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Smad2 and 3 Mediate Transforming Growth Factor-ß1-Induced Inhibition of Chondrocyte Maturation¹

Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Address all correspondence and requests for reprints to: Regis J. O’Keefe, M.D., Box 665, Department of Orthopaedics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642. E-mail: regis_okeefe{at}urmc.rochester.edu

Transforming growth factor-ß (TGF-ß) is a multifunctional regulator of a variety of cellular functions, including proliferation, differentiation, matrix synthesis, and apoptosis. In growth plate chondrocytes, TGF-ß slows the rate of maturation. Because the current paradigm of TGF-ß signaling involves Smad proteins as downstream regulators of target genes, we have characterized their role as mediators of TGF-ß effects on chondrocyte maturation. Both Smad2 and 3 translocated to the nucleus upon TGF-ß1 signaling, but not upon BMP-2 signaling. Cotransfection experiments using the TGF-ß responsive and Smad3 sensitive p3TP-Lux luciferase reporter demonstrated that wild-type Smad3 potentiated, whereas dominant negative Smad3 inhibited TGF-ß1 induced luciferase activity. To confirm the role of Smad2 and 3 as essential mediators of TGF-ß1 effects on chondrocyte maturation, we overexpressed both wild-type and dominant negative Smad2 and 3 in virally infected chondrocyte cultures. Overexpression of both wild-type Smad2 and 3 potentiated the inhibitory effect of TGF-ß on chondrocyte maturation, as determined by colx and alkaline phosphatase activity, whereas dominant negative Smad2 and 3 blocked these effects. Wild-type and dominant negative forms of Smad3 had more pronounced effects than Smad2. Our results define Smad2 and 3 as key mediators of the inhibitory effect of TGF-ß1 signaling on chondrocyte maturation.

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