Noncanonical NF-kappaB Pathway Research Articles

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

Hematopoietic stem cell transplantation for RelB deficiency

Hematopoietic stem cell transplantation for RelB deficiency

Mediation of transitional B cell maturation in the absence of functional Bruton\u2019s tyrosine kinase

X-linked immune-deficient (Xid) mice, carrying a mutation in Bruton’s tyrosine kinase (Btk), have multiple B cell lineage differentiation defects. We now show that, while Xid mice showed only mild reduction in the frequency of the late transitional (T2) stage of peripheral B cells, the defect became severe when the Xid genotype was combined with either a CD40-null, a TCRbeta-null or an MHC class II (MHCII)-null genotype. Purified Xid T1 and T2 B cells survived poorly in vitro compared to wild-type (WT) cells. BAFF rescued WT but not Xid T1 and T2 B cells from death in culture, while CD40 ligation equivalently rescued both. Xid transitional B cells ex vivo showed low levels of the p100 protein substrate for non-canonical NF-kappaB signalling. In vitro, CD40 ligation induced equivalent activation of the canonical but not of the non-canonical NF-kappaB pathway in Xid and WT T1 and T2 B cells. CD40 ligation efficiently rescued p100-null T1 B cells from neglect-induced death in vitro. These data indicate that CD40-mediated signals, likely from CD4 T cells, can mediate peripheral transitional B cell maturation independent of Btk and the non-canonical NF-kappaB pathway, and thus contribute to the understanding of the complexities of peripheral B cell maturation.

A decrease in cellular microRNA-27a content is involved in azacytidine-induced P-glycoprotein expression in SKM-1 cells

We established an azacytidine (AzaC)-resistant human acute myeloid leukemia (AML) cell line (SKM-1/AzaC) by culturing SKM-1 cells in the presence of increasing amounts of AzaC for six months. Because AzaC is not a substrate of P-glycoprotein (a product of the ABCB1 gene; ABCB1), ABCB1 was not responsible for AzaC resistance; nevertheless, it was notably upregulated in SKM-1/AzaC cells. In addition, the transcription of the Nfkb1 gene, which encodes a member of the canonical NF-kappaB regulatory pathway, was downregulated, and the transcription of the Nfkb2 gene, which encodes a member of the non-canonical NF-kappaB regulatory pathway, was upregulated in SKM-1/AzaC cells. Here, we investigate whether miRNA-27a and miRNA-138 (both of which are known to be regulators of ABCB1 expression) are involved in the regulation of ABCB1 expression in SKM-1/AzaC cells. We observed decreased levels of miRNA-27a but of not miRNA-138 in SKM-1/AzaC cells compared with SKM-1 cells. The transfection of SKM-1/AzaC cells with a miRNA-27a mimic induced the downregulation of the ABCB1 mRNA. This was associated with an increase in Nfkb1 and a decrease in Nfkb2 transcript levels in SKM-1/AzaC cells. Taken together, these data indicate that the downregulation of miRNA-27a is involved in the upregulation of ABCB1 expression in SKM-1/AzaC cells, and this effect is associated with a switch between the canonical and non-canonical NF-kappaB pathways.

Mechanism Study of Matrix Metalloproteinase 13 Effects on Osteoclast Activation and Lytic Bone Lesions in Multiple Myeloma

