Wnt3-frizzled 1 chimera as a model to study canonical Wnt signaling

Abstract

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low-density lipoprotein receptor-related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF-luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF-luciferase activation. DKK-1 protein blocks upregulation of the TCF-luciferase reporter by the Wnt3-Fz1 chimera, suggesting involvement of LRP in Wnt3-Fz1 signaling. Expression of a Wnt3-Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3-Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt-Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases.