Cross-linking of SIGNR1 activates JNK and induces TNF-α production in RAW264.7 cells that express SIGNR1

In this study, we evaluated the signaling ability of SIGNR1 in murine macrophage-like RAW264.7 cells that stably expressed FLAG-tagged SIGNR1 (SIGNR1-FLAG). Cross-linking of SIGNR1-FLAG expressed on the cells by an anti-FLAG antibody induced JNK phosphorylation without induction of phosphorylation of ERK1/2 and p38 MAP kinase, and led to phosphorylations of Src family kinases (SFKs) and Akt. The SIGNR1-FLAG molecules in the cells were found in lipid raft-enriched membrane fractions, and the tyrosine kinases Lyn, Hck, and Fgr co-precipitated with SIGNR1-FLAG in the lipid raft fractions. The antibody-induced JNK phosphorylation was inhibited by inhibitors of SFKs and tyrosine kinases. Furthermore, cross-linking of SIGNR1 led to production of TNF-α, and the JNK inhibitor inhibited the antibody-induced TNF-α production. These results show that cross-linking of SIGNR1 triggers phosphorylation of SFKs, which leads to activation of the JNK pathway and induction of TNF-α production in macrophage-like RAW264.7 cells.     

TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts

The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-α and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iα)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-α and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iα). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth.     

Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A¹, A², I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A¹, A² and I (25-200 μg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A¹, A² and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A¹, A² and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A¹, A² and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.     

The glucosamine-mediated induction of CHOP reduces the expression of inflammatory cytokines by modulating JNK and NF-κB in LPS-stimulated RAW264.7 cells

Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles in inflammation in early-stage rheumatoid arthritis (RA). This study investigated whether glucosamine hydrochloride (GlcN), a nonsteroidal anti-inflammatory agent (NSAID) widely used to treat arthritis, affects the expression of inflammatory cytokines via the unfolded protein response (UPR) in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells). Pretreatment with GlcN reduced the expression of inflammatory cytokines and inhibited cell differentiation. Moreover, GlcN treatment increased the expression of CHOP and BiP/Grp78, the UPR target genes, in the presence or absence of LPS. Indeed, knockdown of CHOP using siRNAs prevented the GlcN-mediated reduction of inflammatory cytokines in LPS-stimulated RAW264.7 cells. Finally, we found that GlcN-mediated induction of CHOP reduced the phosphorylation of JNK and NF-?B in LPS-stimulated RAW264.7 cells. Combined, these results suggest that the GlcN-mediated induction of CHOP negatively regulates the inflammatory response by modulating JNK and NF-?B in LPS-stimulated RAW264.7 cells.     

Orexin-A Promotes Cell Migration in Cultured Rat Astrocytes via Ca2+-Dependent PKCα and ERK1/2 Signals

Orexin-A is an important neuropeptide involved in the regulation of feeding, arousal, energy consuming, and reward seeking in the body. The effects of orexin-A have widely studied in neurons but not in astrocytes. Here, we report that OX1R and OX2R are expressed in cultured rat astrocytes. Orexin-A stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and then induced the migration of astrocytes via its receptor OX1R but not OX2R. Orexin-A-induced ERK1/2 phosphorylation and astrocytes migration are Ca²⁺-dependent, since they could be inhibited by either chelating the extracellular Ca²⁺ or blocking the pathway of store-operated calcium entry (SOCE). Furthermore, both non-selective protein kinase C (PKC) inhibitor and PKCα selective inhibitor, but not PKCδ inhibitor, prevented the increase in ERK1/2 phosphorylation and the migration of astrocytes, indicating that the Ca²⁺-dependent PKCα acts as the downstream of the OX1R activation and mediates the orexin-A-induced increase in ERK1/2 phosphorylation and cell migration. In conclusion, these results suggest that orexin-A can stimulate ERK1/2 phosphorylation and then facilitate the migration of astrocytes via PLC-PKCα signal pathway, providing new knowledge about the functions of the OX1R in astrocytes.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

The GRB2 associated binder 1 (GAB1) is an essential docking/adaptor protein for transmitting intracellular signals of the MET tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). We found that in response to hours of HGF/SF treatment, the GAB1 protein level is degraded by a mechanism involving MET activity and the proteasomal machinery. We also showed that GAB1 is both multi- and poly-ubiquitinated in a CBL-dependent manner. A long term exposure to HGF/SF caused a more sustained down-regulation of GAB1 than of MET, associated with a loss of reactivation of the ERK MAP kinases to subsequent acute ligand treatment. These data demonstrate that GAB1 is ubiquitinated by CBL and degraded by the proteasome, and plays a role in negative-feedback regulation of HGF/SF–MET signaling.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.     

Inhibitory mechanism of pure curcumin on Wilms' tumor 1 (WT1) gene expression through the PKCα signaling pathway in leukemic K562 cells

The aim of this study was to investigate the inhibitory mechanism of pure curcumin on WT1 expression in leukemic K562 cells. Pure curcumin suppressed WT1 expression, independent of effects on protein degradation or WT1 mRNA stability. Chromatin immunoprecipitation and reporter gene assays indicate that pure curcumin treatment attenuates WT1 auto-regulation. Interestingly, PKCα inhibition mimicks the repressive effects of pure curcumin in K562 cells. Conversely, myristoylated PKCα over-expression increased WT1 expression and reversed the inhibitory effect of pure curcumin. Our study indicates that pure curcumin attenuates WT1 auto-regulatory function through inhibition of PKCα signaling in K562 cells.     