Background: We have recently shown that Matrix metalloproteinase-13 (MMP-13) is highly expressed in human multiple myeloma (MM) cells and MMP-13 increases osteoclast (OCL) fusion and bone resorption independently of its metalloproteinase activity. In a mouse model of MM bone disease, knockdown (KD) of MMP-13 in MM cells inhibited development of bone lytic lesions, suggesting that MMP-13 is a potential therapeutic target for the treatment of MM bone disease (MMBD). However, the effects of MMP-13 on the MM bone marrow (BM) niche and underlying mechanism by which MMP-13 induces OCL fusion remains undefined.Methods and Results: To delineate the effects of MMP-13 on MM BM microenvironment, we investigated the effects of MMP-13 on cell proliferation and differentiation of pre-OCL, pre-osteoblast and MM cells. WT or Mmp-13-/- mouse CD11b+ BM cells were cultured with or without MMP-13 for 3 days and pre-OCL proliferation was evaluated by WST-1 assay. Neither MMP-13 treatment nor Mmp-13 deficiency affected pre-OCL proliferation, indicating MMP-13 induces OCL activation rather by promoting cell fusion than by affecting cell proliferation. To test the effects of MMP-13 on osteoblast (OB) differentiation and mineralization, mouse pre-OB cell line MC3T3-E1 and primary pre-OB cells from WT or Mmp-13-/- mice BM were cultured with or without MMP-13 for 7-14d. Neither exogenous MMP-13 nor Mmp-13 knockout affected OB differentiation or mineralization in vitro. Consistent with the in vitro observation, our Rag2-/- mouse 5TGM1 MMBD model provided in vivo evidence that knockdown of MMP-13 in MM cells does not affect MM induced OB inhibition or impairment of bone formation. Further, the effects of MMP-13 on MM cell proliferation were also assessed both in vitro and in vivo. Treatment of human MM cell lines RPMI-8266, OPM2 and MM.1S, as well as mouse MM cell line 5TGM1 with MMP-13 for up to 3 days did not induce in vitro cell proliferation. Similarly, MM cell sensitivity against anti-MM agents bortezomib and pomalidomide was not affected by MMP-13 in vitro. But, MMP-13 KD in mouse 5TGM1 MM cell line caused significantly less in vivo tumor burden in Rag2-/- intratibial MMBD model compared to 5TGM1 empty vector control cells, which we speculate occurs as a consequence of effects secondary to decreased OCL activation within the bone microenvironment following MMP-13 silencing.Our previous experiments showed that MMP-13 induces OCL fusion independent of its enzymatic activity, however the underlying mechanism is unknown. To further analyze the mechanism of action immunofluorescence staining and cell fraction-immunoblotting assay demonstrated that extracellular MMP-13 undergoes cell surface binding and subsequent active intracellular translocation into the perinuclear area, further supporting the enzymatic activity independent mechanism of MMP-13 on OCL induction. Furthermore, analysis of MMP-13-induced signaling pathways mediating osteoclastogenesis revealed that MMP-13 activates multiple cell signaling pathways including MAPK, PI3K and NF-kappaB independent of its enzymatic activity. Interestingly, the noncanonical NF-kappaB pathway is selectively activated by either MMP-13 WT or an E223A catalytically-inactive mutant, but not any by any other MMPs or MMP-13 truncated non-functional mutants, indicating that signaling through the noncanonical NF-kappaB pathway in OCL is specific for MMP-13 and may play a role in MMP-13-driven OCL fusion. Further studies are underway to screen for MMP-13 receptors and binding partners in an effort to delineate the processes underlying MMP-13 signaling.Conclusion: Our results demonstrate that MM produced MMP-13 induces OCL activation and bone resorption without affecting OB inhibition and bone formation impairment as well as anti-neoplastic response of MM cells. MMP-13 silencing in MM protects bone structure, and inhibits MM progression in vivo, which may be secondary to decreased OCL activation. MMP-13 specifically acts on OCL fusion. Signaling study showed noncanonical NF-kappaB signaling is selectively activated by MMP-13 independently of its enzymatic activity, and may be critical for MMP-13-induced OCL fusion and activation. Further screening the potential cellular receptor and intracellular transportation mechanism of MMP-13 in OCL is ongoing. DisclosuresRoodman:Amgen: Consultancy; Eli Lilly: Research Funding. Lentzsch:Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

MicroRNAs modulate the noncanonical transcription factor NF-kappaB pathway by regulating expression of the kinase IKKalpha during macrophage differentiation.

MicroRNAs are key regulators in many biological processes including cell differentiation. Here we show that during human monocyte-macrophage differentiation, the expression of the microRNAs miR-223, miR-15a, and miR-16 is dramatically decreased, leading to increased expression of the serine-threonine kinase IKKα in macrophages. In macrophages, higher IKKα expression in conjunction with stabilization of the kinase NIK induces elevated p52. Due to low RelB transcription factor expression in untreated macrophages, high p52 expression represses the basal level of both canonical and noncanonical NF-κB target genes. However, proinflammatory stimuli in macrophages result in greater induction of noncanonical NF-κB target genes. Thus a decrease in certain microRNAs likely prevents macrophage hyperaction yet primes the macrophage for certain responses to proinflammatory stimuli.

Requiem Protein Links RelB/p52 and the Brm-type SWI/SNF Complex in a Noncanonical NF-κB Pathway

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.

Characterization of the t(7;12) chromosomal translocation in infant AMLs: coexpression of T-cell markers, distinct expression of homeobox genes and inhibition of the non-canonical Nf-kappaB pathway

Introduction: The chromosomal translocation t(7;12) was described in MLL negative acute myeloid leukemias of infancy associated with a very poor prognosis. The rearrangement involves the homeobox gene HLXB9 (7q36) and ETV6 (12p13).

