ARTHRITIS & RHEUMATISM Vol. 62, No. 1, January 2010, pp 105–116 DOI 10.1002/art.25060 © 2010, American College of Rheumatology Involvement of MAPKs and NF-B in Tumor Necrosis Factor –Induced Vascular Cell Adhesion Molecule 1 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts Shue-Fen Luo, 1 Rou-Yi Fang, 1 Hsi-Lung Hsieh, 2 Pei-Ling Chi, 1 Chih-Chung Lin, 1 Li-Der Hsiao, 1 Chi-Chuan Wu, 1 Jong-Shyan Wang, 1 and Chuen-Mao Yang 1 Objective. To investigate the roles of MAPKs and NF-B in tumor necrosis factor  (TNF)–induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fi- broblasts (RASFs). Methods. Human RASFs were isolated from syno- vial tissue obtained from patients with RA who under- went knee or hip surgery. The involvement of MAPKs and NF-B in TNF-induced VCAM-1 expression was investigated using pharmacologic inhibitors and trans- fection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase–polymerase chain reaction, and gene promoter assay. NF-B trans- location was determined by Western blot and immuno- fluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. Results. TNF-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-B were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-B (helenalin) or by transfec- tion with their respective shRNA. TNF-stimulated translocation of NF-B into the nucleus and NF-B promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNF in RASFs transfected with VCAM-1-Luc, and this promoter activity was in- hibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 in- creased the adhesion of lymphocytes to the RASF mono- layer, and this adhesion was attenuated by pretreatment with helenalin, U0126, SP600125, or SB202190 prior to exposure to TNF or by anti–VCAM-1 antibody before the addition of lymphocytes. Conclusion. In RASFs, TNF-induced VCAM-1 expression is mediated through activation of the p42/ p44 MAPK, p38 MAPK, JNK, and NF-B pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches. Rheumatoid arthritis (RA) is a chronic inflam- matory disease characterized by thickening of the syno- vial lining layer and infiltration of the synovial mem- brane by inflammatory cells. The cellular infiltration is due to up-regulation of adhesion molecules including intercellular adhesion molecule 1 (ICAM-1) and vascu- lar cell adhesion molecule 1 (VCAM-1) (1–4). The induction of cell adhesion molecules mediates the tight adhesiveness of polymorphonuclear cells (PMNs) and thus facilitates PMN migration across the vascular en- dothelial barrier (5–7). RA synovial fibroblasts (RASFs) are versatile cells with the potential to activate an array of genes that are able to initiate and propagate inflam- mation in RA-affected joints. Up-regulation of Drs. Luo, Lin, and Yang’s work was supported by the Chang Gung Medical Research Foundation (grant CMRPG350642 to Dr. Luo, grant CMRPG350652 to Dr. Lin, and grants CMRPD150253 and CMRPD180061 to Dr. Yang). Drs. Luo and Yang’s work was also supported by the National Science Council, Taiwan (grants NSC96- 2314-B-182-012-MY3 and NSC95-2320-B-182-047-MY3, respectively). 1 Shue-Fen Luo, MD, Rou-Yi Fang, MS, Pei-Ling Chi, MS, Chih-Chung Lin, MD, PhD, Li-Der Hsiao, MS, Chi-Chuan Wu, MD, Jong-Shyan Wang, PhD, Chuen-Mao Yang, PhD: Chang Gung Uni- versity and Chang Gung Memorial Hospital, Kwei-San, Tao-Yuan, Taiwan; 2 Hsi-Lung Hsieh, PhD: Chang Gung Institute of Technology, Kwei-San, Tao-Yuan, Taiwan. Address correspondence and reprint requests to Chuen-Mao Yang, PhD, Department of Pharmacology, Chang Gung University, 259 Wen-Hwa First Road, Kwei-San, Tao-Yuan 33302, Taiwan. E- mail: chuenmao@mail.cgu.edu.tw. Submitted for publication July 22, 2009; accepted in revised form September 25, 2009. 105