Figure 5

Effect of Stat3 inhibitors on Stat3 activation and osteoclast formation in CIA models. (a–d) Arthritis was induced in 5-week-old wild-type DBA/1 J male mice by injection of type II collagen and CFA on day -21 followed by the second injection on day 0. Vehicle, CP690550 or meloxicam (each 15 mg/kg/day) was administered IP once a day for 2 weeks starting at day 0. Specimens of ankle joints derived from CIA mice administered indicated drugs were subjected to immunofluorescence staining for pStat3 (a) or Cathepsin K 14 days after the second injection (c). Nuclei were visualized by DAPI. Bar, 100 µm. Data in (b) and (d) represent mean relative areas ± SD of Stat3-positive (b) and Cathespsin K-positive (d) cells, respectively (n = 3 each, **P < 0.01). (e) Total RNA was prepared from NIH3T3 cells treated 8 h with or without IL-6 (100 ng/ml) plus sIL-6R (100 ng/ml) in the presence or absence of vehicle, CP690550 or Meloxicam (1 μM). RANKL expression was then analyzed by realtime PCR. Data represents mean RANKL expression relative to β actin ± SD (n = 3, *P < 0.05, NS not significant). (f) M-CSF-dependent wild-type bone marrow cells (1 × 10⁵ cells per well) were cultured with osteoblastic MC3T3E1 cells (1 × 10⁴ cells per well) plus IL-6 (100 ng/ml) and sIL-6R (100 ng/ml) in the presence or absence of indicated drugs (1 μM each). Eight days later, mRNA was harvested for osteoclastic marker analysis. Representatives of at least two independent experiments are shown.