TRAF2 and TRAF3 independently mediate Ig class switching driven by CD40

The isotype switch defect in CD40(-/-) mice is corrected by wild-type (WT) CD40 transgene, but not by a mutant CD40 transgene that does not bind tumor necrosis factor receptor-associated factors (TRAF) 2 and 3. To define the individual roles of TRAF2 and TRAF3 in CD40 activation of B cells, we introduced mutant CD40 transgenes that selectively lack the ability to bind TRAF2 (DeltaTR2), TRAF3 (DeltaTR3) or both (DeltaTR2,3) into B cells of CD40(-/-) mice. Serum IgG1 and IgE levels, IgG1 antibody response to sub-optimal doses of the T cell-dependent antigen keyhole limpet hemocyanin, germinal center formation, CD40-mediated proliferation, isotype switching and activation of the non-canonical NF-kappaB pathway were partially diminished in DeltaTR2 and DeltaTR3 mice and virtually absent in DeltaTR2,3 mice. These results suggest that TRAF2 and TRAF3 can each independently mediate class switch recombination (CSR) driven by CD40, but both are required for optimal CD40-driven isotype switching.

HOX-Gene Expression and Inhibition of the Non-Canonical NF-KappaB Pathway in t(7;12) Leukemias

The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age<2 years, no MLL/AF9, MLL/AF4 or MLL/ENL rearrangement in RT-PCR, no chromosomal 11q23 rearrangement and no trisomy 21. Positive samples were cloned and sequenced. A fusion transcript was found in approx. ~17% of patients. Common features of HLXB9/ETV6 positive leukemias in this cohort are poorly differentiated FAB subtypes, trisomy 19 karyotype of the leukemic cells, coexpression of the T-cell markers CD4 and CD7 as well as high expression of the un-rearranged HLXB9 gene. Quantitative PCR showed no upregulation of HOXA9 and MEIS1 in HLXB9/ETV6 positive patients as reported for MLL+ leukemias. The mechanism of HLXB9/ETV6 induced leukemogenesis remains unknown. We show that in vitro retroviral transduction of murine hematopoietic precursor cells with either HLXB9/ETV6 or HLXB9 or ETV6/RUNX1 was not transforming. Furthermore altered activity of the transcription factor family NF-kappaB has been demonstrated for various types of cancer including acute myeloid leukemias. Therefore we analysed the impact of the HLXB9/ETV6 fusion on the NF-kappaB signaling pathway in a 293T cell-system. HLXB9/ETV6 and HLXB9 showed inhibition of the NF-kappaB dependent transcription signal in a reporter assay, whereas the typical ALL-fusion ETV6/RUNX1 showed no influence. Analysis of nuclear extracts by western blot confirmed, that the inhibition of the NF-kappaB signal is due to a reduced nuclear translocation of the non-canonical NF-kappaB components p52 and RelB, whereas the canonical pathway remains not influenced.Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.

RelB-Dependent Stromal Cells Promote T-Cell Leukemogenesis

BackgroundThe Rel/NF-κB transcription factors are often activated in solid or hematological malignancies. In most cases, NF-κB activation is found in malignant cells and results from activation of the canonical NF-κB pathway, leading to RelA and/or c-Rel activation. Recently, NF-κB activity in inflammatory cells infiltrating solid tumors has been shown to contribute to solid tumor initiation and progression. Noncanonical NF-κB activation, which leads to RelB activation, has also been reported in breast carcinoma, prostate cancer, and lymphoid leukemia.Methodology/Principal FindingsHere we report a novel role for RelB in stromal cells that promote T-cell leukemogenesis. RelB deficiency delayed leukemia onset in the TEL-JAK2 transgenic mouse model of human T acute lymphoblastic leukemia. Bone marrow chimeric mouse experiments showed that RelB is not required in the hematopoietic compartment. In contrast, RelB plays a role in radio-resistant stromal cells to accelerate leukemia onset and increase disease severity.Conclusions/SignificanceThe present results are the first to uncover a role for RelB in the crosstalk between non-hematopoietic stromal cells and leukemic cells. Thus, besides its previously reported role intrinsic to specific cancer cells, the noncanonical NF-κB pathway may also play a pro-oncogenic role in cancer microenvironmental cells.

