title: The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFβ1.
abstract: OBJECTIVE
  In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorß1 (TGFβ1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFβ1 signaling was also studied.
  
  
  METHODS
  Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFβ1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFβ/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line.
  
  
  RESULTS
  In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFβ1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFβ/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFβ1.
  
  
  CONCLUSIONS
  This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFβ1. This observation underlines the importance of TGFβ1 in maintenance of chondrocyte phenotype.
authors: Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: A
authors: A
authors: van Caam
authors: Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: E
authors: E
authors: Blaney Davidson
authors: Department of Molecular Cell Biology and Cancer Genomics Centre Netherlands, Leiden University Medical Center, Leiden, The Netherlands.
authors: A
authors: A
authors: Garcia de Vinuesa
authors: Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: E
authors: E
authors: van Geffen
authors: Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: W
authors: W
authors: van den Berg
authors: Department of Molecular Cell Biology and Cancer Genomics Centre Netherlands, Leiden University Medical Center, Leiden, The Netherlands.
authors: M-J
authors: MJ
authors: Goumans
authors: Department of Molecular Cell Biology and Cancer Genomics Centre Netherlands, Leiden University Medical Center, Leiden, The Netherlands.
authors: P
authors: P
authors: ten Dijke
authors: Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address: Peter.vanderkraan@radboudumc.nl.
authors: P
authors: P
authors: van der Kraan
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D055427:Growth Differentiation Factor 2 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006984:Hypertrophy; D008024:Ligands; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D051898:Smad1 Protein / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D051902:Smad5 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D000099222:betaIG-H3 Protein
chemical_list: D016326:Extracellular Matrix Proteins; D055427:Growth Differentiation Factor 2; D008024:Ligands; D051898:Smad1 Protein; D051899:Smad2 Protein; D051902:Smad5 Protein; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein
keywords: BMP-9; Chondrocyte; Hypertrophy; Smad signaling; TGF-beta1