Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.     

Effects of gut barrier dysfunction and NF‐κB activation on aggravating mechanism of severe acute pancreatitis

OBJECTIVE: To study the effects of gut‐derived endotoxin translocation and NF‐κB activation on the aggravating mechanism of severe acute pancreatitis (SAP) and of treatment with pyrrolidine dithiocarbamate (PDTC) on rats with SAP. METHODS: SD rats were randomly divided into sham operation group (SO), SAP group, SAP + lipopolysaccharide(LPS) group, pyrrolidine dithiocarbamate (PDTC) treatment group and LPS group. Biochemical parameters and cytokines were examined in the serum. Multiple organs pathological slices were examined. Expression of NF‐κB mRNA in the liver tissue was detected by RT‐PCR. Activation of NF‐κB by the method of streptomycin avidin‐peroxidase (SP) and expression of NF‐κB p65 protein and its binding activity were analyzed by Western blot and electrophoretic mobidity shift assay (EMSA). RESULTS: Compared with sham operation group, the concentration of TNF‐α, alanine aminotransferase (ALT), and diamine oxidase (DAO) in serum significantly increased in SAP + LPS group (P < 0.05). Pathological changes were markedly observed in tissues and the expression of NF‐κB mRNA in the liver significantly increased (P < 0.05) also, the activation of NF‐κB and binding activity of NF‐κB p65 protein in the liver markedly increased (P < 0.01) in SAP + LPS group. Treatment with PDTC markedly reduced concentration of ALT, DAO and TNF‐α, and the expression of NF‐κB, and the pathologic scores, as well as significantly decreased the expression of NF‐κB p65 protein. CONCLUSION: The activation and overexpression of NF‐κB may participate in the aggravating mechanism of SAP. Treatment with PDTC has a protective effect on multiple organs damage in SAP.     

Tumor Necrosis Factor α–Induced MicroRNA‐18a Activates Rheumatoid Arthritis Synovial Fibroblasts Through a Feedback Loop in NF‐κB Signaling

Objective

To elucidate whether the microRNA (miRNA) cluster miR‐17–92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

RASFs were stimulated with tumor necrosis factor α (TNFα), and the expression and regulation of the miR‐17–92 cluster were studied using real‐time quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR‐17–92 and the expression of matrix‐degrading enzymes and cytokines was measured by quantitative PCR and enzyme‐linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF‐κB signaling was determined by reporter gene assays.

Results

We found that TNFα induces the expression of miR‐17–92 in RASFs in an NF‐κB–dependent manner. Transfection of RASFs with precursor molecules of single members of miR‐17–92 revealed significantly increased expression levels of matrix‐degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR‐18a (pre‐miR‐18a)–transfected RASFs. Using reporter gene assays, we identified the NF‐κB pathway inhibitor TNFα‐induced protein 3 as a new target of miR‐18a. In addition, pre‐miR‐18a–transfected RASFs showed stronger activation of NF‐κB signaling, both constitutively and in response to TNFα stimulation.

Conclusion

Our data suggest that the miR‐17–92–derived miR‐18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF‐κB signaling, with concomitant up‐regulation of matrix‐degrading enzymes and mediators of inflammation in RASFs.

     

Immunohistochemical study of nuclear factor‐κB expression in esophageal squamous cell carcinoma: prognostic significance and sensitivity to treatment with 5‐FU

Nuclear factor‐κB (NF‐κB) is expressed in many types of cancers. It has been suggested that the expression of NF‐κB is associated with poor prognosis and resistance to chemoradiation therapies. This study evaluated the relationship between the expression of NF‐κB and the prognosis and sensitivity of esophageal squamous cell carcinoma (ESCC) to chemotherapy. One hundred and nine ESCC specimens, from patients who had undergone radical esophagectomy, were divided into two groups depending on the expression of NF‐κB. Surgical data and prognosis were compared between the two groups. NF‐κB‐positive tumors were detected in 61.5% of the cases. In 69 patients with stage II and III disease, 41 patients who were NF‐κB‐positive showed poor survival. The sensitivity of esophageal squamous cell carcinoma cell lines to 5‐fluorouracil (5‐FU) was analyzed by their NF‐κB expression, and the effect of 5‐FU was evaluated on the proliferation and activity of two cell lines of cultured ESCCs expressing NF‐κB. ESCCs with activated NF‐κB had poor sensitivity to 5‐FU. These results suggest that the increased expression of NF‐κB is associated with poor prognosis in patients with ESCC. NF‐κB may be a target for ESCC therapy because of its selective expression in this type of cancer.     

Interleukin‐18 enhances monocyte tumor necrosis factor α and interleukin‐1β production induced by direct contact with T lymphocytes: Implications in rheumatoid arthritis

Objective

At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up‐regulation of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNFα). We examined the regulatory effects of IL‐18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA).