Cytomegalovirus induces abnormal chondrogenesis and osteogenesis during embryonic mandibular development

BackgroundHuman clinical studies and mouse models clearly demonstrate that cytomegalovirus (CMV) disrupts normal organ and tissue development. Although CMV is one of the most common causes of major birth defects in humans, little is presently known about the mechanism(s) underlying CMV-induced congenital malformations. Our prior studies have demonstrated that CMV infection of first branchial arch derivatives (salivary glands and teeth) induced severely abnormal phenotypes and that CMV has a particular tropism for neural crest-derived mesenchyme (NCM). Since early embryos are barely susceptible to CMV infection, and the extant evidence suggests that the differentiation program needs to be well underway for embryonic tissues to be susceptible to viral infection and viral-induced pathology, the aim of this study was to determine if first branchial arch NCM cells are susceptible to mCMV infection prior to differentiation of NCM derivatives.ResultsE11 mouse mandibular processes (MANs) were infected with mouse CMV (mCMV) for up to 16 days in vitro. mCMV infection of undifferentiated embryonic mouse MANs induced micrognathia consequent to decreased Meckel's cartilage chondrogenesis and mandibular osteogenesis. Specifically, mCMV infection resulted in aberrant stromal cellularity, a smaller, misshapen Meckel's cartilage, and mandibular bone and condylar dysmorphogenesis. Analysis of viral distribution indicates that mCMV primarily infects NCM cells and derivatives. Initial localization studies indicate that mCMV infection changed the cell-specific expression of FN, NF-κB2, RelA, RelB, and Shh and Smad7 proteins.ConclusionOur results indicate that mCMV dysregulation of key signaling pathways in primarily NCM cells and their derivatives severely disrupts mandibular morphogenesis and skeletogenesis. The pathogenesis appears to be centered around the canonical and noncanonical NF-κB pathways, and there is unusual juxtaposition of abnormal stromal cells and surrounding matrix. Moreover, since it is critically important that signaling molecules are expressed in appropriate cell populations during development, the aberrant localization of components of relevant signaling pathways may reveal the pathogenic mechanism underlying mandibular malformations.

Promiscuous Mutations Activate the Noncanonical NF-κB Pathway in Multiple Myeloma

Activation of NF-kappaB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kappaB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2 and activation of NFKB1, NFKB2, CD40, LTBR, TACI, and NIK that result primarily in constitutive activation of the noncanonical NF-kappaB pathway, with the single most common abnormality being inactivation of TRAF3. These results highlight the critical importance of the NF-kappaB pathway in the pathogenesis of multiple myeloma.

Introduction to NF-κB: players, pathways, perspectives

This article serves as an introduction to the collection of reviews on nuclear factor-kappaB (NF-kappaB). It provides an overview of the discovery and current status of NF-kappaB as a research topic. Described are the structures, activities and regulation of the proteins in the NF-kappaB family of transcription factors. NF-kappaB signaling is primarily regulated by inhibitor kappaB (IkappaB) proteins and the IkappaB kinase complex through two major pathways: the canonical and non-canonical NF-kappaB pathways. The organization and focus of articles included in the following reviews are described, as well as likely future areas of research interest on NF-kappaB.

Akt Regulates Basal and Induced Processing of NF-κB2 (p100) to p52

NF-kappaB is a family of transcription factors important for innate and adaptive immunity. NF-kappaB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-kappaB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IkappaB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKalpha and IKKbeta. The preferred substrate for IKKbeta is IkappaBalpha, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKalpha activates a non-canonical NF-kappaB pathway in which p100 (NF-kappaB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKalpha and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-kappaB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-kappaB activity.

Differential regulation of CCL21 in lymphoid/nonlymphoid tissues for effectively attracting T cells to peripheral tissues.

CC chemokine ligand 21 (CCL21)/secondary lymphoid chemokine (SLC), a ligand for CC chemokine receptor 7 (CCR7), has been demonstrated to play a vital role in the homing and localization of immune cells to lymphoid tissues, but its role in nonlymphoid tissues largely remains undefined. Here, we provide evidence that CCL21 in lymphoid and nonlymphoid tissues is differentially regulated by lymphotoxin-dependent (LT-dependent) and -independent mechanisms, respectively. This differential regulation is due to the selective regulation of the CCL21-Ser/CCL21a but not the CCL21-Leu/CCL21b gene by the LT and noncanonical NF-kappaB pathways. This alternate pathway, not dependent on LT or lymphocytes, leading to constitutive expression of CCL21 in nonlymphoid tissues, is critical for the initial recruitment of T lymphocytes to peripheral effector sites. CCL21 expression is subsequently further enhanced in a LT-dependent fashion following airway challenge, potentially facilitating a positive feedback loop to attract additional CCR7+ effector cells. These findings establish an essential role for CCL21 in the recruitment of effector T cells to peripheral tissues and suggest that LT-dependent and -independent regulation of CCL21 plays a role in balancing the central and peripheral immune responses between lymphoid and nonlymphoid tissues.