Actinomycin D renders cultured synovial fibroblasts susceptible to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis.
OBJECTIVE
To investigate the expression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors in cultured synovial fibroblasts from rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and to examine their susceptibility to TRAIL-induced apoptosis in the presence or absence of metabolic inhibitors.


METHODS
The expression of TRAIL receptors in synovial fibroblasts was examined by Western blot and immunohistochemistry. Expression of TRAIL-receptor 1 (TRAIL-R1), FLICE-inhibitory protein (Fas-associating protein with death domain-like interleukin-1-converting enzyme), and Bcl-2 was assessed by Western blot. Synovial cell viability was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay (XTT), and apoptosis was determined both by DNA content analysis after propidium iodide staining and Annexin V stain.


RESULTS
TRAIL-R1 was constitutively expressed on cultured synovial fibroblasts from RA and OA, however, expression of TRAIL-R2 and TRAIL-R3 was not observed by immunohistochemistry and Western blot. Cultured synovial fibroblasts were resistant to apoptosis by TRAIL alone, but combined treatment of TRAIL with actinomycin D (ActD: 200 ng/mL), cycloheximide (CHX: 10 microg/mL), or proteasome inhibitor (MG132: 20 microM) induced apoptosis in a dose-dependent manner. The apoptosis was completely or partially inhibited by various caspase inhibitors, implicating an involvement of caspase pathway in TRAIL-induced apoptosis in the presence of these metabolic inhibitors. Expression of TRAIL-R1, FLIPL, and Bcl-2 did not account for the apoptosis by the combined treatment of TRAIL with ActD.


CONCLUSIONS
Although TRAIL-R1 was constitutively expressed; cultured synovial fibroblasts were resistant to apoptosis by TRAIL. ActD, CHX, and MG132 rendered cultured synovial fibroblasts susceptible to TRAIL-induced apoptosis by a caspase-dependent mechanism. However, the exact mechanism of sensitization by these metabolic inhibitors remains to be determined.
Park
Y W
YW
Division of Rheumatology, The Hospital for Rheumatic Diseases, Hanyang University, Seoul, Korea.
Ji
J D
JD
Lee
J S
JS
Ryang
D W
DW
Yoo
D H
DH
D017209:Apoptosis; D001172:Arthritis, Rheumatoid / Q000473:pathology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology*; D058851:GPI-Linked Proteins; D006801:Humans; D007150:Immunohistochemistry; D018836:Inflammation Mediators / Q000032:analysis; D010003:Osteoarthritis / Q000473:pathology; D011379:Prognosis; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D053320:Receptors, Tumor Necrosis Factor, Member 10c; D012494:Sampling Studies; D012680:Sensitivity and Specificity; D013583:Synovial Membrane / Q000166:cytology; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D058851:GPI-Linked Proteins; D018836:Inflammation Mediators; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D053320:Receptors, Tumor Necrosis Factor, Member 10c; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C108661:TNFRSF10C protein, human; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin