title: Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation.
abstract: Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation.
  
  
  BACKGROUND
  Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures.
  
  
  METHODS
  Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.
  
  
  RESULTS
  Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor.
  
  
  CONCLUSIONS
  These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.
authors.affiliation: Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Tian-Fang
authors.initials: TF
authors.lastname: Li
authors.forename: Michael
authors.initials: M
authors.lastname: Darowish
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zuscik
authors.forename: Di
authors.initials: D
authors.lastname: Chen
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: Randy N
authors.initials: RN
authors.lastname: Rosier
authors.forename: Hicham
authors.initials: H
authors.lastname: Drissi
authors.forename: Regis J
authors.initials: RJ
authors.lastname: O'Keefe
mesh_terms: D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002357:Cartilage Diseases / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
chemical_list: D019485:Bone Morphogenetic Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D016212:Transforming Growth Factor beta