Figure 4
Interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) induce Synoviolin (SYN) expression via the Erk pathway. (a) Synovial fibroblasts were partially starved for 24 hours by cultivation of these cells with media that contain 0.5% fetal bovine serum and then cultured with 10 ng/ml of IL-1β. Cells were also treated with each of the MAPK inhibitors. The concentrations of each inhibitor used were as follows: Erk inhibitor PD98059, 20 μM; JNK inhibitor SP600125, 10 μM; p38 inhibitor SB202190, 10 μM; and NF-κB inhibitor SN50, 20 μM. The expression of SYN was examined by Western blotting (top panel). The protein level of actin was reprobed as a control (bottom panel). (b) The activation of Erk was analyzed by anti-p-Erk antibody (top panel). The same membrane was reprobed by anti-Erk antibody (bottom panel). (c) p38 activation was analyzed by anti-p-ATF2 antibody (top panel). The same membrane was reprobed by anti-ATF2 antibody (bottom panel). (d) JNK1 activation was analyzed with anti-phospho-Jun antibody (top panels). The total protein levels of c-Jun were examined using anti-Jun (bottom panels). (e) Mouse synovial fibroblasts were starved for 24 hours and then cultured with 10 ng/ml of TNF-α without or with Erk inhibitor. The expression of SYN was analyzed by Western blotting (top panel). The activation of Erk was determined by anti-phosphorylated Erk antibody (middle panel). The protein level of Erk was analyzed by anti-Erk antibody (bottom panel). (f) The effect of NF-κB inhibitor on SYN expression. Mouse synovial fibroblasts were transfected with NF-κB-luc reporter, which contains firefly luciferase gene under control of NF-κB. The control plasmid pRL-TK encoding renillar luciferase was also included to correct transfection efficiency. Transfected cells were starved and then cultivated in the presence of 10 ng/ml of IL-1β or TNF-α without or with SN50 for 24 hours. The expression of SYN in these cells was analyzed by Western blotting (top panel), and the same membrane was reprobed with anti-actin (middle panel). (g) Parallel prepared cell lysates from (f) were used for testing the NF-κB-driven luciferase activity (bottom panel). Error bars represent three different experiments (mean ± standard deviation). Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B.