Connection between the intrinsic and extrinsic apoptotic pathways in RA FLS There is some indication that RA FLS are type II cells in relation to apoptosis because Bid was cleaved after anti Fas stimulation. We have confirmed these results showing that after incubation with anti Fas the detectable full Bid protein is significantly decreased in all RA FLS lines analy sed. Furthermore, we wanted to know whether the cleavage of Bid is essential for apoptosis in RA FLS. To this end, Bid was suppressed in RA FLS from five different patients and the efficiency of Bid silencing is shown in Fig ures 2b and 2c. Interestingly, suppression of Bid completely abrogated Fas induced apoptosis.

In contrast, transfection with control siRNA did not alter Fas induced apoptosis, indicating the relevance of the Bid protein in apoptosis induced by anti Fas, and consequently the con nection between intrinsic and extrinsic pathways. Regulation of Bid cleavage by the PI3K/Akt pathway Given the above results, it seemed possible that RA FLS could resemble human prostate cancer lines, in which the PI3K/Akt pathway interferes with TRAIL mediated apop tosis by inhibiting the cleavage of Bid. To test whether a similar mechanism was at play in RA FLS, we analysed the effect of Akt inhibition on Bid expression. For this, RA FLS from six different patients were treated with the PI3 kinase inhibitor Wort for one hour before the addi tion of anti Fas antibody. As shown in Figure 3, this treat ment significantly reduced the level of Akt phosphorylation and markedly increased the cleavage of Bid in comparison to that observed after anti Fas alone.

This later effect was demonstrated by a marked reduction of cellular Bid protein expression. Relevance of Bid cleavage for Akt contribution to Fas induced apoptosis resistance To further assess the contribution that regulation of Bid cleavage by Akt has on the Fas mediated resistance to apoptosis in RA FLS, we used siRNA suppression of Bid. RA FLS non transfected and transfected with control or Bid siRNA were pre treated with the PI3 kinase inhibitors LY or Wort before Fas stimulation and apoptosis rate was determined. Neither treatment with LY nor treatment with Wort alone induced apoptosis in RA FLS, whereas Fas stimulation after pre treatment with any of these two inhibitors induced significant apoptosis compared with Fas only treatment.

The same result was observed in cells transfected with control siRNA, but not in cells trans fected with the specific Entinostat Bid siRNA, where full resistance to Fas induced apoptosis was found both with and without Wort treatment. Bid availability limits Fas induced apoptosis in RA FLS The high cleavage of Bid shown after blocking Akt phos phorylation was accompanied by a modest increase in Fas induced apoptosis.