title: Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification.
abstract: The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.
authors.affiliation: Department of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Catharine A
authors.initials: CA
authors.lastname: Hellingman
authors.forename: Esmeralda N Blaney
authors.initials: EN
authors.lastname: Davidson
authors.forename: Wendy
authors.initials: W
authors.lastname: Koevoet
authors.forename: Elly L
authors.initials: EL
authors.lastname: Vitters
authors.forename: Wim B
authors.initials: WB
authors.lastname: van den Berg
authors.forename: Gerjo J V M
authors.initials: GJ
authors.lastname: van Osch
authors.forename: Peter M
authors.initials: PM
authors.lastname: van der Kraan
mesh_terms: D000328:Adult; D052117:Benzodioxoles / Q000494:pharmacology; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects*; D024043:Collagen Type II / Q000378:metabolism; D024121:Collagen Type X / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007150:Immunohistochemistry; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011720:Pyrazoles / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051898:Smad1 Protein / Q000378:metabolism*; D051902:Smad5 Protein / Q000378:metabolism*; D051904:Smad8 Protein / Q000378:metabolism*; D055815:Young Adult
chemical_list: C519132:2-(5-benzo(1,3)dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride; D052117:Benzodioxoles; D024043:Collagen Type II; D024121:Collagen Type X; D007093:Imidazoles; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; C497399:SMAD1 protein, human; C497797:SMAD5 protein, human; C107514:SMAD9 protein, human; D051898:Smad1 Protein; D051902:Smad5 Protein; D051904:Smad8 Protein; C516138:dorsomorphin; D053509:Matrix Metalloproteinase 13