Presentation: Signaling Pathways Involved in Trail- Induced Ra Synovial Fibroblast Cell Death, Proliferation, and Survival (2007)

144 Signaling Pathways Involved in Trail- Induced Ra Synovial Fibroblast Cell Death, Proliferation, and Survival

Rational: A hallmark of rheumatoid arthritis (RA) is the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLS), and the RAFLS have been proposed as a therapeutic target. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor on RAFLS and, therefore, suggested as a potential drug. We have previously showed that TRAIL induced apoptosis only in a portion of RAFLS. In the surviving cells, TRAIL induced RAFLS proliferation and complicating then the proposed strategy to use TRAIL in the treatment of RA. To evaluate the possibilities to overcome TRAIL resistance in RAFLS, we study the intracellular pathways implicated in TRAIL-induced signaling.
Methods: To evaluate signaling pathways involved in TRAIL induced in RA synovial fibroblast cell death, proliferation, and survival, RAFLS were pre-treated for one hour with specific chemical inhibitors and cultured with TRAIL for 24 hours. Apoptosis was then analyzed by flow cytometry using annexin V-FITC/TOPRO-3. Proliferation was measured using incorporation of tritiated thymidine. Activation of MAPK and Akt pathways and p21 and p27 expression levels were evaluated by Western Blot.
Results: Inhibition of MAP or PI3 kinases decreased significantly TRAIL-induced proliferation of RAFLS. More importantly, Akt inhibitors not only abrogated TRAIL-mediated RAFLS proliferation, but also increased cell death induced by TRAIL (4.9±1.3 fold increased, n=6). Pan-caspases inhibitor almost completely blocked TRAIL-induced apoptosis, but also blocked TRAIL-induced proliferation (n=4; p<0.05). Because MAPK and Akt pathways are involved in TRAIL induced proliferation, we tested the participation of the caspases on MAPK and Akt pathways activation after TRAIL treatment. Inhibition with pan caspases inhibitor did not have any effect on TRAIL-induced MAP and PI3 kinases activation (n=5). Among substrates for caspases, some proteins such as p21 and p27 negatively regulated cell cycle when there are not cleaved. We observed that TRAIL induced p21 and p27 cleavage (respectively 33%±7 and 25±6%, n=4) which was inhibited by pan-caspases inhibitor. This suggests that cleavage of p21 and p27 by caspases, following TRAIL activation, could be an additional mechanism to induce cell cycle progression and subsequently RA FLS proliferation.
We have identified different signaling pathways involved in cell death, proliferation and survival. Taken together, the use of Akt inhibitors in combination with TRAIL might be a useful strategy, in order to induce RAFLS cell death and to control synovial hyperplasia.

 R. Audo, None; H. Michael, None; B. Combe, None; J. Morel, None.