Citrullinated vimentin stimulates proliferation, pro-inflammatory cytokine secretion, and PADI4 and RANKL expression of fibroblast-like synoviocytes in rheumatoid arthritis

Abstract

Objectives: We aimed to investigate the possible effects of vimentin (Vim) and citrullinated Vim (cVim) on proliferation capacity, pro-inflammatory cytokine secretion, and the expression of peptidylarginine deiminase type 4 (PADI4) and receptor activator of nuclear factor kappa B ligand (RANKL) in cultured fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis (RA) and osteoarthritis (OA) patients.

Method: Human native Vim was citrullinated with rabbit PAD in vitro and detected using a Western blot assay with anti-modified citrulline antibody (anti-MC Ab). FLSs from RA or OA synovial samples were stimulated with Vim or cVim. Cell proliferation capacity was determined using the Celltiter 96 AQueous cell proliferation assay. The concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-1, and IL-17 were measured by enzyme-linked immunosorbent assay (ELISA). The expression of PADI4 and RANKL was measured by real-time polymerase chain reaction (RT-PCR) and a Western blot assay.

Results: Our Western blot assay with anti-MC Ab indicated that the amount of cVim increased significantly after Vim had been incubated with rabbit PAD in vitro. The proliferation capacity and secretion of TNF-α and IL-1 were significantly enhanced in the FLSs of RA patients when treated with cVim. However, when treated with Vim, an inhibitory effect on the proliferation capacity was noted in the FLSs from RA and also from OA patients. cVim significantly increased the expression of PADI4 and RANKL in the FLSs from RA patients.

Conclusion: cVim seems to have remarkable biological effects on RA as confirmed by the stimulation of proliferation capacity, pro-inflammatory cytokine secretion, and PADI4 and RANKL expression in the FLSs of RA patients.

Acknowledgements

This project was supported by the Shanghai Municipal Science and Technology Commission for International Cooperation (No. 09410705000), the Bureau of Shanghai Pudong New Area (No. PW2009D-1), and the National Natural Science Foundation of China (NSFC) (No. 81072467) to Prof. L. Y. Fan.