Abstract
Transforming growth factor-beta (TGF-beta) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-beta rapidly induced beta-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-beta-increased beta-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3(-/-) chondrocytes treated with TGF-beta. SMAD3 inhibited beta-transducin repeat-containing protein-mediated degradation of beta-catenin and immunoprecipitated with beta-catenin following TGF-beta treatment. Both SMAD3 and beta-catenin co-localized to the nucleus after TGF-beta treatment. Although both TGF-beta and beta-catenin stimulated cyclin D(1) expression in chondrocytes, the effect of TGF-beta was inhibited with beta-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-beta stimulates cyclin D(1) expression at least in part through activation of beta-catenin signaling.
Highlights
In this study, we investigated the interaction between TGF- and -catenin signaling in chondrocytes
The interaction of TGF- and -catenin signaling in chondrocytes has not been previously described
We have demonstrated that TGF- activated -catenin signaling through SMAD3
Summary
Cell Culture—Smad3Ϫ/Ϫ mice derived from a C57/B6 lineage, in which exon 8 of the Smad gene is deleted The anterior rib cage and sternum were harvested en bloc, washed with sterile phosphate-buffered saline (PBS), and 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 Dulbecco’s modified Eagle’s medium; Roche Applied Science) for 90 min at 37 °C. After excess serum was removed, mouse anti--catenin and rabbit anti-SMAD3 antibodies diluted to 1:50 in PBS containing 10% goat serum and 0.1% saponin (Research Organics, Inc., Cleveland, OH) were applied to slides and incubated overnight at 4 °C. After thorough washes with PBS, the slides were incubated in the dark for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:100 in PBS containing 0.1% saponin and 10% goat serum. All BrdUrd-positive chondrocytes in the growth plate were counted, and at least three samples from each group were used
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