Methods

Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)–stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFα, IL‐1β, IL‐10, and IL‐18 were measured by enzyme‐linked immunosorbent assay. Expression of adhesion molecules, IL‐18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF‐κB p65, phosphorylated IκBα, and phosphatidylinositol 3‐kinase (PI 3‐kinase) p110 was analyzed by Western blotting.

Results

IL‐18 dose‐dependently enhanced the production of IL‐1β and TNFα, but not IL‐10, by monocytes following contact with RA synovial T cells or PHA‐prestimulated T cells. NF‐κB inhibitors N‐acetyl‐L ‐cysteine and Bay 11‐7085 and PI 3‐kinase inhibitor LY294002 inhibited the enhancing effects of IL‐18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF‐κB in the nucleus, phosphorylated IκB, and PI 3‐kinase were confirmed in monocytes cocultured with PHA‐prestimulated T cells, and the levels were further increased by stimulation with IL‐18. Neutralizing antibody to IL‐18 inhibited monocyte activation induced by direct contact with PHA‐prestimulated T cells. Via cell–cell contact, PHA‐prestimulated T cells increased autocrine production of IL‐18 by monocytes, which was mediated by activation of the NF‐κB and PI 3‐kinase pathways, and up‐regulated the expression of the IL‐18 receptor in monocytes. IL‐18 up‐regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) on monocytes. Blocking the binding of the TNF receptors VCAM‐1 or ICAM‐1 on monocytes to their ligands on stimulated T cells suppressed the IL‐18–enhanced production of TNFα and IL‐1β in monocytes induced by contact with PHA‐prestimulated T cells.

Conclusion

IL‐18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF‐κB and PI 3‐kinase pathways. IL‐18 up‐regulates the expression of the TNF receptors VCAM‐1 and ICAM‐1 on monocytes, which mediate the enhancing effects of IL‐18 on T cell–monocyte contact.

     

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

Objective

Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl‐1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor α (TNFα)–induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG) in down‐regulating Mcl‐1 expression and its mechanism of RA synovial fibroblast sensitization to TNFα‐induced apoptosis.

Methods

EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP‐ribose) polymerase (PARP), Mcl‐1, Bcl‐2, Akt, and nuclear translocation of NF‐κB.

Results

In RA synovial fibroblasts, EGCG (5–50 μM) inhibited constitutive and TNFα‐induced Mcl‐1 protein expression in a concentration‐ and time‐dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl‐1 expression in RA synovial fibroblasts and affected Mcl‐1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl‐1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down‐regulation of the TNFα‐induced Akt and NF‐κB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF‐κB inhibitors. Interestingly, Mcl‐1 degradation by EGCG sensitized RA synovial fibroblasts to TNFα‐induced PARP cleavage and apoptotic cell death.

Conclusion

Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFα‐induced apoptosis by specifically blocking Mcl‐1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

     

Anti-inflammatory effects of moxifloxacin on rat airway smooth muscle cells exposed to allergen: Inhibition of extracellular-signal-regulated kinase and nuclear factor-κB activation and of interleukin-8 and eotaxin synthesis

Background and objective: Moxifloxacin (MXF) has been shown to possess immunomodulatory properties in addition to its antimicrobial effects. We investigated the effects of MXF on cytokine secretion and signal transduction mechanisms in naive control and allergen‐exposed airway smooth muscle cell (ASMC) stimulated with tumour necrosis factor (TNF)‐α. Methods: An animal model was established. ASMC was derived from rat airway tissue and cultured in vitro, then incubated with 10 ng/mL of TNF‐α. Interleukin (IL)‐8 and eotaxin secretion were measured by enzyme‐linked immunosorbent assay, and activation of extracellular‐signal‐regulated kinase (ERK)1/2 and nuclear factor (NF)‐κB p65 was measured by western blotting, with or without the addition of MXF (20 µg/mL) and/or dexamethasone (DXM) (10^?6 M). Results: Baseline IL‐8 and eotaxin secretion did not differ between control and allergen‐exposed cells. Stimulation with TNF‐α increased IL‐8 and eotaxin secretion, with increased IL‐8 secretion by allergen‐exposed compared with naive control ASMC, post‐TNF‐α stimulation (P = 0.001). Baseline phosphorylation of ERK1/2 (p‐ERK1/2) and NF‐κB p65 was higher in allergen‐exposed than in control ASMC. TNF‐α increased p‐ERK1/2 and NF‐κB p65 levels, with higher levels in allergen‐exposed ASMC, post‐TNF‐α stimulation (P < 0.001). MXF and the combination of MXF with DXM suppressed the secretion of IL‐8 and eotaxin, but DXM alone did not affect IL‐8, post‐TNF‐α stimulation (P > 0.05). MXF, DXM and the combination of MXF with DXM inhibited TNF‐α‐stimulated p‐ERK1/2 and NF‐κB p65 levels by 34, 40 and 62%, and 33, 38 and 64%, respectively. Conclusions: MXF suppressed the secretion of pro‐inflammatory cytokines by allergen‐exposed rat ASMC, partly by inhibiting NF‐κB and ERK activation. DXM may have additional or synergistic effects with MXF.     

Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

NF‐κB protects Behçet's disease T cells against CD95‐induced apoptosis up‐regulating antiapoptotic proteins

Objective

To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up‐regulation of antiapoptotic factors.

Methods

The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF‐κB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down‐regulate NF‐κB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF‐κB small interfering RNA.

Results

Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95‐induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up‐regulation of the short form of cellular FLIP (cFLIP) and Bcl‐x_L in BD activated T cells, while levels of Bcl‐2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IκB was up‐regulated, whereas NF‐κB translocated to the nucleus in BD T cells, suggesting that NF‐κB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF‐κB small interfering RNA down‐regulated cFLIP and Bcl‐x_L expression levels and sensitized BD activated T cells to CD95‐induced apoptosis.

Conclusion

Taken together, these results indicate that NF‐κB contributes to the regulation of the apoptosis‐related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF‐κB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune‐mediated disease.

     

Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor‐α in galactosamine‐sensitized mice

Abstract: Background/Aims: To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor‐α (TNF‐α), caspase cascade and ceramide formation were investigated in the liver of D‐galactosamine (GalN)‐sensitized mice treated with TNF‐α. Methods: Seven‐week‐old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF‐α (0.5 μg/mouse). Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using chromogenic peptides as substrates. Ceramide content was determined using Escherichia coli diacylglycerol kinase. Results: Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF‐α (GalN/TNF‐α), but not GalN or TNF‐α alone. Activation of caspases‐9 and ‐3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF‐α. In a cell‐free system, processing of procaspases‐9 and ‐3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF‐α‐treated mice, but not in that from control mice. Processing of procaspase‐3 was inhibited by a caspase‐9 inhibitor, but not by inhibitor for caspase‐8 or ‐2. In a reconstitution assay system, procaspase‐9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF‐α. Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation. Conclusion: Cytochrome c release and caspase‐9 activation are required for the activation of executor caspase‐3 in TNF‐α‐induced hepatocyte apoptosis, but caspases‐8 and ‐2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process.     

NF‐κB–regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α–mediated apoptosis

Objective

Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor α (TNFα) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFα. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFα‐induced NF‐κB controls the expression of FLIP long (FLIP_L) and FLIP short (FLIP_S) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFα‐induced apoptosis.

Methods

RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super‐repressor IκBα (srIκBα). The cells were stimulated with TNFα or a control vehicle, and expression of FLIP_L and FLIP_S was determined by isoform‐specific real‐time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3.

Results

TNFα induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIP_L was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFα induced the expression of FLIP_L and FLIP_S mRNA and protein. The TNFα‐induced, but not the basal, expression of FLIP was regulated by NF‐κB. When NF‐κB activation was suppressed by the expression of srIκBα, TNFα‐mediated apoptosis was induced. TNFα‐induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP_L or the caspase 8 inhibitor CrmA.

Conclusion

The TNFα‐induced, but not the basal, expression of FLIP is regulated by NF‐κB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFα‐induced apoptosis is mediated by the NF‐κB–regulated expression of FLIP. These observations support the role of NF‐κB and FLIP as attractive therapeutic targets in RA.

     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

Clinicopathological significance of nuclear factor‐κB activation in hepatocellular carcinoma

Aim: Nuclear factor‐κB (NF‐κB) is a critical signaling mediator in inflammation, apoptosis resistance and oncogenesis. It has been reported that NF‐κB is activated in several cancers, including hepatocellular carcinoma (HCC). Studies of genetic disruptions in mice also suggest that NF‐κB plays critical roles in hepatocarcinogenesis. The aim of the present study is to characterize NF‐κB activation and correlate it with the degree of malignancy in HCC. Methods: To examine the correlation between the positivity of the nuclear p50 subunit and HCC recurrence, we analyzed immunostaining of the NF‐κB p50 subunit in two groups of HCC samples with known prognosis and Akt phosphorylation status: 49 patients showing early recurrence within 6 months (group A) and 50 patients who were recurrence‐free for at least for 3 years (group B). Results: In group A, positive nuclear staining of p50 was shown in 18 cases (36.7%), whereas only one case (2.0%) in group B had positive nuclear staining of p50 (P = 2.48839 × 10^–5). This suggests a positive relationship between nuclear p50 and early recurrence and advanced HCC in humans. The presence of phosphorylated Akt correlated with nuclear staining of p50 in HCCs in group A (R² = 0.213, P < 0.001). Conclusion: Our results indicate that nuclear staining of p50 was clearly associated with early recurrent HCC, and the Akt pathway might play a role in NF‐κB activation in a subset of early recurrent HCC.