Figure 6
Erk inhibition suppresses ETS1 activation and hyperproliferation of mouse synovial fibroblasts from collagen-induced arthritis (CIA) mice. (a) Synovial fibroblasts isolated from CIA or normal mice were cultured without or with Erk inhibitor. The expression of Synoviolin (SYN) was determined by Western blotting by using anti-SYN antibody (top panel). The activation and protein levels of Erk in the parallel prepared cell lysates were detected by anti-phospho-Erk and anti-Erk, respectively (the second and third panels). Similarly, the activation and protein levels of ETS1 in the parallel prepared cell lysates were detected by anti-phospho-ETS1 and anti-ETS1, respectively (the bottom two panels). (b) Synovial cells were cultured without or with Erk inhibitor in 12-well plates for 24 hours. One microcurie of 3H-tymidine was added to each well of plated cells and further cultured for 16 hours. 3H-tymidine incorporation was analyzed as described previously [18]. Error bars represent three independent experiments (mean ± standard deviation). (c) Cell cycle analysis of Erk inhibitor-treated synovial fibroblasts. Cells treated with or without the Erk-specific inhibitor were collected and fixed in cold methanol and then stained with propidium iodide (PI) in the presence of RNase. PI-stained cells were washed once with phosphate-buffered saline and then analyzed by flow cytometry. The cell death was significantly increased when CIA synovial fibroblasts were treated with Erk inhibitor, PD98059 (PD). CPM, counts per minute; Erk, extracellular signal-regulated kinase; FBS, fetal bovine serum.