title: Wnt-dependent osteogenic commitment of bone marrow stromal cells using a novel GSK3β inhibitor.
abstract: Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) can differentiate into multiple lineages including osteogenic and adipogenic cells. Wnt signalling has been implicated in controlling BMSC fate, but the mechanisms are unclear and apparently conflicting data exist. Here we show that a novel glycogen synthase kinase 3β inhibitor, AR28, is a potent activator of canonical Wnt signalling using in vitro β-catenin translocation studies and TCF-reporter assays. In vivo, AR28 induced characteristic axis duplication and secondary regions of chordin expression in Xenopus laevis embryos. Using human BMSCs grown in adipogenic medium, we confirmed that AR28-mediated Wnt signalling caused a significant (p<0.05) dose-dependent reduction of adipogenic markers. In osteogenic media, including dexamethasone, AR28 caused significant (p<0.05) decreases in alkaline phosphatase (ALP) activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when excluding dexamethasone from the osteogenic media, increases in both ALP and mineralisation were identified following AR28 treatment, which was blocked by mitomycin C. Pre-treatment of BMSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. Furthermore, BMP2-induced osteogenic differentiation was strongly enhanced by AR28 addition within 3 days, but without concomitant changes in cell number, therefore revealing BMP-dependent and independent mechanisms for Wnt-induced osteogenesis.
authors.affiliation: Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Cook
authors.affiliation: Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: Simon W
authors.initials: SW
authors.lastname: Fellgett
authors.affiliation: Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Pownall
authors.affiliation: New Opportunities, AstraZeneca R&D, Alderley Park, Cheshire, UK.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: O'Shea
authors.affiliation: Department of Biology, University of York, York YO10 5DD, UK. Electronic address: paul.genever@york.ac.uk.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Genever
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D010012:Osteogenesis / Q000187:drug effects; D051153:Wnt Proteins / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000187:drug effects; D014982:Xenopus laevis
chemical_list: D055396:Bone Morphogenetic Protein 2; D004791:Enzyme Inhibitors; D051153:Wnt Proteins; D003907:Dexamethasone; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3