Wnt signaling during BMP‐2 stimulation of mesenchymal chondrogenesis

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Abstract
Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. A key component of the Wnt signaling pathway is the cytosolic protein, β‐catenin. We have recently shown that the chondrogenic activity of BMP‐2 in vitro involves the action of the cell–cell adhesion protein, N‐cadherin, which functionally complexes with β‐catenin. The aim of this study is to test the hypothesis that Wnts may be involved in BMP‐2 induced chondrogenesis, using an in vitro model of high‐density micromass cultures of the murine multipotent mesenchymal cell line, C3H10T1/2. Expression of a number of Wnt members was detected in these cultures, including Wnt‐3A and Wnt‐7A, whose levels were up‐ and downregulated, respectively, by BMP‐2. To assess the functional involvement of Wnt signaling in BMP‐2 induced chondrogenesis, cultures were treated with lithium chloride, a Wnt‐7A mimetic that acts by inhibiting the serine/threonine phosphorylation activity of glycogen synthase kinase‐3β (GSK‐3β). Lithium treatment significantly inhibited BMP‐2 stimulation of chondrogenesis as well as GSK‐3β enzymatic activity, and decreased the levels of N‐cadherin protein and mRNA. Furthermore, lithium decreased BMP‐2 upregulation of total and nuclear levels of LEF‐1 and β‐catenin as well as their interaction during later chondrogenesis; similarly, the interaction of β‐catenin with N‐cadherin was also decreased. Interestingly, lithium treatment did not affect the ability of BMP‐2 to decrease ubiquitination of β‐catenin, although it did reduce the interaction of β‐catenin with GSK‐3β during late chondrogenesis (days 9–13). We suggest that the chondro‐inhibitory effect of lithium on BMP‐2 induced chondrogenesis indicates antagonism between lithium‐like Wnts and BMP‐2 during mesenchymal condensation. J. Cell. Biochem. 84: 816–831, 2002.