FIGURE 5.

EGFR signaling stimulates the expression of Mmp9 and -13 and Rankl through β-catenin-dependent and -independent pathways. A, TGFα stimulates the expression of Mmp9 and -13 and Rankl in mouse chondrocytes through the EGFR pathway. Mouse primary epiphyseal chondrocytes from either WT or Egfr CKO mice were treated with TGFα for 48 h followed by qRT-PCR analysis. ***, p < 0.001 versus vehicle-treated WT cells. B and C, rat primary epiphyseal chondrocytes were pretreated with either control (DMSO, 0.1% v/v) or pathway-specific inhibitors for 30 min followed by TGFα treatment for 48 h. The total RNA and protein were prepared for qRT-PCR analysis (B) and immunoblotting (C), respectively. con, DMSO; SB, SB203580; IWR, IWR-endo; wort, wortmannin; JNK, JNK II. #, p < 0.01; &, p < 0.001 versus DMSO- and vehicle-treated cells; *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus DMSO- and TGFα-treated cells. a, proMMP9 (92 kDa); b, proMMP13 (54 kDa). D, TGFα stimulates the phosphorylation of ERK (p-ERK) and p38 kinase (p-p38) in chondrocytes through activating EGFR. Rat primary epiphyseal chondrocytes were treated with DMSO or EGFR inhibitor gefitinib (gef) for 30 min followed by 15 min of TGFα treatment. Cell lysates were collected for Western blotting for phosphorylated and total ERK and p38 kinase. E and F, activation of the β-catenin pathway stimulates the expression of Mmp9 (E) and Rankl (F) in chondrocytes. Rat primary epiphyseal chondrocytes were treated with Wnt3a (100 ng/ml), LiCl (20 mm), or BIO (0.5 μg/ml) for 48 h. RNA was harvested for qRT-PCR analysis of Mmp9 and Rankl mRNA levels. *, p < 0.05, **, p < 0.01, ***, p < 0.001 versus control (con).