Figure 4

Shp2 deficiency impairs Erk activation but promotes Ihh and Pthrp expression. a, (Left panel) qRT-PCR showing increased Col2α1, Col10α1, Ihh, and Pthrp expression in laser-captured cartilaginous cells from exostoses in 4 mice/genotype, compared with normal articular cartilage cells (mean±S.D; *p<0.05, 2-tailed Student’s t test). (Right panel) Immunostaining of representative paraffin sections from Perichondrial Groove of Ranvier region of Ctsk-KO and Control mice. Note the decreased number of p-Erk+ cells (75.4% in Ctsk-Control vs 32.2% in Ctsk-KO; n=3 mice), but increased Ihh expression in Ctsk-KO, compared with Control, mice. b, (Left Panel) Immunoblot showing Shp2 in ATDC5 cells stably expressing shRNAs against mouse Ptpn11 (ATDC5-KD1, ATDC5-KD2, respectively) or scrambled control hairpin. (Right panels). Representative blot showing that Shp2 deficiency decreases Erk activation in response to Fgf18 (top); data from multiple experiments (n=3) showing pErk levels (compared with control at 5 minutes, mean±S.D.; p<0.05, 2-tailed Student’s t test) are quantified below. qRT-PCR (bottom left) shows increased Ihh and Pthrp expression in Shp2-deficient ATDC5 cells (mean ± S.D.; n=3, *p<0.05, 2-tailed Student’s t test). c, FGFR (PD173074, 10nM) or MEK (UO126, 1μM) inhibitor treatment of parental ATDC5 cells enhances Ihh and Pthrp expression, as shown by qRT-PCR (mean±S.D; n=3, *p<0.05, 2-tailed Student’s t test). d, Faxitron radiographs showing that Hedgehog pathway blockade following administration of the Smoothened inhibitor PF-04449913 (100μg/g body weight) to Ctsk-KO mice ameliorates tumor formation, compared with vehicle control (0.5% methylcellulose)-treated mice. Images of representative posterior paws (i–iv) and knees (v–vii) taken pre- (i,iii,v,vii) and post-treatment with Vehicle (ii,vi) or Smoothened inhibitor (SMOi) (iv,viii) for 4 weeks. Note continued development of exostoses and endochromas in Vehicle-treated mice, and their amelioration in SMOi-treated group (arrows). Also, see Figs. S4–S7 and Supplemental video clips 1,2.