ATF-2 cooperates with Smad3 to mediate TGF-beta effects on chondrocyte maturation.
This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-beta. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-beta, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-beta-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-beta. However, in combination the effects were additive and similar to the inhibitory effects of TGF-beta on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-beta on colX, while together the blockade was complete. Similar effects were observed with another TGF-beta-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-beta signaling. In contrast, although TGF-beta does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-beta and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-beta signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-beta or BMP-2.
Ionescu
Andreia M
AM
Center for Musculoskeletal Research, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA.
Schwarz
Edward M
EM
Zuscik
Michael J
MJ
Drissi
Hicham
H
Puzas
J Edward
JE
Rosier
Randy N
RN
O'Keefe
Regis J
RJ
D051698:Activating Transcription Factor 2; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020239:Receptor Cross-Talk; D015398:Signal Transduction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases
D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases