Wnt/␤-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 ␤-catenin induced the expression of alkaline phosphatase (ALP). Wnt3a-mediated ALP induction was inhibited by overexpression of either Xdd1, dickkopf 1 (dkk1), or LRP5⌬C, indicating that canonical ␤-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.

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A large number of similarities have previously been noted between the blood and milk clotting phenomena [Jollès, P. (1975) Mol. Cell. Biochem. 7, 73–85; Jollès, P. & Henschen, A. (1982) Trends Biochem. Sci. 7, 325–328]: some analogous features have also been found between fibrinogen and k‐casein. In this connection, the effect of a natural and a synthetic peptide derived from k‐casein on platelet function was studied: the undecapeptide Met‐Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp‐Lys (residues 106 → 116 of cow k‐casein) inhibited both aggregation of ADP‐treated platelets and binding of 125I‐fibrinogen to ADP‐treated platelets: its behaviour was similar to that of the structurally related C‐terminal dodecapeptide of human fibrinogen γ‐chain.