Transforming Growth Factor-β Stimulates Cyclin D1 Expression through Activation of β-Catenin Signaling in Chondrocytes

Abstract

Transforming growth factor-␤ (TGF-␤) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-␤ rapidly induced ␤-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-␤-increased ␤-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3 ؊/؊ chondrocytes treated with TGF-␤. SMAD3 inhibited ␤-transducin repeat-containing protein-mediated degradation of ␤-catenin and immunoprecipitated with ␤-catenin following TGF-␤ treatment. Both SMAD3 and ␤-catenin co-localized to the nucleus after TGF-␤ treatment. Although both TGF-␤ and ␤-catenin stimulated cyclin D 1 expression in chondrocytes, the effect of TGF-␤ was inhibited with ␤-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-␤ stimulates cyclin D 1 expression at least in part through activation of ␤-catenin signaling.Endochondral bone formation involves condensation and differentiation of mesenchymal cells into chondrocytes, followed by chondrocyte proliferation, maturation, hypertrophic differentiation, and apoptosis. Eventually, the calcified cartilage tissue formed in the growth plate is replaced by bone tissue (1). Each step of the endochondral bone formation process is precisely regulated by local growth factors. Among these factors, transforming growth factor-␤ (TGF-␤) 3 plays important roles in chondrocyte proliferation and hypertrophy. TGF-␤ promotes chondrocyte proliferation but inhibits chondrocyte differentiation and hypertrophy (2-6). The mechanism of TGF-␤-induced chondrocyte proliferation remains undefined.In this study, we investigated the interaction between TGF-␤ and ␤-catenin signaling in chondrocytes. We found that TGF-␤ activates ␤-catenin signaling through SMAD3. SMAD3 interacted with ␤-catenin and increased ␤-catenin nuclear translocation and signaling. Although TGF-␤ stimulated cyclin D 1 expression in chondrocytes, this effect was abolished by inhibition of ␤-catenin signaling. These results demonstrate for the first time that TGF-␤ stimulates cyclin D 1 expression in chondrocytes at least in part through activation of ␤-catenin signaling. These findings provide novel insight regarding the mechanism through which TGF-␤ regulates chondrocyte proliferation. MATERIALS AND METHODS Cell Culture-Smad3Ϫ/Ϫ mice derived from a C57/B6 lineage, in which exon 8 of the Smad3 gene is deleted (a kind gift from Dr. C. X. Deng, National Institutes of Health, Bethesda, MD) (7), were bred using heterozygote pairs. 3-day-old neonatal mice were killed and genotyped using tail tissues obtained at the time of death. The anterior rib cage and sternum were harvested en bloc, washed with sterile phosphate-buffered saline (PBS), and then digested with Pronase (Roche Applied Science) dissolved in PBS (2 mg/ml) in a 37°C water bath with continuous shaking for 60 min. This was followed by incubation in a solution of collagenase D (3 mg/ml dissolved in serum-free Dulbec...

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Polarity of Response to Transforming Growth Factor-β1 in Proximal Tubular Epithelial Cells Is Regulated by β-Catenin

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Temporal Activation of β-Catenin Signaling in the Chondrogenic Process of Mesenchymal Stem Cells Affects the Phenotype of the Cartilage Generated

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