression per cell acquired upon mTEC-high differentiation.

Lack of nuclear factor-kappa B2/p100 causes a RelB-dependent block in early B lymphopoiesis.

Nuclear factor-kappaB (NF-kappaB) transcription factors regulate B-cell development and survival. However, whether they also have a role during early steps of B-cell differentiation is largely unclear. Here, we show that constitutive activation of the alternative NF-kappaB pathway in p100(-/-) knockin mice resulted in a block of early B-cell development at the transition from the pre-pro-B to the pro-B-cell stage due to enhanced RelB activity. Expression of the essential B-cell transcription factors EBF and in particular Pax5 was reduced in p100(-/-) B-cell precursors in a RelB-dependent manner, resulting in reduced mRNA levels of B lineage-specific genes. Moreover, enhanced RelB function in p100(-/-) B-cell precursors was accompanied by increased expression of B lineage-inappropriate genes, such as C/EBP alpha, correlating with a markedly increased myeloid differentiation potential of p100(-/-) progenitor B cells. Ectopic expression of Pax5 in hematopoietic progenitors restored early B-cell development in p100(-/-) bone marrow, suggesting that impaired early B lymphopoiesis in mice lacking the p100 inhibitor may be due to down-regulation of Pax5 expression. Thus, tightly controlled p100 processing and RelB activation is essential for normal B lymphopoiesis and lymphoid/myeloid lineage decision in bone marrow.

A novel mutation in the Nfkb2 gene generates an NF-kappa B2 "super repressor".

The noncanonical NF-kappaB pathway regulates the development and function of multiple organs and cell lineages. We have generated mice harboring a novel mutation in Nfkb2 that prevents the processing of the inhibitory precursor, p100, into the active subunit, p52. Mutant mice express a complex phenotype with abnormalities in a variety of tissues, and with a spectrum that is more severe than in mice carrying a targeted deletion of Nfkb2. Signaling through the noncanonical pathway is ablated due to the absence of p52, resulting in disorganized splenic architecture and disrupted B cell development. The inhibitory precursor form of NF-kappaB2 interacts with RelA, preventing activation of RelA dimers in response to both canonical and noncanonical stimuli, which in combination with p52 deficiency, results in defective lymph node formation and bone homeostasis. These findings demonstrate a key role for NF-kappaB2 in the regulation of RelA activation and suggest overlap in the function of NF-kappaB members in canonical and noncanonical pathway signaling.

NF-kappaB p50 and p52 regulate receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor-induced osteoclast precursor differentiation by activating c-Fos and NFATc1.

Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-kappaB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-kappaB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-kappaB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-kappaB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-kappaB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-kappaB should prevent RANKL- and TNF-induced bone resorption.

A transgenic model reveals important roles for the NF-kappa B alternative pathway (p100/p52) in mammary development and links to tumorigenesis.

A regulated pattern of nuclear factor kappaB (NF-kappaB) activation is essential for normal development of the mammary gland. An increase in NF-kappaB activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-kappaB pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-kappaB p100/p52 subunit in mammary epithelium using the beta-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-kappaB activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.

Rescue of TRAF3-null mice by p100 NF-kappa B deficiency.

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.

Regulation of naive T cell function by the NF-kappaB2 pathway.

T cell activation involves the orchestration of several signaling pathways, including that of the 'classical' transcription factor NF-kappaB components NF-kappaB1-RelA. The function of the 'nonclassical' NF-kappaB2-RelB pathway is less clear, although T cells lacking components of this pathway have activation defects. Here we show that mice deficient in NF-kappaB-inducing kinase have a complex phenotype consisting of immunosuppression mediated by CD25(-)Foxp3(-) memory CD4(+) cells and, in the absence of those cells, hyper-responsive naive CD4(+) T cells, which caused autoimmune lesions after adoptive transfer into hosts deficient in recombination-activating genes. Biochemical studies indicated involvement of a cell-intrinsic mechanism in which NF-kappaB2 (p100) limits nuclear translocation of NF-kappaB1-RelA and thereby functions as a regulatory 'brake' for the activation of naive T cells.