Figure 11.

Periostin activates β-catenin signaling in human chondrocytes. A) Western blot analysis of β-catenin in human articular chondrocytes treated with periostin (1–5 µg/ml) for the indicated times. Tubulin is shown as a loading control. IL-1, a known activator of the MAPK and Wnt signaling pathways, was used as a positive control. Representative blots of 3 independent chondrocyte cultures are shown. Age, sex, and basal periostin levels of the 3 OA chondrocyte cultures are indicated above the blots. B) Human OA chondrocytes from 3 independent patients pretreated with inhibitors of either MEK1/2 (ERK1/2-PD98059), p38 (SB202190), or NF-кB (MG-132) (10 µM) or control vehicle (DMSO) for 2 h were incubated with recombinant periostin (5 µg/ml) in serum free-medium for an additional 24 h, and the supernatant was analyzed for MMP-13 by ELISA. For control vs. periostin-treated culture supernatants, P < 0.01 by 1-way ANOVA with post-Bonferroni multiple comparison test. C) TOPflash activity in vehicle- and periostin-treated human C28/I2 chondrocytes. Cells were transfected with TOPflash luciferase reporter and cotransfected with pSEAP vector as a control for transfection efficiency. Means ± sd of 3 independent experiments are shown. Nonparametric Student's 2-tailed t test. D) Human OA chondrocytes (n = 3) pretreated with the Wnt inhibitor CCT031374 hydrobromide (10–50 µM) or control vehicle for 2 h were incubated with recombinant periostin (5 µg/ml) in serum free-medium for an additional 24 h. The supernatant was analyzed for MMP-13, and the cells were used for total RNA isolation. Data are means ± sd from 3 independent chondrocyte cultures. P = 0.025, control vs. periostin-treated; P = 0.042, periostin-treated with Wnt inhibitor vs. without Wnt inhibitor. Nonparametric Student's 2-tailed t test. E) Human OA chondrocytes from 3 patients were pretreated with the Wnt inhibitor CCT031374 (50 µM) or control vehicle (DMSO) for 2 h. Recombinant periostin (5 µg/ml) was added in serum free-medium for 24 h. Total RNA was isolated and analyzed for MMP-13, ADAMTS4, ADAMTS5, and Col2A1 expression by RT-qPCR. *P < 0.05.