Involvement of MAPKs and NF-kappaB in tumor necrosis factor alpha-induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts.
OBJECTIVE
To investigate the roles of MAPKs and NF-kappaB in tumor necrosis factor alpha (TNFalpha)-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-kappaB in TNFalpha-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-kappaB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay.


RESULTS
TNFalpha-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-kappaB were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-kappaB (helenalin) or by transfection with their respective shRNA. TNFalpha-stimulated translocation of NF-kappaB into the nucleus and NF-kappaB promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNFalpha 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 TNFalpha or by anti-VCAM-1 antibody before the addition of lymphocytes.


CONCLUSIONS
In RASFs, TNFalpha-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches.
Luo
Shue-Fen
SF
Chang Gung University, Chang Gung Memorial Hospital, Kwei-San, Tao-Yuan, Taiwan.
Fang
Rou-Yi
RY
Hsieh
Hsi-Lung
HL
Chi
Pei-Ling
PL
Lin
Chih-Chung
CC
Hsiao
Li-Der
LD
Wu
Chi-Chuan
CC
Wang
Jong-Shyan
JS
Yang
Chuen-Mao
CM
D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism*; D014178:Translocation, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases