Fig. 6. Wnt3a and β-catenin activate BMP signaling. (A) Primary chondrocytes were treated with or without Wnt3a (100 ng/ml) for 24 hours and the expression of Bmp2 and Bmp4 was examined by real-time RT-PCR. Wnt 3a upregulates mRNA expression of Bmp2 and Bmp4 in chondrocytes. (B) RCJ3.1C5.18 chondrocytes were transfected with the BMP signaling reporter (12xSBE-Luc) and control vector, and treated with BMP2 (100 ng/ml), BIO (1 µg/ml) or Wnt3a (100 ng/ml). To determine the effect of β-catenin on BMP signaling, RCJ3.1C5.18 cells were also co-transfected with BMP signaling reporter and constitutively active β-catenin (S33Y). Luciferase activity was measured using cell lysates 48 hours after transfection. β-catenin, BIO and Wnt3a stimulated BMP-reporter activity in chondrocytes. BMP2 was used as a positive control. (C) BMP signaling is required for Wnt3a-induced colX expression. Primary chondrocytes isolated from WT mice were treated with Wnt3a (100 ng/ml) for 4 and 6 days with or without noggin (300 ng/ml). Type X collagen (colX) mRNA levels were measured by real-time RT-PCR and were normalized to β-actin levels. The expression of colX was completely inhibited by the BMP antagonist noggin. (D) Expression of Bmp2 and Bmp4 mRNA was examined by real-time RT-PCR using primary chondrocytes isolated from Col2a1-ICAT transgenic mice and WT littermates after cells were cultured for 2 days. Expression of Bmp2 and Bmp4 was significantly decreased in Col2a1-ICAT transgenic mice. ^*P<0.05, unpaired t-test, n=4 (A-D).