TNFR-associated factor 2 deficiency in B lymphocytes predisposes to chronic lymphocytic leukemia/small lymphocytic lymphoma in mice

We have previously shown that transgenic (tg) mice expressing in B lymphocytes both BCL-2 and a TNFR-associated factor 2 (TRAF2) mutant lacking the really interesting new gene and zinc finger domains (TRAF2DN) develop small lymphocytic lymphoma and chronic lymphocytic leukemia with high incidence (Zapata et al. 2004. Proc. Nat. Acad. Sci. USA 101: 16600-16605). Further analysis of the expression of TRAF2 and TRAF2DN in purified B cells demonstrated that expression of both endogenous TRAF2 and tg TRAF2DN was negligible in Traf2DN-tg B cells compared with wild-type mice. This was the result of proteasome-dependent degradation, and rendered TRAF2DN B cells as bona fide TRAF2-deficient B cells. Similar to B cells with targeted Traf2 deletion, Traf2DN-tg mice show expanded marginal zone B cell population and have constitutive p100 NF-κB2 processing. Also, TRAF3, X-linked inhibitor of apoptosis, and Bcl-X(L) expression levels were increased, whereas cellular inhibitors of apoptosis 1 and 2 levels were drastically reduced compared with those found in wild-type B cells. Moreover, consistent with previous results, we also show that TRAF2 was required for efficient JNK and ERK activation in response to CD40 engagement. However, TRAF2 was deleterious for BCR-mediated activation of these kinases. In contrast, TRAF2 deficiency had no effect on CD40-mediated p38 MAPK activation but significantly reduced BCR-mediated p38 activation. Finally, we further confirm that TRAF2 was required for CD40-mediated proliferation, but its absence relieved B cells of the need for B cell activating factor for survival. Altogether, our results suggest that TRAF2 deficiency cooperates with BCL-2 in promoting chronic lymphocytic leukemia/small lymphocytic lymphoma in mice, possibly by specifically enforcing marginal zone B cell accumulation, increasing X-linked inhibitor of apoptosis expression, and rendering B cells independent of B cell activating factor for survival.     

Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L), inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.     

Overexpression of BCL2 blocks TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells

The tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL or Apo2L) and its receptors are members of the tumor necrosis factor superfamily. TRAIL triggers apoptosis by binding to its two proapoptotic receptors DR4 and DR5, a process which is negatively regulated by binding of TRAIL to its two decoy receptors TRID and TRUNDD. Here, we show that TRAIL effectively induces apoptosis in H460 human non-small-cell lung carcinoma cells via cleavage of caspases 8, 9, 7, 3, and BID, release of cytochrome c from the mitochondria, and cleavage of poly (ADP-ribose) polymerase (PARP). However, overexpression of Bcl2 blocked TRAIL-induced apoptosis in H460 cells, which correlated with the Bcl2 protein levels. Importantly, the release of cytochrome c and cleavage of caspase 7 triggered by TRAIL were considerably blocked in Bcl2 overexpressing cells as compared to vector control cells. Moreover, inhibition of TRAIL-mediated cytochrome c release and caspase 7 activation by Bcl2 correlated with the inability of PARP to be cleaved and the inability of the Bcl2 transfectants to undergo apoptosis. Thus, these results suggest that Bcl2 can serve an anti-apoptotic function during TRAIL-dependent apoptosis by inhibiting the release of cytochrome c and activation of caspase 7, thereby blocking caspase 7-dependent cleavage of cellular substrates.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.     

Inhibition of nuclear factor-kappaB activation is essential for membrane-associated TNF-alpha-induced apoptosis in HL-60 cells

The killing of tumour cells that are resistant to soluble TNF-alpha (sTNF-alpha) by the membrane-bound form of TNF-alpha (mTNF-alpha) suggests that different intracellular signalling pathways are involved. We found that mTNF-alpha induced apoptosis in HL-60 cells and failed to cause degradation of inhibitor of kappa B alpha (IkappaB-alpha) and translocation and activation of nuclear factor kappa B (NF-kappaB), whereas sTNF-alpha failed to induce apoptosis, but lowered cytoplasmic inhibitor of kappa B alpha, induced translocation of NF-kappaB to the nucleus and experimentally increased activity of the regulated luciferase. Furthermore, mTNF-alpha upregulated the expression of TNF receptor associated factor (TRAF) 1 and failed to induce TRAF1 and TRAF2 membrane translocation, but led to cytoplasmic colocalization. In contrast, sTNF-alpha stimulated the expression of TRAF1 and TRAF2, recruiting both molecules onto the cell membrane poststimulation. These results suggest that the increased susceptibility of HL-60 cells to mTNF-alpha may be due to the failure of TRAF2 membrane translocation caused by the upregulation of TRAF1 expression and formation of a TRAF1/TRAF2 complex in the cytoplasm, thereby inhibiting NF-kappaB activation and inducing apoptosis.     

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.     

TNF receptor-associated factor 3 is required for T cell-mediated immunity and TCR/CD28 signaling

We recently reported that TNFR-associated factor (TRAF)3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. In this article, we report the generation and characterization of T cell-specific TRAF3(-/-) mice, in which the traf3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell lineage did not affect CD4 or CD8 T cell populations in secondary lymphoid organs or the numbers or proportions of CD4(+),CD8(+) or double-positive or double-negative thymocytes, except that the T cell-specific TRAF3(-/-) mice had a 2-fold increase in FoxP3(+) T cells. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3-deficient mice exhibited defective IgG1 responses to a T-dependent Ag, as well as impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon costimulation and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity.     

