Soluble CXCL16 promotes TNF‐α‐induced apoptosis in DLBCL via the AMAD10‐NF‐κB regulatory feedback loop

We had previously identified that the co‐expression of transmembrane CXCL16 (TM‐CXCL16) and its receptor CXCR6 is an independent risk factor for poor survival in patients with diffuse large B‐cell lymphoma (DLBCL). However, the impact of the soluble form of CXCL16 (sCXCL16) on the pathogenesis of DLBCL remains unknown. In the present study, the synergistic effect of sCXCL16 and tumor necrosis factor α (TNF‐α) on apoptosis in DLBCL cell lines (OCI‐LY8 and OCI‐LY10) was investigated in vitro. sCXCL16 reinforced TNF‐α‐mediated inhibition of DLBCL cell proliferation, as determined by the cell counting kit‐8 assay. The results of annexin V staining showed that sCXCL16 enhanced TNF‐α‐induced apoptosis in OCI‐LY8 and OCI‐LY10 cells through a death receptor‐caspase signaling pathway. The results of gene microarray suggested a significant upregulation of differentially expressed genes in the TNF signaling pathway. sCXCL16 increased the concentration of extracellular TNF‐α by binding to CXCR6 to activate the nuclear factor‐κB (NF‐κB) signaling pathway. TNF‐α also induced the secretion of sCXCL16 by increasing the expression of ADAM10, which is known to cleave TM‐CXCL16 to yield sCXCL16. Moreover, bioinformatics analysis revealed that elevated TNF‐α and ADAM10 expression levels in tumor tissues predicted better survival in patients with DLBCL. Thus, our study suggests that sCXCL16 enhances TNF‐α‐induced apoptosis of DLBCL cells, which may involve a positive feedback loop consisting of TNF‐α, ADAM10, sCXCL16, and members of the NF‐κB pathway. sCXCL16 and TNF‐α may be used as prognostic markers in the clinic, and their combinational use is a promising approach in the context of DLBCL therapy.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

TNF‐α increases αvβ3 integrin expression and migration in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumour with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Tumour necrosis factor (TNF)‐α is a key cytokine involved in inflammation, immunity, cellular homeostasis and tumour progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. However, the effects of TNF‐α in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that TNF‐α increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of MAPK kinase (MEK), extracellular signal‐regulating kinase (ERK) and nuclear factor‐κB (NF‐κB) pathways after TNF‐α treatment were demonstrated, and TNF‐α‐induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK and NF‐κB cascades. Taken together, our results indicated that TNF‐α enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the MEK/ERK/NF‐κB signal transduction pathway. J. Cell. Physiol. 226: 792–799, 2011. © 2010 Wiley‐Liss, Inc.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

FAK activation is required for TNF‐α‐induced IL‐6 production in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. IL‐6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL‐6 production stimulated by TNF‐α in cultured myoblasts. TNF‐α caused concentration‐dependent increases in IL‐6 production. TNF‐α‐mediated IL‐6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3‐kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF‐κB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IκB protease inhibitor (L ‐1‐tosylamido‐2‐phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF‐α. TNF‐α increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF‐α activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, p65 phosphorylation, and κB‐luciferase activity. TNF‐α mediated an increase of κB‐luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF‐α increased IL‐6 production in myoblasts via the FAK/PI3K/Akt and NF‐κB signaling pathway. J. Cell. Physiol. 223: 389–396, 2010. © 2010 Wiley‐Liss, Inc.     

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF‐α–induced injury via inhibiting NF‐κB and JNK signals

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.     

Yes‐associated protein promotes tumour necrosis factor α–treated cementoblast mineralization partly by inactivating NF‐κB pathway

Cementum regeneration, as one of the most difficult challenges of periodontal regeneration, is influenced by inflammatory factors. Inflammation may hamper or promote periodontal tissue repair under different circumstances, as it is found to do in dentin‐pulp complex and bone tissue. Our team demonstrated that YAP promotes mineralization of OCCM, a cementoblast cell line. However, the effect of YAP on its mineralization under inflammatory microenvironment is unclear. In this study, cementogenesis in vitro was up‐regulated after transient TNF‐α treatment for 30 minutes. YAP expression also was increased by TNF‐α treatment. YAP overexpression promoted OCCM mineralization after the cells were transiently treated with TNF‐α because YAP overexpression inhibited NF‐κB pathway activity, while YAP knockdown elevated it. The inhibited mineralization potential and activated NF‐κB pathway activity by YAP knockdown also were partly rescued by the application of the NF‐κB inhibitor Bay 11‐7082. These results demonstrated that YAP plays a positive role in the mineralization of TNF‐α transiently treated cementoblast, partly by inhibiting the NF‐κB pathway activity.     

