Background Recent studies have shown that Akt signaling is activative in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and plays an important role of anti-apoptosis in the pathogenesis of RA. Akt can be fully activated by phosphorylation at Ser473 by mTORC2 (mammalian target of rapamycin complex 2), one of whose key components is RICTOR (rapamycin-insensitive companion of mTOR). We assumed that mTORC2/Akt might involve in the pathological processes of RA-FLS.
Objectives To investigate the influence of mTORC2/Akt on the cell viability in RA-FLS.
Methods FLS were isolated from the primary synovial tissue obtained from three patients with RA. After being transfected with RICTOR siRNA by cationic liposome for 48h, RA-FLS was treated with or without 10ng/ml TNF-alpha for 24h and 48h. The expression of RICTOR and Akt and the level of phosphorylated Akt at Ser473 were evaluated by western blotting, and the cell viability was analyzed by methyl thiazol tetrazolium (MTT) assay. After treatment with Akt pathway blocker MK-2206 at the dose of 0uM, 1uM, 2uM, 4uM and 8uM for 24h and 48h, the expression of Akt and the level of phosphorylated Akt at Ser473 were detected by western blotting. RA-FLS was incubated with MK-2206 at the dose of 0uM, 4uM and 8uM, and the cell viability was analyzed by MTT after 1.5h, 3h, 6h, 12h, 24h and 48h.
Results 1) After the silence of RICTOR, the level of phosphorylated Akt at Ser473 increased and the cell viability decreased in RA-FLS, no matter with or without TNF-alpha for 48h later. But the expression of Akt was not influenced by the transfection with RICTOR siRNA. 2) In comparison with the control group, treatment of RA-FLS with MK-2206 at doses of 4uM, 8uM for 48h inhibited the expression of Akt protein and the level of phosphorylated Akt at Ser473. 3) The effect of MK-2206 on the cell viability of RA-FLS was time- and dose-dependent, since the cell viability significantly decreased after the treatment with MK-2206 for more than 3h at the dose of 8uM.
Conclusions There is an unexpected result that silencing RICTOR (thus mTORC2) causes the increasing level of phosphorylated Akt at Ser473 of RA-FLS in vitro. But at the same time, inhibiting the activation of mTORC2 and Akt can both reduce the cell viability of RA-FLS. These results indicate that the abnormity of mTORC2/Akt may contribute to the stable activation of RA-FLS.
Nissim Hay, Nahum Sonenberg. Upstream and downstream of mTOR. Genes Dev. 2004,18: 1926-1945.
Disclosure of Interest None Declared