Phosphorylation-dependent inactivation of FoxO transcription factors by the proto-oncogene product protein kinase B (PKB) plays a central role in promoting cellular survival, proliferation, and activation mediated by PI3-kinase. PI3-kinase-dependent activation of PKB has been observed in rheumatoid arthritis (RA) synovial tissue, and blocking PKB activation has a protective effect in animal models of arthritis. However, the molecular mechanisms by which activation of PKB promotes arthritis have not been elucidated.
To determine whether FoxO transcription factors (FoxO1, FoxO3a, and FoxO4) are specifically inactivated in RA synovial tissue, to identify cell types in RA synovial tissue in which FoxO proteins are inactivated, and to identify inflammatory stimuli relevant to RA that inactivate FoxO transcription factors in cultured RA fibroblast-like synoviocytes (FLS) in vitro.
Expression and PKB-dependent phosphorylation of FoxO1, FoxO3a, and FoxO4 were determined using specific antibodies in immunohistochemical and computer-assisted quantitative digital analysis of synovial tissue sections obtained from 12 RA and nine osteoarthritis (OA) patients. Double labelling with cell-specific antibodies was performed to identify FoxO expression and inactivation in specific cell populations. In vitro, cultured RA (n = 3) and OA (n = 2) FLS were stimulated with tumour necrosis factor (TNF) alpha, transforming growth factor (TGF) beta, or soluble CD154 prior to lysis and detection of FoxO phosphorylation by immunoblotting. FLS proliferative responses to these stimuli were determined in the presence and absence of the PI3-kinase inhibitor LY294002 by crystal violet staining.
Phosphorylated inactive FoxO1, FoxO3a, and FoxO4 were observed in both RA and OA synovial tissue. Inactivation of FoxO1, FoxO3a, and FoxO4 was restricted to FLS, T lymphocytes, and synovial macrophages, respectively. Inactivation of FoxO1 and FoxO4 was significantly enhanced (P < 0.05) in RA synovial tissue compared with OA synovial tissue. No significant differences in the inactivation of T lymphocyte FoxO3a were observed between RA and OA synovial tissue. In vitro, PI3-kinase-dependent inactivation of FoxO1 was observed in RA FLS following stimulation with TNF, TGF, and soluble CD154. Inactivation of FoxO1 was not sufficient to promote FLS proliferation, as only TNF and TGF but not soluble CD154 stimulated PI3-kinase-dependent FLS proliferation. Our studies demonstrate that inactivation of FoxO1 and FoxO4 is enhanced in RA compared with OA, provide the first (patho)physiological evidence of FoxO4 inactivation in vivo, and suggest important roles for FoxO1 and FoxO4 in maintaining, respectively, FLS and macrophage activation and survival in RA synovial tissue.