Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF-κB activation

The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.     

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.     

The intact strontium ranelate complex stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-κB activation

Strontium ranelate, a pharmaceutical agent shown in clinical trials to be effective in managing osteoporosis and reducing fracture risk in postmenopausal women, is relatively unique in its ability to both blunt bone resorption and stimulate bone formation. However, its mechanisms of action are largely unknown. As the nuclear factor-kappa B (NF-κB) activation antagonists both stimulate osteoblastic bone formation and repress osteoclastic bone resorption, we hypothesized that strontium ranelate may achieve its anabolic and anti-catabolic activities by modulating NF-κB activation in bone cells. In this study, osteoclast and osteoblast precursors were treated with intact strontium ranelate or its individual components sodium ranelate and/or strontium chloride, and its effect on in vitro osteoclastogenesis and osteoblastogenesis and on NF-κB activation quantified. Although the activity of strontium ranelate has been attributed to the release of strontium ions, low dose intact strontium ranelate complex, but not sodium ranelate and/or strontium chloride, potently antagonized NF-κB activation in osteoclasts and osteoblasts in vitro, and promoted osteoblast differentiation while suppressing osteoclast formation. Taken together, our data suggest a novel centralized mechanism by which strontium ranelate promotes osteoblast activity and suppresses osteoclastogenesis, based on suppression of NF-κB signal transduction. We further demonstrate that the biological actions of strontium ranelate may be related to low dose of the intact molecule rather than dissociation and release of strontium ions, as previously thought. These data may facilitate the development of additional novel pharmacological agents for the amelioration of osteoporosis, based on NF-κB blockade.     

NF-κB targeting for overcoming tumor resistance and normal tissues toxicity

Radiotherapy and chemotherapy are two famous modalities in tumor-targeted therapy that lead to systemic and local toxicities for normal tissues. Moreover, several studies have confirmed that exposure of the tumor to radiation or chemotherapy drugs stimulate some signaling pathways in the tumor microenvironment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. Nuclear factor kappa B (NF-κB) plays a central role in the regulation of inflammatory responses in both normal tissues and tumors via the release of several cytokines, regulation of prostaglandins, reduction/oxidation (redox) reactions, angiogenesis, and cell death. Upregulation of NF-κB in normal tissues causes an appearance of inflammatory reactions and oxidative stress, whereas it regulates angiogenesis and suppresses apoptosis, leading to resistance to subsequent doses of radiation or chemotherapy. Selective inhibition of NF-κB in experimental studies has shown promising results for tumor sensitization via apoptosis induction, inhibition of angiogenesis, and increasing delay of tumor growth. The use of some agents for NF-κB inhibition has been shown to alleviate radiation/chemotherapy toxicities in normal cells/ tissues. In this current review, we explained the pivotal role of NF-κB in both normal tissue toxicity and tumor resistance. We also discussed the promising strategies for overcoming these problems with regard to chemotherapy and radiotherapy.     

JNK-binding protein 1 regulates NF-κB activation through TRAF2 and TAK1

The mitogen-activated protein kinase (MAPK) cascades, including c-Jun N-terminal kinase (JNK), are composed of a MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Previously, we reported that JNK-binding protein 1 (JNKBP1) enhances JNK activation induced by the TGF-β-activated kinase1 (TAK1) MAPKKK in transfected cells. We have investigated whether JNKBP1 functions as an adaptor protein for nuclear factor (NF)-κB activation mediated by TAK1 in COS-7 cells. Co-expression experiments showed that JNKBP1 interacted with not only TAK1, but also with its upstream regulators, TNF-receptor associated factors 2 and 6 (TRAF2 and TRAF6). An endogenous interaction between JNKBP1 and TRAF2 or TAK1 was confirmed by immunoprecipitation analysis. We also found that JNKBP1 could enhance the NF-κB activation induced by TAK1 and TRAF2, and could promote TRAF2 polyubiquitination. These results suggest a scaffolding role for JNKBP1 in the TRAF2-TAK1-NF-κB signaling pathway.     

Tumor necrosis factor (TNF) receptor-associated factor 7 is required for TNFα-induced Jun NH2-terminal kinase activation and promotes cell death by regulating polyubiquitination and lysosomal degradation of c-FLIP protein

The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH(2)-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIP(L) and demonstrate that degradation of c-FLIP(L) also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIP(L) expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death.     

Protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α pathway attenuates tumor necrosis factor alpha-induced apoptosis in nucleus pulposus cells by activating autophagy

Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.     

