title: EB1 levels are elevated in ascorbic Acid (AA)-stimulated osteoblasts and mediate cell-cell adhesion-induced osteoblast differentiation.
abstract: Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.
authors.affiliation: Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario M1C 1A4, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Pustylnik
authors.forename: Cara
authors.initials: C
authors.lastname: Fiorino
authors.forename: Noushin
authors.initials: N
authors.lastname: Nabavi
authors.forename: Tanya
authors.initials: T
authors.lastname: Zappitelli
authors.forename: Rosa
authors.initials: R
authors.lastname: da Silva
authors.forename: Jane E
authors.initials: JE
authors.lastname: Aubin
authors.forename: Rene E
authors.initials: RE
authors.lastname: Harrison
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D000975:Antioxidants; D015820:Cadherins; D050659:Core Binding Factor Alpha 1 Subunit; C405804:EB1 microtubule binding proteins; C584107:EB1 protein, mouse; D008869:Microtubule-Associated Proteins; D051176:beta Catenin; D019938:Cyclin D1; D001205:Ascorbic Acid
keywords: Cell Biology; Cell Contacts; E-cadherin; EB1; Microtubules; Osteoblasts; beta-Catenin