Wnt/␤-catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation. Introduction:The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism. Materials and Methods:In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1 +/− animals. Results: Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1 +/− mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold. Conclusions: We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.

Low density lipoprotein receptor-related protein 5 (LRP5) has been identified as a Wnt co-receptor involved in the activation of the ␤-catenin signaling pathway. To improve our understanding of the molecular mechanisms by which LRP5 triggers the canonical Wnt signaling cascade, we have screened for potential partners of LRP5 using the yeast two-hybrid system and identified Frat1 as a protein interacting with the cytoplasmic domain of LRP5. We demonstrate here that LRP5/Frat1 interaction is involved in ␤-catenin nuclear translocation and TCF-1 transcriptional activation. The addition of Wnt3a or overexpression of constitutively active truncated LRP5 (LRP5C) induces Frat1 recruitment to the cell membrane. Overexpression of a dominant negative form of disheveled (Dvl) shows that this protein positively affects LRP5/Frat1 interaction. Furthermore, the fact that dominant negative Dvl does not interfere with LRP5C/Frat1 interaction can explain how LRP5C is capable of acting independently of this major Wnt signaling player. Axin, which has been shown to interact with LRP5 and to be recruited to the membrane through this interaction, was found to co-immunoprecipitate with Frat1 and LRP5. We propose that recruitment of Axin and Frat1 to the membrane by LRP5 leads to both Axin degradation and Frat1-mediated inhibition of glycogen synthase kinase-3. As a consequence, ␤-catenin is no longer bound to Axin or phosphorylated by glycogen synthase kinase-3, resulting in TCF-1 activation.

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