Substantiation of the role of Bid in the Fas induced apoptosis was obtained by transfection of RA FLS with the full length Bid vector. Additional evidence for the involvement of the intrinsic pathway in Fas induced apoptosis was gathered by the experiments of inhibition of caspase 9. Direct activation of caspase 3 by caspase 8 seemed insufficient to RA FLS cell death. Therefore, our results demonstrated the connection between the intrinsic and extrinsic apoptotic pathways in Fas mediated apoptosis in RA FLS cells. In mice, Scatizzi and colleagues recently showed the importance of Bid for arthritis. In K/BxN serum transfer induced arthritis, mice lacking Bid developed severe arthritis and joint destruction. Synovial analysis showed fewer apoptotic cells in Bid deficient mice than in control mice.

In addition, our work points to the PI3 kinase/Akt path way as a novel molecular mechanism explaining the Fas mediated resistance in RA FLS. Previous observations in RA FLS and other cell types are alike. In RA FLS, Zhang and colleagues reported that inhibition of endogenous Akt phosphorylation sensitized RA FLS to TNF induced apoptosis. Moreover, Miyashita and col leagues showed that Akt inhibition by siRNA technol ogy significantly increased TRAIL mediated apoptosis in RA FLS. However, the molecular mechanism has not been investigated. Recently, Audo and colleagues have shown that inhibition of PI3 kinase/Akt pathway sensitizes RA FLS to TRAIL induced apoptosis by reduction of expression of the anti apoptotic proteins Mcl 1, XIAP, and RIP, and increase of the cell cycle inhibitor p21.

Of interest in our work is that the Akt dependent resistance to apopto sis is due to its inhibition of Bid cleavage in RA FLS cells. Therefore, Akt links the death receptor and the mitochon drial pathways in these cells. This mechanism of resistance to apoptosis has been previously reported in prostate cancer cells. Although it is unknown how Akt regulates Bid cleavage, it is conceivable that activated Akt could phosphorylate Bid, inhibiting its cleavage by caspase 8. Indeed, it has been demonstrated that phosphorylation Dacomitinib of Thr59, a residue localized near to the caspase 8 cleavage site, inhibits Bid cleavage by this caspase. However, Akt inhibits apoptosis through several other mechanisms including activation of nuclear factor kB, phosphorylation of Bad, Bax, and inhibition of pro apop totic p53. It seems that different cells types have different mechanisms leading to the Akt dependent resistance to apoptosis. Conclusions Our results show, for the first time, that endogenous phos phorylation of Akt protects RA FLS against the apoptosis induced by Fas through inhibition of Bid cleavage and point to PI3 kinase/Akt pathway as potential therapeutic target in RA.