TY - JOUR
T1 - Actinomycin D renders cultured synovial fibroblasts susceptible to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis
AU - Park, Y. W.
AU - Ji, J. D.
AU - Lee, J. S.
AU - Ryang, D. W.
AU - Yoo, D. H.
N1 - Funding Information:
This work was supported by the fund HMP-00-CH-08-0007 from the Ministry of Health and Social Affairs of Korea.
PY - 2003
Y1 - 2003
N2 - 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 μg/mL), or proteasome inhibitor (MG132: 20 μM) 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-R1was 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.
AB - 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 μg/mL), or proteasome inhibitor (MG132: 20 μM) 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-R1was 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.
KW - Actinomycin-D
KW - Apoptosis
KW - Synovial fibroblast
KW - TRAIL
UR - http://www.scopus.com/inward/record.url?scp=0842308921&partnerID=8YFLogxK
U2 - 10.1080/03009740410005025
DO - 10.1080/03009740410005025
M3 - Article
C2 - 15080267
AN - SCOPUS:0842308921
VL - 32
SP - 356
EP - 363
JO - Scandinavian Journal of Rheumatology
JF - Scandinavian Journal of Rheumatology
SN - 0300-9742
IS - 6
ER -