Wnt/β‐catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP‐2 in pre‐osteoblastic cells.

Introduction: Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). Because LRP5 acts as a coreceptor for Wnt proteins, these findings suggest a crucial role for Wnt signaling in bone biology.

Materials and Methods: We have investigated the involvement of the Wnt/LRP5 cascade in osteoblast function by using the pluripotent mesenchymal cell lines C3H10T1/2, C2C12, and ST2 and the osteoblast cell line MC3T3‐E1. Transfection experiments were carried out with a number of elements of the Wnt/LRP5 pathway. Measuring osteoblast and adipocyte differentiation markers addressed the effect of this cascade on osteoblast differentiation.

Results: In mesenchymal cells, only Wnt's capable of stabilizing β‐catenin induced the expression of alkaline phosphatase (ALP). Wnt3a‐mediated ALP induction was inhibited by overexpression of either Xdd1, dickkopf 1 (dkk1), or LRP5ΔC, indicating that canonical β‐catenin signaling is responsible for this activity. The use of Noggin, a bone morphogenic protein (BMP) inhibitor, or cyclopamine, a Hedgehog inhibitor, revealed that the induction of ALP by Wnt is independent of these morphogenetic proteins and does not require de novo protein synthesis. In contrast, blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP‐2 and Shh to induce ALP in mesenchymal cells. Moreover, BMP‐2 enhanced Wnt1 and Wnt3a expression in our cells. In MC3T3‐E1 cells, where endogenous ALP levels are maximal, antagonizing the Wnt/LRP5 pathway led to a decrease of ALP activity. In addition, overexpression of dkk1 reduced extracellular matrix mineralization in a BMP‐2‐dependent assay.

Conclusions: Our data strongly suggest that the capacity of BMP‐2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP‐2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. These results may help to explain the phenotype of OPPG patients and HBM.