Abstract
Objective
To investigate the roles of MAPKs and NF‐κB in tumor necrosis factor α (TNFα)–induced expression of vascular cell adhesion molecule 1 (VCAM‐1) in human rheumatoid arthritis synovial fibroblasts (RASFs).
Methods
Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF‐κB in TNFα‐induced VCAM‐1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase–polymerase chain reaction, and gene promoter assay. NF‐κB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM‐1 was evaluated by lymphocyte adhesion assay.
Results
TNFα‐induced VCAM‐1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF‐κB were attenuated by the inhibitors of MEK‐1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF‐κB (helenalin) or by transfection with their respective shRNA. TNFα‐stimulated translocation of NF‐κB into the nucleus and NF‐κB promoter activity were blocked by Bay11‐7082, but not by U0126, SB202190, or SP600125. VCAM‐1 promoter activity was enhanced by TNFα in RASFs transfected with VCAM‐1‐Luc, and this promoter activity was inhibited by Bay11‐7082, U0126, SB202190, and SP600125. Moreover, up‐regulation of VCAM‐1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with helenalin, U0126, SP600125, or SB202190 prior to exposure to TNFα or by anti–VCAM‐1 antibody before the addition of lymphocytes.
Conclusion
In RASFs, TNFα‐induced VCAM‐1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF‐κB pathways. These results provide new insights into the mechanisms underlying cytokine‐initiated joint inflammation in RA and may inspire new targeted therapeutic approaches.