We examined the role of Wnt/β‐catenin signaling in successive stages of osteoblast differentiation. It has been shown that Wnt signaling in mature osteoblasts needs to be downregulated to enable the formation of a mineralized matrix. Using RNA interference, we showed that this is, at least in part, accomplished by upregulation of the Wnt antagonists Dickkopf‐1 and ‐2.

Introduction: The role of Wnt signaling in the initiation of osteoblast differentiation has been well studied. However, the role during late‐stage differentiation is less clear. We have examined the role of Wnt/β‐catenin signaling in successive stages of osteoblast differentiation.

Materials and Methods: We treated murine bone marrow and mesenchymal stem cell‐like KS483 cells with either LiCl or Wnt3A during several stages of osteoblast differentiation. In addition, we generated stable KS483 cell lines silencing either the Wnt antagonist Dkk‐1 or ‐2

Results: Activation of Wnt signaling by LiCl inhibits the formation of a mineralized bone matrix in both cell types. Whereas undifferentiated KS483 cells respond to Wnt3A by inducing nuclear β‐catenin translocation, differentiated cells do not. This is at least in part accomplished by upregulated expression of Dkk‐1 and ‐2 during osteoblast differentiation. Using RNA interference, we showed that Dkk‐1 plays a crucial role in blunting the BMP‐induced alkaline phosphatase (ALP) response and in the transition of an ALP⁺ osteoblast in a mineralizing cell. In contrast, Dkk‐2 plays a role in osteoblast proliferation and the initiation of osteoblast differentiation.

Conclusions: Our data suggest that Wnt signaling in maturing osteoblasts needs to be downregulated to enable the formation of a mineralized bone matrix. Furthermore, they suggest that Dkk‐1 and Dkk‐2 may have distinct functions in osteoblast differentiation.