Abstract
Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling.
Highlights
Wnt/b-catenin signaling orchestrates embryogenesis and adult stem cell maintenance in mammals [1]
We have previously described a cell-based assay that measures the nuclear translocation of bcatenin using enzyme fragment complementation (EFC) [10]
We tested 2300 drug-like compounds with activity towards G protein-coupled receptors (GPCR) or kinases for their ability to activate Wnt/b-catenin signaling in a human osteosarcoma U2OS cell line genetically engineered to couple nuclear entry of b-catenin to increases in b-galactosidase complementation [10] and found three hits
Summary
Wnt/b-catenin signaling orchestrates embryogenesis and adult stem cell maintenance in mammals [1]. It is initiated when Wnt ligands bind to seven transmembrane receptors of the Frizzled family and to representatives of the single-pass low-density lipoprotein receptor-related protein family (LRP5 or -6) [2,3,4]. In non-stimulated cells, b-catenin protein stability is compromised by the glycogen synthase kinase 3 (GSK3)-mediated phosphorylation of b-catenin on several conserved N-terminal residues. These phosphorylations serve as cues for proteasomal degradation of b-catenin. Quiescent cells typically contain low levels of cytoplasmic and nuclear b-catenin
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