Finally, to test whether the mitochondrial pathway is the only one involved in these effects, we used the caspase 9 inhibitor, Z LE HD FMK before Fas stimulation. Treatment of cells with LEHD alone had no effect on cell viability. However, as shown in Figure 4c, caspase 9 inhibition completely blocked apoptosis induced by treatment with anti Fas and Wort even in Bid transfected cells. This was shown by the apoptotic rate that decreased near to basal levels in all RA FLS groups. It has been recently described that memFasL stimulation leads to more effective apoptosis than anti Fas antibody due to different organization of DISC, leading to more efficient caspase 8 activation. Then, to exclude that the Bid requirement in Fas mediated apoptosis of RA FLS was linked to signalling with anti Fas antibody, apoptosis was induced by treatment with memFasL.

RA FLS from seven patients were treated with 1, 10 or 100 ng/ml mFasL and the 100 ng/ml was chosen as the most efficient. As shown in Figure 5a, induction of apoptosis was similar to that obtained after treatment with anti Fas antibody. These results confirm that Bid is a limiting factor in Fas mediated apoptosis of RA FLS under a more physiological stimulus. We also explored by western blot the expression of cas pase 9 in Bid overexpressing and parental RA FLS after treatment with anti Fas or anti Fas and Wort. Our results showed that PI3 kinase inhibition pro motes caspase 9 cleavage that was significantly more marked in overexpressing FLS treated with Bid, confirming the mitochondrial pathway involvement.

Discussion Resistance of RA FLS to Fas mediated apoptosis is of great interest not only from a scientific point Drug_discovery of view but also for its practical implications. The synovial hyperplasia charac teristic of RA is facilitated by the resistance of FLS to apop tosis. It has been demonstrated that only a small percentage of cultured FLS undergo apoptosis after Fas stimulation despite their expression of functional Fas. Furthermore, ex vivo studies of RA synovial tissues show that apoptotic cells are rare, although Fas receptors in FLS and its ligand in co localized macrophages and T cells are seen. Therefore, to elucidate the molecular mechanisms of this resistance to apoptosis, and to clarify the steps of the Fas pathway in this specific type of cells is required. Our exper iments confirm that RA FLS are type II cells, in which death receptor induced apoptosis requires activation of the mitochondrial pathway through Bid cleavage. This has already been suggested in a previous work. We have also shown that constitutive Akt phosphorylation mediates the resistance to Fas induced apoptosis in these cells. Inter estingly, the effect is mediated by inhibition of the cleavage of Bid.