AKT regulates TNF-alpha-mediated apoptosis of rheumatoid arthritis synovial fibroblasts

Objective

To determine whether TNF-α driven proliferation of rheumatoid synovial fibroblasts is associated with upregulation of the serine/threonine kinase B (PKB)/AKT activity and RA synovial fibroblast survival.

Method

Staining of phosphorylated AKT was done using antiphosphorylated Thr308-AKT antibody. Phosphorylated AKT was analyzed by Western blot, and AKT activity was analyzed using a kinase assay. The cytotoxicity of TNF-α treatment or TNF-α plus AKT activity inhibitor wortmannin, TNF-α plus dominant mutant (AdAkt-DN) or AdPTEN was analyzed using ATPLite assay.

Results

The levels of phosphorylated-Akt are higher in RASF than in OASF, as demonstrated by immunohistochemical staining, immunoblot analysis and an Akt kinase assay. The levels of phosphorylated Akt and Akt kinase activity were increased by stimulation of primary RASF with TNF-α (10 ng/ml). Treatment of RASF with the PI 3-kinase inhibitor, wortmannin (50 nM), plus TNF-α resulted in apoptosis of 75 ± 8% of RASF within 24 h. This proapoptosis effect was specific for Akt, as equivalent levels of apoptosis were observed upon TNF-α treatment of RASF trans-fected with adenovirus expressing a dominant negative-Akt (AdAkt-DN) and with an adenovirus expressing PTEN (AdPTEN), which opposes the action of Akt.

Conclusion

These results indicate that phosphorylated Akt acts as a survival signal in RASF and contributes to the stimulatory effect of TNF-α on these cells by inhibiting the apoptosis response. This effect was not observed in OSAF, and may reflect the pathophysiologic changes associated with the proliferating synovium in rheumatoid arthritis.