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

Shaochun Bai, Hongtao Liu, Kun Hung Chen, Polikseni Eksarko, Harris Perlman, Terry L. Moore, Richard M. Pope

Research output: Contribution to journalArticle

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Abstract

Objective. Little apoptosis has been observed in rheumatoid arthritis (MA) 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 adenoviras 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 fiα broblasts to TNFα-induced apoptosis is mediated by the NF-κB-reguIated expression of FLIP. These observations support the role of NF-κB and FLIP as attractive therapeutic targets in RA.

Original languageEnglish (US)
Pages (from-to)3844-3855
Number of pages12
JournalArthritis and Rheumatism
Volume50
Issue number12
DOIs
StatePublished - Dec 2004
Externally publishedYes

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CASP8 and FADD-Like Apoptosis Regulating Protein
Rheumatoid Arthritis
Tumor Necrosis Factor-alpha
Fibroblasts
Apoptosis
Caspase 8
Protein Isoforms
Joints
Western Blotting
RNA Isoforms
Caspase Inhibitors
Annexin A5
DNA Fragmentation
Caspase 3

ASJC Scopus subject areas

  • Immunology
  • Rheumatology

Cite this

NF-κB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α-mediated apoptosis. / Bai, Shaochun; Liu, Hongtao; Chen, Kun Hung; Eksarko, Polikseni; Perlman, Harris; Moore, Terry L.; Pope, Richard M.

In: Arthritis and Rheumatism, Vol. 50, No. 12, 12.2004, p. 3844-3855.

Research output: Contribution to journalArticle

Bai, Shaochun ; Liu, Hongtao ; Chen, Kun Hung ; Eksarko, Polikseni ; Perlman, Harris ; Moore, Terry L. ; Pope, Richard M. / NF-κB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α-mediated apoptosis. In: Arthritis and Rheumatism. 2004 ; Vol. 50, No. 12. pp. 3844-3855.
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abstract = "Objective. Little apoptosis has been observed in rheumatoid arthritis (MA) 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 adenoviras 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 fiα broblasts to TNFα-induced apoptosis is mediated by the NF-κB-reguIated expression of FLIP. These observations support the role of NF-κB and FLIP as attractive therapeutic targets in RA.",
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T1 - NF-κB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α-mediated apoptosis

AU - Bai, Shaochun

AU - Liu, Hongtao

AU - Chen, Kun Hung

AU - Eksarko, Polikseni

AU - Perlman, Harris

AU - Moore, Terry L.

AU - Pope, Richard M.

PY - 2004/12

Y1 - 2004/12

N2 - Objective. Little apoptosis has been observed in rheumatoid arthritis (MA) 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 adenoviras 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 fiα broblasts to TNFα-induced apoptosis is mediated by the NF-κB-reguIated expression of FLIP. These observations support the role of NF-κB and FLIP as attractive therapeutic targets in RA.

AB - Objective. Little apoptosis has been observed in rheumatoid arthritis (MA) 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 adenoviras 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 fiα broblasts to TNFα-induced apoptosis is mediated by the NF-κB-reguIated expression of FLIP. These observations support the role of NF-κB and FLIP as attractive therapeutic targets in RA.

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