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Reduction of tumor necrosis factor induced nuclear factor-kappaB nuclear translocation and DNA binding by
Thomas Lehmann1, Christian Murphy, David G Zahra
1St Vincent's Hospital Clinical School, University of New South Wales, Sydney, Australia.
Objective:
The antiinflammatory effects of glucocorticoids are mediated by several mechanisms, including inhibition of nuclear factor-kappaB (NF-kappaB) nuclear translocation and DNA binding. This mechanism is not evident in some cell types, including endothelial cells and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). We determined the effect of glucocorticoids and tumor necrosis factor (TNF) on nuclear localization and DNA binding of the transcription factor NF-kappaB in osteoarthritic (OA) synovial tissue.
Methods:
Explants of synovial tissue from patients undergoing joint replacement surgery for arthritis were placed in culture and treated with dexamethasone 10(-6) M for 18 h and again at 30 min prior to stimulation with TNF for a further 30 min. NF-kappaB and AP-1 DNA binding activities were determined by electrophoretic mobility shift analysis of nuclear extracts prepared from 6 whole tissue explants. Nuclear localization of NF-kappaB was determined by quantitative immunohistochemistry for Rel-A(p65) in thin sections of 5 synovial tissue explants.
Results:
TNF induced NF-kappaB nuclear translocation and DNA binding in all OA synovial tissue explants, although there were no consistent effects on AP-1 DNA binding. Dexamethasone reduced TNF stimulated nuclear translocation of RelA(p65) in all 5 OA synovial explants analyzed by immunohistochemistry. Dexamethasone partially decreased NF-kappaB DNA binding in 5 of 6 TNF stimulated explants and 4 of 6 unstimulated explants. In cultured rheumatoid arthritis and OA fibroblast-like synoviocytes and Mono Mac 6 cells the effects of dexamethasone on NF-kappaB DNA binding were not evident.
Conclusion:
Dexamethasone partially inhibits TNF induced NF-kappaB DNA binding in human synovial tissue. It is feasible to use explants of intact fresh human synovium as a substrate for the action of antirheumatic drugs targeting a transcription factor.
Insights
Glucocorticoids like dexamethasone partially inhibit tumor necrosis factor (TNF)-induced nuclear factor-kappaB (NF-kappaB) DNA binding in osteoarthritic synovial tissue. This study demonstrates the feasibility of using synovial explants to test anti-rheumatic drugs.
Area of Science:
- Rheumatology
- Molecular Biology
- Immunology
Background:
- Glucocorticoids possess anti-inflammatory properties mediated by inhibiting nuclear factor-kappaB (NF-kappaB) nuclear translocation and DNA binding.
- This inhibitory mechanism is not consistently observed in all cell types, such as endothelial cells and rheumatoid arthritis fibroblast-like synoviocytes.
- The specific effects of glucocorticoids and tumor necrosis factor (TNF) on NF-kappaB activity in osteoarthritic (OA) synovial tissue require further investigation.
Purpose of the Study:
- To investigate the impact of glucocorticoids and TNF on the nuclear localization and DNA binding of the transcription factor NF-kappaB.
- To assess these effects specifically within human osteoarthritic synovial tissue explants.
Main Methods:
- Synovial tissue explants from patients undergoing joint replacement for arthritis were cultured.
- Explants were treated with dexamethasone and/or TNF.
- NF-kappaB and AP-1 DNA binding activities were analyzed using electrophoretic mobility shift assays, and NF-kappaB nuclear localization was quantified via immunohistochemistry for Rel-A(p65).
Main Results:
- TNF stimulated NF-kappaB nuclear translocation and DNA binding in all OA synovial tissue explants.
- Dexamethasone significantly reduced TNF-induced nuclear translocation of RelA(p65) and partially decreased NF-kappaB DNA binding in most explants.
- Dexamethasone's effects on NF-kappaB DNA binding were not evident in cultured fibroblast-like synoviocytes or Mono Mac 6 cells.
Conclusions:
- Dexamethasone demonstrates a partial inhibitory effect on TNF-induced NF-kappaB DNA binding within human synovial tissue.
- Intact human synovium explants serve as a viable model for evaluating the efficacy of antirheumatic drugs targeting transcription factors.