Negative regulation of NF‐κB action by Set9‐mediated lysine methylation of the RelA subunit

Proper regulation of NF‐κB activity is critical to maintain and balance the inflammatory response. Inactivation of the NF‐κB complex relies in part on the proteasome‐mediated degradation of promoter‐bound NF‐κB, but the detailed molecular mechanism initiating this process remains elusive. Here, we show that the methylation of the RelA subunit of NF‐κB has an important function in this process. Lysine methyltransferase Set9 physically associates with RelA in vitro and in vivo in response to TNF‐α stimulation. Mutational and mass spectrometric analyses reveal that RelA is monomethylated by Set9 at lysine residues 314 and 315 in vitro and in vivo. Methylation of RelA inhibits NF‐κB action by inducing the proteasome‐mediated degradation of promoter‐associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites prolongs DNA binding of NF‐κB and enhances TNF‐α‐induced expression of NF‐κB target genes. Together, these findings unveil a novel mechanism by which methylation of RelA dictates the turnover of NF‐κB and controls the NF‐κB‐mediated inflammatory response.     

TNF‐α promotes survival and migration of MSCs under oxidative stress via NF‐κB pathway to attenuate intimal hyperplasia in vein grafts

The oxidative stress caused by endothelial injury is involved in intimal hyperplasia (IH) in vein grafts. Mesenchymal stem cells (MSCs) can home to injured intima and promote endothelial repair. However, MSC apoptosis is increased accompanied by decreased functional activity under oxidative stress. Thus, we investigate whether tumour necrosis factor‐α (TNF‐α) can promote the survival and activity of MSCs under oxidative stress to reduce IH more effectively, and establish what role the NF‐κB pathway plays in this. In this study, we preconditioned MSCs with TNF‐α (^{TNF‐α‐PC}MSCs) for 24 hrs and measured the activation of the IKK/NF‐κB pathway. EdU and transwell assays were performed to assess proliferation and migration of ^{TNF‐α‐PC}MSCs. Apoptosis and migration of ^{TNF‐α‐PC}MSCs were evaluated in conditions of oxidative stress by analysis of the expression of Bcl‐2 and CXCR4 proteins. ^{TNF‐α‐PC}MSCs were transplanted into a vein graft model, so that cell homing could be tracked, and endothelial apoptosis and IH of vein grafts were measured. The results demonstrated that TNF‐α promotes proliferation and migration of MSCs. Furthermore, survival and migration of ^{TNF‐α‐PC}MSCs under oxidative stress were both enhanced. A greater number of MSCs migrated to the intima of vein grafts after preconditioning with TNF‐α, and the formation of neointima was significantly reduced. These effects could be partially abolished by IKK XII (NF‐κB inhibitor). All these results indicate that preconditioning with TNF‐α can promote survival and migration of MSCs under oxidative stress via the NF‐κB pathway and thus attenuate IH of vein grafts.     

Dose-specific effects of tumor necrosis factor alpha on osteogenic differentiation of mesenchymal stem cells

Objectives: To investigate tumor necrosis factor alpha (TNF‐α)‐induced changes in osteogenic differentiation from mesenchymal stem cells (MSCs). Materials and methods: Blockade of nuclear factor‐κB (NF‐κB) was achieved in ST2 murine MSCs via overexpression of the NF‐κB inhibitor, IκBα. Osteogenic differentiation was induced in IκBα‐overexpressing ST2 cells and normal ST2 cells when these cells were treated with TNF‐α at various concentrations. Expression levels of bone marker genes were determined using real time RT‐PCR and ALP activity assay. In vitro mineralization was performed to determine long‐term exposure to TNF‐α on mineral nodule formation. MTT assay was used to determine the changes in cell proliferation/survival. Results: Levels of Runx2, Osx, OC and ALP were up‐regulated in cell cultures treated with TNF‐α at lower concentrations, while down‐regulated in cell cultures treated with TNF‐α at higher concentrations. Blockade of NF‐κB signaling reversed the inhibitory effect observed in cell cultures treated with TNF‐α at higher concentrations, but showed no effect on cell cultures treated with TNF‐α at lower concentrations. In contrast, long‐term treatment of TNF‐α at all concentrations induced inhibitory effects on in vitro mineral nodule formation. MTT assay showed that TNF‐α inhibits proliferation/survival of mesenchymal stem cells when the NF‐κB signaling pathway is blocked. Conclusions: The binding of TNF‐α to its receptors results in the activation of multiple signaling pathways, which actively interact with each other to regulate the differentiation, proliferation, survival and apoptosis of MSCs.     

Inhibition of NF‐κB activity by HIV‐1 Vpr is dependent on Vpr binding protein

Numerous studies have reported that Vpr alters NF‐κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF‐κB, however, this effect is dependent on the particular manner of NF‐κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF‐α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF‐κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF‐α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV‐1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF‐α mediated viral replication, perhaps suggesting that other HIV‐1 components may antagonize these anti‐NF‐κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF‐κB pathways. J. Cell. Physiol. 228: 781–790, 2013. © 2012 Wiley Periodicals, Inc.     

TNF‐α induces matrix metalloproteinase‐9 expression in A549 cells: Role of TNFR1/TRAF2/PKCα‐dependent signaling pathways

Matrix metalloproteinases (MMPs), in particular MMP‐9, have been shown to be induced by cytokines, including TNF‐α and contributes to airway inflammation. However, the mechanisms underlying TNF‐α‐induced MMP‐9 expression in human A549 cells remain unclear. Here, we report that TNF‐α‐induced MMP‐9 gene expression was mediated through the TNFR1/TRAF2/PKCα‐dependent signaling pathways in A549 cells, determined by zymographic, RT‐PCR, and Western blotting analyses. TNF‐α‐induced MMP‐9 expression was reduced by pretreatment with a TNFR Ab. Furthermore, TNF‐α‐induced TNFR1 and TRAF2 complex formation was revealed by immunoprecipitation using an anti‐TNFR1 Ab followed by Western blot analysis against an anti‐TRAF2 or anti‐TNFR1 Ab. In addition, TNF‐α‐induced MMP‐9 expression was also reduced by pretreatment with the inhibitor of PKCα (Gö6983), c‐Src (PP1), EGFR (AG1478), or PI3K (LY294002) or transfection with siRNAs of PKCα, Src, EGFR, Akt, p65, p300, and c‐Jun. On the other hand, TNF‐α stimulated the phosphorylation of c‐Src, EGFR, Akt, JNK1/2, and c‐Jun, which were inhibited by pretreatment with Gö6983. We also showed that TNF‐α induced Akt translocation and the formation of an Akt/p65/p300 complex. Pretreatment with the inhibitor of JNK1/2 (SP600125) but not the inhibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or PI3K (LY294002), markedly inhibited TNF‐α‐induced c‐Jun mRNA levels. Taken together, these data suggest that in A549 cells, TNF‐α induces MMP‐9 expression via the TNFR1/TRAF2/PKCα‐dependent JNK1/2/c‐Jun and c‐Src/EGFR/PI3K/Akt pathways. J. Cell. Physiol. 454–464, 2010. © 2010 Wiley‐Liss, Inc.     

Interleukin‐12 P40 induces the expression of TNF‐α in microglia and macrophages

Recently, it has been found that overproduction of IL‐12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL‐12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF‐α in microglia. Interestingly, we have found that IL‐12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose‐dependently induced the production of TNF‐α in BV‐2 microglial cells. This induction of TNF‐α production was accompanied by an induction of TNF‐α mRNA. In addition to BV‐2 glial cells, p70, p402 and p40 also induced the production of TNF‐α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF‐κB and C/EBPb is important for the expression of TNF‐α in microglial cells, we investigated the effect of p40 on the activation of NF‐κB as well as C/EBPb. Activation of NF‐κB as well as C/EBPb by p40 and inhibition of p40‐induced expression of TNF‐α by Dp65, a dominant‐negative mutant of p65, and DC/EBPb, a dominant‐negative mutant of C/EBPb, suggests that p40 induces the expression of TNF‐α through the activation of NF‐κB and C/EBPb. This study delineates a novel role of IL‐12 p40 in inducing the expression of TNF‐α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases. Acknowledgements: This study was supported by NIH grants (NS39940 and AG19487).     

Z‐FA.FMK activates duodenal epithelial cell proliferation through oxidative stress,NF‐κB and IL‐1β in d‐GalN/TNF‐α‐administered mice

This study was designed to evaluate the effect of Z‐FA.FMK (benzyloxycarbonyl‐l ‐phenylalanyl‐alanine‐fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z‐FA.FMK, the third group received d ‐GalN (d ‐galactosamine) and TNF‐α (tumour necrosis factor‐α) and the fourth group was given both d ‐GalN/TNF‐α and Z‐FA.FMK. When d ‐GalN/TNF‐α was administered alone, we observed an increase in IL‐1β‐positive and active NF‐κB‐positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)‐positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z‐FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d ‐GalN/TNF‐α injection. On the contrary, the Z‐FA.FMK pretreatment before d ‐GalN/TNF‐α blocked these effects. Based on these findings, we suggest that Z‐FA.FMK might act as a proliferative mediator which is controlled by IL‐1β through NF‐κB and oxidative stress in duodenal epithelial cells of d ‐GalN/TNF‐α‐administered mice.