MAP kinase subtypes and Akt regulate diosgenin-induced apoptosis of rheumatoid synovial cells in association with COX-2 expression and prostanoid production.
In the present study, we investigated the signalling pathways involved in diosgenin-induced apoptosis in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro with particular interest on Akt and MAPKs activation in relation to arachidonic acid metabolism via COX-2 pathway. MAPK activation was measured by ELISA quantification in diosgenin-treated human RA FLS. Expression of Akt and phospho-Akt was analyzed by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) translocation was evaluated by electromobility shift assay. The prostanoid production (COX-2 activity) was measured by quantitative ELISA. Diosgenin-induced apoptosis in the presence of MAPK or Akt inhibitors was detected by a quantitative determination of DNA fragmentation. Treatment of human RA FLS with 40 microM diosgenin caused an activation of p38 and JNK and an inhibition of ERK phosphorylation. Akt and NF-kappaB are potentially required for diosgenin-induced apoptosis in human RA FLS because 40 microM diosgenin abrogated Akt phosphorylation which correlated with an inhibition of NF-kappaB nuclear translocation. SB203580 and SP600125 (p38 and JNK inhibitors) reduced diosgenin-induced DNA fragmentation whereas U0126 and LY294002 (MEK and PI3 kinase/Akt inhibitors) caused an amplification of proapoptotic effect of diosgenin. Diosgenin increased COX-2 activity resulting in PGE2 and 6-keto-PGF1alpha overproduction in human RA FLS. All MAPK inhibitors markedly reduced diosgenin-induced PGE2 and 6-keto-PGF1alpha synthesis except for SP600125 on 6-keto-PGF1alpha production. These results provide, for the first time, strong evidence that a combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS.
Liagre
Bertrand
B
Laboratoire de Biochimie, UPRES EA 4021, Faculté de Pharmacie, 87025 Limoges Cedex, France. bertrand.liagre@unilim.fr
Leger
David Y
DY
Vergne-Salle
Pascale
P
Beneytout
Jean Louis
JL
D000368:Aged; D017209:Apoptosis; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D004144:Diosgenin / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000378:metabolism*; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D011453:Prostaglandins / Q000096:biosynthesis*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D013583:Synovial Membrane / Q000166:cytology
D008565:Membrane Proteins; D016328:NF-kappa B; D011453:Prostaglandins; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D004144:Diosgenin