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Matilde Todaro Monica Zerilli Giovanni Triolo Flora Iovino Mariella Patti Antonina Accardo‐Palumbo Francesca di Gaudio Maria Caterina Turco Antonello Petrella Ruggero de Maria Giorgio Stassi 《Arthritis \u0026amp; Rheumatology》2005,52(7):2179-2191
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‐xL 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‐xL 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.2.
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Francesco Grassi Anna Piacentini Sandra Cristino Stefania Toneguzzi Andrea Facchini Gina Lisignoli 《Arthritis \u0026amp; Rheumatology》2004,50(2):498-506
Objective
Cartilage homeostasis dysregulation during osteoarthritis (OA) has been linked to an increased rate of apoptosis of chondrocytes, the only cell type resident in the cartilage. In addition, the CD95–CD95 ligand (the Fas system) has emerged as one of the major pathways of cell death in the cartilage. We undertook the present study to investigate the role of interferon‐γ (IFNγ) in the regulation of the Fas system by analyzing the modulation of intracellular signaling molecules (FLICE inhibitory protein [FLIP] and caspases 3 and 8) in primary cultures of human OA chondrocytes.Methods
CD95‐induced apoptotic death of human OA chondrocytes was analyzed in the presence or absence of IFNγ using cell death immunoassay for apoptosis, real‐time polymerase chain reaction for FLIP and caspase 8 expression, Western blotting for FLIP, and proteolytic activity for caspases 3 and 8.Results
CD95‐induced apoptotic death of human OA chondrocytes was strongly counteracted by IFNγ treatment, although the surface expression of CD95 was slightly up‐regulated by this cytokine. The messenger RNA (mRNA) expression of FLIP and caspase 8, mediators involved in CD95 signaling, revealed that FLIP expression in human OA chondrocytes was significantly up‐regulated (2‐fold increase) by IFNγ treatment. Moreover, the FLIP:caspase 8 mRNA ratio increased significantly. FLIP up‐regulation by IFNγ was confirmed at the protein level. Caspase 8 and caspase 3 proteolytic activities, both induced in these cells by stimulation with anti‐CD95, were also significantly down‐modulated by IFNγ.Conclusion
These findings suggest that IFNγ impairs CD95‐mediated signaling and apoptotic death in human chondrocytes. Its mechanism of action involves down‐regulation of caspase 8 and caspase 3 activities and increased expression of the antiapoptotic protein FLIP, suggesting an interesting mechanism for the inhibition of chondrocyte apoptosis.4.
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Shaochun Bai Hongtao Liu Kun‐Hung Chen Polikseni Eksarko Harris Perlman Terry L. Moore Richard M. Pope 《Arthritis \u0026amp; Rheumatology》2004,50(12):3844-3855
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 (FLIPL) and FLIP short (FLIPS) 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 FLIPL and FLIPS 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, FLIPL was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFα induced the expression of FLIPL and FLIPS 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 FLIPL 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.9.
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Franck Grall Xuesong Gu Lujian Tan Je‐Yoel Cho Mehmet Sait Inan Allison R. Pettit Usanee Thamrongsak Bob K. Choy Cathy Manning Yasmin Akbarali Luiz Zerbini Susan Rudders Steven R. Goldring Ellen M. Gravallese Peter Oettgen Mary B. Goldring Towia A. Libermann 《Arthritis \u0026amp; Rheumatology》2003,48(5):1249-1260
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Hanshi Xu Peng Liu Liuqin Liang Farhad R. Danesh Xiuyan Yang Yijin Ye Zhongping Zhan Xueqing Yu Hui Peng Lin Sun 《Arthritis \u0026amp; Rheumatology》2006,54(11):3441-3451
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.19.
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Teruhisa Hirayama Simon Dai Sabiha Abbas Yasuhiro Yamanaka Yousef Abu‐Amer 《Arthritis \u0026amp; Rheumatology》2005,52(9):2719-2729