Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-β 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-β deficiency and is required for accelerated chondrocyte maturation.
Disruption of TGF-β 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.
Materials and 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-β and BMP signaling were examined. Chondrocytes were treated with TGF-β and BMP-2, and effects on maturation-related genes and BMP/TGF-β responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.
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-β 1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-β toward BMP signaling. In Smad3−/− chondrocytes, alternative TGF-β signaling pathways remained responsive, as shown by luciferase assays. These non–Smad3-dependent TGF-β pathways reduced colX expression and alkaline phosphatase activity in TGF-β–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.
These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-β signaling with important implications for osteoarthritis and cartilage diseases.