Bone remodeling, which plays an important role in maintaining bone homeostasis and its structural adaptation to mechanical environment, is regulated by various kinds of molecular signals. Wnt signaling is one of the most important factors in bone remodeling, particularly in bone formation. To reveal the Wnt signaling and its inhibition mechanisms, we measured the interaction between Wnt signal receptor, LRP5, and its signaling molecules, Wnt3a, Dkk1, and Sclerostin, by using Atomic Force Microscope (AFM). In this study, we calculated the dissociation constants of Wnt3a/LRP5, sclerostin/LRP5, and Dkk1/LRP5, and it was proved that the dissociation constant of Dkk1/LRP5 is about 10-times smaller than that of sclerostin/LRP5, that is, the bond life time of Dkk1/LRP5 was about 10-times longer than that of sclerostin/LRP5. As the results, we quantitatively verified a previous suggestion that Dkk1 may function as a main regulator of Wnt signaling, and that sclerostin, which is more selective in its activity and restricted in its expression, may function as a more refined regulator of Wnt signaling.
Selection of bone cells, particularly osteoblasts and osteocytes, for analysis of cellular processes and differentiation is a very important issue because bone remodeling is a highly complex and harmonized process, which includes molecular and cellular interactions and communications. In this study, we introduce a novel osteoblast and osteocyte selection method that uses atomic force microscopy and OB7.3, an antibody of Phex, which is a specific protein marker expressed on the surface of osteocytes. The elasticity and Phex expression levels were simultaneously detected by force spectroscopy using the OB7.3-modified atomic force microscopy probe on the bone cell surface. The elastic modulus was different between osteoblasts and osteocytes. Phex expression level was analyzed by the distribution of Phex-OB7.3 rupturing.
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