Smad2 and 3 mediate transforming growth factor-beta1-induced inhibition of chondrocyte maturation.
Transforming growth factor-beta (TGF-beta) is a multifunctional regulator of a variety of cellular functions, including proliferation, differentiation, matrix synthesis, and apoptosis. In growth plate chondrocytes, TGF-beta slows the rate of maturation. Because the current paradigm of TGF-beta signaling involves Smad proteins as downstream regulators of target genes, we have characterized their role as mediators of TGF-beta effects on chondrocyte maturation. Both Smad2 and 3 translocated to the nucleus upon TGF-beta1 signaling, but not upon BMP-2 signaling. Cotransfection experiments using the TGF-beta responsive and Smad3 sensitive p3TP-Lux luciferase reporter demonstrated that wild-type Smad3 potentiated, whereas dominant negative Smad3 inhibited TGF-beta1 induced luciferase activity. To confirm the role of Smad2 and 3 as essential mediators of TGF-beta1 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-beta 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-beta1 signaling on chondrocyte maturation.
Ferguson
C M
CM
Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Schwarz
E M
EM
Reynolds
P R
PR
Puzas
J E
JE
Rosier
R N
RN
O'Keefe
R J
RJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects; D003094:Collagen / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D008156:Luciferases / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D003094:Collagen; D008156:Luciferases; D000469:Alkaline Phosphatase