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.

Targeting Wnt pathway in mantle cell lymphoma-initiating cells

“…Our results showed up-regulated expression of canonical ligand Wnt3 [ 30 ] but not of the non-canonical ligands such as Wnt4, Wnt5, and Wnt11 [ 31 ] in MCL-ICs compared to MCL-non-ICs. Immunostaining results also confirmed the differentially higher staining of active unphosphorylated β-catenin in MCL-ICs, which is required for canonical but not for non-canonical Wnt pathway [ 32 ].…”

Wnt3 function in the epiblast is required for the maintenance but not the initiation of gastrulation in mice

“…Wnt3 has been suggested to act through the canonical Wnt pathway using morphological analysis and reporter assays in vitro (Bhat et al, 2010; Shimizu et al, 1997). In order to test whether Wnt3 expression is sufficient to activate the canonical Wnt pathway and to further understand the molecular basis of Wnt3 signaling during gastrulation we assessed the expression of primitive streak markers in mouse embryonic fibroblasts (MEFs) after treatment with Wnt3-conditioned media.…”

Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors

“…S2). Interestingly, each of these receptors has been previously implicated in canonical Wnt signaling (12)(13)(14)(15)(16)(17). The epitope on the Fzd proteins bound by OMP-18R5 was mapped through a survey of introduced amino acid substitutions within FZD8, several of which eliminated OMP-18R5 binding ( Fig.…”