Omentin inhibits TNF-α-induced expression of adhesion molecules in endothelial cells via ERK/NF-κB pathway

In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-α-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-α-activated signal pathway of nuclear factor-κB (NF-κB) by preventing NF-κB inhibitory protein (IκBα) degradation and NF-κB/DNA binding activity. Omentin pretreatment significantly inhibited TNF-α-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-α-induced NF-κB activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α. These results suggest that omentin may inhibit TNF-α-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-κB pathway.     

Phytoestrogens directly inhibit TNF-α-induced bone resorption in RAW264.7 cells by suppressing c-fos-induced NFATc1 expression

TNF-α-induced osteoclastogenesis is central to post-menopausal and inflammatory bone loss, however, the effect of phytoestrogens on TNF-α-induced bone resorption has not been studied. The phytoestrogens genistein, daidzein, and coumestrol directly suppressed TNF-α-induced osteoclastogenesis and bone resorption. TRAP positive osteoclast formation and resorption area were significantly reduced by genistein (10(-7) M), daidzein (10(-5) M), and coumestrol (10(-7) M), which was prevented by the estrogen antagonist ICI 182,780. TRAP expression in mature TNF-α-induced osteoclasts was also significantly reduced by these phytoestrogen concentrations. In addition, in the presence of ICI 182,780 genistein and coumestrol (10(-5) -10(-6) M) augmented TNF-α-induced osteoclast formation and resorption. However, this effect was not observed in the absence of estrogen antagonist indicating that genistein's and coumestrol's ER-dependent anti-osteoclastic action normally negates this pro-osteoclastic effect. To determine the mechanism mediating the anti-osteoclastic action we examined the effect of genistein, coumestrol, and daidzein on caspase 3/7 activity, cell viability and expression of key genes regulating osteoclast differentiation and fusion. While anti-osteoclastic phytoestrogen concentrations had no effect on caspase 3/7 activity or cell viability they did significantly reduce TNF-α-induced c-fos and NFATc1 expression in an ER dependent manner and also inhibited NFATc1 nuclear translocation. Significant decreases in NFκB and DC-STAMP levels were also noted. Interestingly, constitutive c-fos expression prevented the anti-osteoclastic action of phytoestrogens on differentiation, resorption and NFATc1. This suggests that phytoestrogens suppress TNF-α-induced osteoclastogenesis via inhibition of c-fos-dependent NFATc1 expression. Our data provides further evidence that phytoestrogens have a potential role in the treatment of post-menopausal and inflammatory bone loss directly inhibiting TNF-α-induced resorption.     

Analysis of isoform specific ERK signaling on the effects of interleukin-1β on COX-2 expression and PGE2 production in human chondrocytes

The MAPK/ERK pathway is involved in IL-1β-induced cyclooxygenase (COX-2) expression and prostaglandin E2 (PGE2) production; two factors that play important roles in OA pathogenesis. In the present study, we find that IL-1β induced COX-2 expression and PGE2 production in human chondrocytes via a process that required the activation of the MAPK/ERK pathway. To evaluate the respective roles and relationship of ERK1 and ERK2 on IL-1β induced COX-2 expression and PGE2 production, small interfering RNA was used to knockdown ERK1, ERK2 or both in human chondrocytes. COX-2 expression and PGE2 production were significantly suppressed to a similar degree by the silencing of ERK1 or ERK2 alone. Moreover, the combined knockdown displayed a synergistic effect. Simultaneously, Western blotting indicated that the knockdown of ERK1 or ERK2 down regulated phospho-ERK1 and ERK1 or phospho-ERK2 and ERK2 levels, respectively. No significant compensatory mechanism through the upregulation of the other phospho-ERK and ERK isoform was observed. The combined silencing suppressed both phospho-ERK1/2 and ERK1/2. In conclusion, each ERK isoform similarly influenced IL-1β-mediated COX-2 expression and PGE2 production in human chondrocytes, and ERK1 and ERK2 displayed synergistic effects. Although, inhibition of both ERK1 and ERK2 would be a more effective, each ERK isoform may sufficiently regulate these effects in human chondrocytes. ERK1 or ERK2 may be potential therapeutic target for the inflammatory process of OA.     

Silencing of TNF-α receptors coordinately suppresses TNF-α expression through NF-κB activation blockade in THP-1 macrophage

Persistently elevated level of TNF-α has been implicated in several inflammatory disorders, however, its autocrine production through TNF-α receptors signaling is poorly understood. Here we report that simultaneous silencing of TNF-receptors, R1 and R2 by DNAzyme or siRNA suppressed TNF-α expression more efficiently than silencing them individually in lipopolysaccharides (LPS) stimulated THP-1 macrophages. Co-silencing of TNF-receptors also inhibited TNF-α induced NF-κB activation to a higher extent. It was further observed that NF-κB inhibitor but not c-Jun N-terminal kinase inhibitor (SP600125) suppressed TNF-α expression. All these results suggest that TNF-α expression is regulated by synergistic signaling of TNF receptors through downstream NF-κB activation.     

Inhibitory effect of TNF-α on malaria pre-erythrocytic stage development: influence of host hepatocyte/parasite combinations

Background

The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages.

Methods and Findings

Human or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.

Conclusions

Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.