Abstract
Objective:
To investigate the anti-apoptotic role of tumor necrosis factor-a (TNF-a) and its signaling pathways in cultured human fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis.
Methods:
FLS were cultured in Dulbecco's modified Eagle's medium. Apoptotic cells were identified by TUNEL assay and Hoechst staining. Cell viability was determined by the MTT method. Expression of phospho-Akt and phospho-BAD was measured by Western blotting.
Results:
A 24-h TNF-a treatment prevented FLS apoptosis induced by nitric oxide (NO) donor sodium nitroprusside dihydrate (SNP), achieving 70% protection. At 1-10 ng x ml-1 concentrations, TNF-a induced phosphorylation of Akt and BAD in a time and concentration-dependent manner. This effect was blocked by treatment with both LY294002 and nuclear factor-kB inhibitor pyrrolidine-dithiocarbamate.
Conclusion:
TNF-a has an anti-apoptotic effect in human FLS. Activation of Akt and BAD may have an important role in this process.
MeSH terms
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Apoptosis / drug effects*
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Cell Survival / drug effects
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Chromones / pharmacology
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Dose-Response Relationship, Drug
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Drug Antagonism
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Drug Combinations
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Fibroblasts / drug effects
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Fibroblasts / metabolism
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Fibroblasts / pathology
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Humans
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Morpholines / pharmacology
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NF-kappa B / antagonists & inhibitors
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Nitric Oxide / metabolism*
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Nitroprusside / pharmacology
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Phosphatidylinositol 3-Kinases / metabolism*
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Phosphorylation
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Proto-Oncogene Proteins c-akt / metabolism*
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Pyrrolidines / pharmacology
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Signal Transduction / drug effects
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Synovial Membrane / drug effects*
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Synovial Membrane / metabolism
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Synovial Membrane / pathology
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Thiocarbamates / pharmacology
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Tumor Necrosis Factor-alpha / pharmacology*
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bcl-Associated Death Protein / metabolism
Substances
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Chromones
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Drug Combinations
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Morpholines
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NF-kappa B
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Pyrrolidines
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Thiocarbamates
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Tumor Necrosis Factor-alpha
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bcl-Associated Death Protein
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Nitroprusside
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pyrrolidine dithiocarbamic acid
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Nitric Oxide
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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Phosphatidylinositol 3-Kinases
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AKT1 protein, human
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Proto-Oncogene Proteins c-akt