Fig. 7. β-catenin signaling is required for BMP2 to activate Vegf and Mmp13 expression. (A-C) Primary chondrocytes isolated from Col2a1-ICAT transgenic mice and WT littermates were treated with BMP2 (100 ng/ml) for 48 hours. The expression of colX, Vegf and Mmp13 was determined by real-time RT-PCR and was normalized to β-actin levels. ALP activity was measured using cell lysates from the same cells. BMP2 stimulated the expression of all marker genes in WT chondrocytes and rescued (A) the colX expression and (B) ALP activity in chondrocyte isolated from Col2a1-ICAT transgenic mice. However, BMP failed to induce the expression of (C) VEGF and (D)Mmp13 in Col2a1-ICAT transgenic chondrocytes. ^*P<0.05, unpaired t-test, n=3. (E) RCJ3.1C5.18 chondrogenic cells were treated with BMP2 (100 ng/ml) for 2 hours with or without the BMP antagonist noggin (300 ng/ml) or Wnt signaling inhibitor Dkk1 (1 µg/ml). Total RNA was extracted and VEGF expression determined by real-time RT-PCR normalized to β-actin levels. The induction of VEGF expression by BMP2 was completely inhibited by noggin and Dkk1. (F) RCJ3.1C5.18 cells were treated with BIO (1 µg/ml) for 2 hours with or without noggin or Dkk1. The expression of VEGF mRNA levels were measured and normalized to β-actin levels. BIO induced VEGF expression within 2 hours. Noggin partially inhibited BIO-induced VEGF expression. ^*P<0.05, unpaired t-test, compared to the untreated group, n=3. ^**P<0.05, unpaired t-test, compared with BMP2 or BIO treatment groups, n=3. (G,H) To further determine changes in VEGF and MMP13 expression in vivo in Col2a1-ICAT transgenic mice, we performed immunostaining using the anti-VEGF and anti-MMP13 antibodies. The results showed that the area and intensity of the expression of VEGF and MMP13 proteins were reduced in Col2a1-ICAT transgenic mice.