title: Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.
abstract: BACKGROUND
  Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs.
  
  
  METHODS
  ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1.
  
  
  RESULTS
  ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis.
  
  
  CONCLUSIONS
  ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.
authors.affiliation: Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands.|Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, the Netherlands.
authors.forename: Laurie M G
authors.initials: LM
authors.lastname: de Kroon
authors.affiliation: Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, the Netherlands.
authors.forename: Roberto
authors.initials: R
authors.lastname: Narcisi
authors.affiliation: Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands.
authors.forename: Esmeralda N
authors.initials: EN
authors.lastname: Blaney Davidson
authors.affiliation: Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, the Netherlands.|Section of Veterinary Clinical Sciences, School of Veterinary Medicine, University College Dublin, Dublin, Ireland.
authors.forename: Mairéad A
authors.initials: MA
authors.lastname: Cleary
authors.affiliation: Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands.
authors.forename: Henk M
authors.initials: HM
authors.lastname: van Beuningen
authors.affiliation: Department of Otorhinolaryngology, Erasmus MC University Medical Center, Rotterdam, the Netherlands.
authors.forename: Wendy J L M
authors.initials: WJ
authors.lastname: Koevoet
authors.affiliation: Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, the Netherlands.|Department of Otorhinolaryngology, Erasmus MC University Medical Center, Rotterdam, the Netherlands.
authors.forename: Gerjo J V M
authors.initials: GJ
authors.lastname: van Osch
authors.affiliation: Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands.
authors.forename: Peter M
authors.initials: PM
authors.lastname: van der Kraan
mesh_terms: D029404:Activin Receptors / Q000235:genetics*; D030301:Activin Receptors, Type II / Q000235:genetics*; D001853:Bone Marrow / Q000502:physiology*; D001854:Bone Marrow Cells / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism / Q000502:physiology; D020219:Chondrogenesis / Q000502:physiology; D024043:Collagen Type II / Q000235:genetics; D015536:Down-Regulation / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000235:genetics*
chemical_list: D024043:Collagen Type II; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; C425364:ACVRL1 protein, human; D029404:Activin Receptors; D030301:Activin Receptors, Type II; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human