Smad2 and 3 mediate transforming growth factor-beta 1-induced inhibition of chondrocyte maturation

Transforming growth factor-beta (TGF-beta) is a multifunctional regulator o f a variety of cellular functions, including proliferation, differentiation , matrix synthesis, and apoptosis. In growth plate chondrocytes, TGF-beta s lows the rate of maturation. Because the current paradigm of TGF-beta signa ling involves Smad proteins as downstream regulators of target genes, we ha ve characterized their role as mediators of TGF-beta effects on chondrocyte maturation. Both Smad2 and 3 translocated to the nucleus upon TGF-beta1 si gnaling, but not upon BMP-2 signaling. Cotransfection experiments using the TGF-beta responsive and Smad3 sensitive p3TP-Lux luciferase reporter demon strated that wild-type Smad3 potentiated, whereas dominant negative Smad3 i nhibited TGF-beta1 induced luciferase activity. To confirm the role of Smad 2 and 3 as essential mediators of TGF-beta1 effects on chondrocyte maturati on, we overexpressed both wild-type and dominant negative Smad2 and 3 in vi rally infected chondrocyte cultures. Overexpression of both wildtype Smad2 and 3 potentiated the inhibitory effect of TGF-beta on chondrocyte maturati on, as determined by colx and alkaline phosphatase activity, whereas domina nt negative Smad2 and 3 blocked these effects. Wild-type and dominant negat ive farms of Smad3 had more pronounced effects than Smad2. Our results defi ne Smad2 and 3 as key mediators of the inhibitory effect of TGF-beta1 signa ling on chondrocyte maturation.