TRAF1 improves cisplatin-induced acute kidney injury via inhibition of inflammation and metabolic disorders

BackgroundCisplatin-induced acute kidney injury (AKI) is a severe clinical complication with no satisfactory therapies in the clinic. Tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) plays a vital role in both inflammation and metabolism. However, the TRAF1 effect in cisplatin induced AKI needs to be evaluated.MethodsWe observed the role of TRAF1 in eight-week-old male mice and mouse proximal tubular cells both treated with cisplatin by examining the indicators associated with kidney injury, apoptosis, inflammation, and metabolism.ResultsTRAF1 expression was decreased in cisplatin-treated mice and mouse proximal tubular cells (mPTCs), suggesting a potential role of TRAF1 in cisplatin-associated kidney injury. TRAF1 overexpression significantly alleviated cisplatin-triggered AKI and renal tubular injury, as demonstrated by reduced serum creatinine (Scr) and urea nitrogen (BUN) levels, as well as the ameliorated histological damage and inhibited upregulation of NGAL and KIM-1. Moreover, the NF-κB activation and inflammatory cytokine production enhanced by cisplatin were significantly blunted by TRAF1. Meanwhile, the increased number of apoptotic cells and enhanced expression of BAX and cleaved Caspase-3 were markedly decreased by TRAF1 overexpression both in vivo and vitro. Additionally, a significant correction of the metabolic disturbance, including perturbations in energy generation and lipid and amino acid metabolism, was observed in the cisplatin-treated mice kidneys.ConclusionTRAF1 overexpression obviously attenuated cisplatin-induced nephrotoxicity, possibly by correcting the impaired metabolism, inhibiting inflammation, and blocking apoptosis in renal tubular cells.General significanceThese observations emphasize the novel mechanisms associated to metabolism and inflammation of TRAF1 in cisplatin-induced kidney injury.     

Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling

TNFR-associated factor 1 (TRAF1) is unique among the TRAF family, lacking most zinc-binding features, and showing marked up-regulation following activation signals. However, the biological roles that TRAF1 plays in immune cell signaling have been elusive, with many reports assigning contradictory roles to TRAF1. The overlapping binding site for TRAFs 1, 2, and 3 on many TNFR superfamily molecules, together with the early lethality of mice deficient in TRAFs 2 and 3, has complicated the quest for a clear understanding of the functions of TRAF1. Using a new method for gene targeting by homologous recombination in somatic cells, we produced and studied signaling by CD40 and its viral oncogenic mimic, latent membrane protein 1 (LMP1) in mouse B cell lines lacking TRAF1, TRAF2, or both TRAFs. Results indicate that TRAFs 1 and 2 cooperate in CD40-mediated activation of the B cell lines, with a dual deficiency leading to a markedly greater loss of function than that of either TRAF alone. In the absence of TRAF1, an increased amount of TRAF2 was recruited to lipid rafts, and subsequently, more robust degradation of TRAF2 and TRAF3 was induced in response to CD40 signaling. In contrast, LMP1 did not require either TRAFs 1 or 2 to induce activation. Taken together, our findings indicate that TRAF1 and TRAF2 cooperate in CD40 but not LMP1 signaling and suggest that cellular levels of TRAF1 may play an important role in modulating the degradation of TRAF2 and TRAF3 in response to signals from the TNFR superfamily.     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

Bcl10 is an essential regulator for A20 gene expression

A20, a tumor suppressor in several types of lymphomas, has been suggested to be an nuclear factor kappa B (NF-κB) target gene; conversely, the deubiquitylation activity of A20 is required for inhibition of Bcl10-mediated activation of NF-κB. BCL10, which is activated in a recurrent chromosomal translocation that causes human mucosa-associated lymphoid tissue lymphomas, is known to be essential for NF-κB activation in B cells. We report here that Bcl10 upregulates endogenous A20 gene expression in B lymphocytes upon B-cell receptor engagement of anti-IgM. Transient transfection assays in HEK 293 cells indicate that Bcl10 can activate the A20 promoter, which contains NF-κB-binding sites. We also construct a theoretical structure of mouse Bcl10 and analyze the structure by molecular modeling and molecular dynamics simulation. Lastly, we found that marginal zone B cells from BCL10-transgenic mice proliferate more readily than wild-type B cells, whereas, surprisingly, the transgenic follicular B cells from these mice proliferate comparably to wild-type cells. Collectively, our results indicate that Bcl10 is an essential regulator of A20 gene expression and B-cell proliferation mediated by B-cell receptor signaling.     

Overexpression of Bcl(XL) in B cells promotes Th1 response and exacerbates collagen-induced arthritis

B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.     

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells

The relationship between the mitogen‐activated protein kinase response, nuclear factor‐κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V‐Fluorescein isothiocyanate assays. Western‐blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine‐specific protein kinase, extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA‐NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl‐2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB‐DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase‐dependent pathway. Our study provides a possible anti‐cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine. Copyright © 2015 John Wiley & Sons, Ltd.