Molecular cloning of rock bream's (Oplegnathus fasciatus) tumor necrosis factor receptor-associated factor 2 and its role in NF-κB activiation

Tumor necrosis factor receptor-associated factor 2 (TRAF2) acts as a transducer of tumor necrosis factor-α (TNF-α)-triggered cell signals which results in inflammation, cell proliferation and antiapoptotic response. In this study, we have cloned cDNA of rock bream (Oplegnathus fasciatus) TRAF2, and analyzed its function in activation of NF-κB. The full length cDNA of rock bream TRAF2 consisted of 95 bp 5' UTR, 335 bp 3' UTR, and 1563 bp ORF encoding 520 amino acids that contained N-terminal RING-type and TRAF-type zinc finger domains and a C-terminal TRAF domain. The deduced amino acid sequence of rock bream TRAF2 showed more than 75% identity with other fish TRAF2s, and even as high as 56% identity with mouse and human TRAF2 proteins. To know whether the rock bream TRAF2 involves in NF-κB activation, Epithelioma papulosum cyprini (EPC) cells harboring an NF-κB reporting vector were transfected with a vector expressing rock bream TRAF2 or a control empty vector. NF-κB activity of EPC cells was significantly increased by exposure to the rock bream recombinant TNF-α. EPC cells transfected with the vector expressing rock bream TRAF2 showed significantly higher NF-κB activity by stimulation with the recombinant TNF-α than cells transfected with a control empty vector, suggesting the present rock bream TRAF2 acts as a transducer of TNF-α-mediated cell signals that enhance NF-κB activation.     

Cross-talk between receptor tyrosine kinase and tumor necrosis factor-α signaling networks regulates apoptosis but not proliferation

Although many of the signaling networks activated by receptor tyrosine kinases (RTKs) and cytokine receptors are well understood, how these networks interconnect is much less clear. We set out to determine how cells respond to simultaneous exposure to opposing signals and how their downstream networks process this information. Using six isogenic cell lines, each stably transfected with a different RTK, we found that, in each case, the cognate growth factor induced proliferation, whereas TNFα induced apoptosis. Surprisingly, when the cells were treated simultaneously with growth factor and TNFα, the growth factor enhanced, rather than antagonized, TNFα-induced cell death. In contrast, TNFα had no effect on growth factor-induced proliferation, suggesting that cross-talk between these networks is unidirectional. A quantitative, system-wide study of signaling at early and late time points corroborated this observation: proteins in the RTK networks were not affected by TNFα treatment, but proteins in the TNFα network were affected by growth factors. These studies also highlighted the stress mitogen-activated protein kinase proteins p38 and c-Jun N-terminal kinase as the key nodes of signal integration, and their activation states at an early time point correlated well with subsequent measurements of apoptosis. Knocking down cRaf reduced the growth factor enhancement of TNFα-induced apoptosis, highlighting its role as a regulator of network cross-talk upstream of p38 and c-Jun N-terminal kinase. Overall, we found that when cells encounter conflicting stimuli, their phenotypic response is determined not by the sum of isolated processes, but by how their signaling networks interconnect. This underscores the need to build mechanistic models of network integration as a first step in predicting cellular behavior in complex settings and in rationally designing combination therapies.     

The MC159 protein from the molluscum contagiosum poxvirus inhibits NF-κB activation by interacting with the IκB kinase complex

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immunoevasion proteins. For example, the MCV MC159 protein inhibits TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show in this article that, in human embryonic kidney 293 T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when using mouse embryonic fibroblasts that lack either IKKα or IKKβ, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ coimmunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159's inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.     

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.     

Sam68 is required for both NF-κB activation and apoptosis signaling by the TNF receptor

The RNA-binding protein Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, and signal transduction. Here we identify a role of Sam68 in TNF-induced NF-κB activation and apoptosis. We found that Sam68 is recruited to the TNF receptor, and its deficiency dramatically reduces RIP recruitment and ubiquitylation. It also impairs cIAP1 recruitment and maintenance of recruited TRAF2 at the TNF receptor. In its absence, activation of the TAK1-IKK kinase complex is defective, greatly reducing signal transduction. Sam68 is also found as a part of the TNF-induced cytoplasmic caspase-8-FADD complex. RIP is not recruited to this complex in Sam68 knockout cells, and caspase activation is virtually absent. These findings delineate previously unknown functions for Sam68 in the TNF signaling pathway, where it acts as a signaling adaptor both in the membrane-associated complex I and in the cytoplasmic complex II, regulating both NF-κB activation and apoptosis.     

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.