Osteogenesis induced by frizzled-related protein (FRZB) is linked to the netrin-like domain

Abnormal Wnt signaling is associated with bone mass disorders. Frizzled-related protein (FRZB, also known as secreted frizzled-related protein-3 (SFRP3)) is a Wnt modulator that contains an amino-terminal cysteine-rich domain (CRD) and a carboxy-terminal Netrin-like (NTN) motif. Frzb(-/-) mice show increased cortical thickness. However, the direct effect of FRZB on osteogenic differentiation and the involvement of the structural domains herein are not fully understood. In this study, we observed that stable overexpression of Frzb in MC3T3-E1 cells increased calcium deposition and osteoblast markers compared with control. Western blot analysis showed that the increased osteogenesis was associated with reduced canonical, but increased non-canonical Wnt signaling. On the contrary, loss of Frzb induced the opposite effects on osteogenesis and Wnt signaling. To translationally validate the positive effects of FRZB on primary human cells, we treated human periosteal and human bone marrow stromal cells with conditioned medium from MC3T3-E1 cells overexpressing Frzb and observed an increase in Alizarin red staining. We further studied the effect of the domains. FrzbNTN overexpression induced similar effects on osteogenesis as full-length Frzb, whereas FrzbCRD overexpressing cells mimicked loss of Frzb experiments. The CRD is considered as the Wnt binding domain, but the NTN domain also has important effects on bone biology. FRZB and other SFRPs or their specific domains may hold surprising potential as therapeutics for bone and joint disorders considering that excess of SFRPs has effects that are not expected under physiological, endogenous expression conditions.