Negative functional interaction of retinoic acid and TGF-beta signaling mediated by TG-interacting factor during chondrogenesis.
We previously showed that atRA (all-trans Retinoic Acid, atRA) inhibites chondrogenesis by downregulation of TGF-beta/Smad signaling. However, the molecular link between RA and TGF-beta signaling is unknown. Using a mouse embryo palate mesenchyme micomass cultures (MMCs) system, we explored interactions of RA and TGF-beta signaling during chondrogenesis. We found that atRA suppressed chondrogenesis and Smad2/3 phosphorylation regardless of the presence of TGF-beta3. Functional assays indicated that TGF-beta3 treatment or co-transfection of expressing Smad2/3 vectors suppressed atRA-induced RARE-tk-Luc activity. Conversely, atRA or RAR-overexpression repressed TGF-beta3-induced transactivation of the TGF-beta-responsive reporter, p3TP-Lux. ChIP assay revealed the binding of the Smad transcriptional co-repressor TGIF (TG-interacting factor, TGIF) to RARbeta promoter in control MMCs, but this association was decreased by the addition of RA and increased by TGF-beta3, respectively. Further examinations revealed that TGIF exerted a pivotal role in regulating crosstalk of RA and TGF-beta signaling, since siRNA knockdown of TGIF partially abolished the ability of atRA to suppress TGF-beta3-induced chondrogenesis, whereas forced expression of TGIF blocked the ability of TGF-beta3 to relieve atRA-mediated the suppression of chondrogenesis. Furthermore, we demonstrated that the effects of atRA on TGF-beta-dependent gene activation and of TGF-beta on RA-dependent gene activation are mediated by TGIF with siRNA to downregulate TGIF. Collectively, these findings indicated a negative functional interplay of RA and TGF-beta signaling mediated by TGIF to modulate chondrogenesis in MMCs.
Zhang
Hang
H
School of Public Health, Zhengzhou University, Zhengzhou, PR, China.
Li
Ning
N
Tang
Yunan
Y
Wu
Weidong
W
Zhang
Qi
Q
Yu
Zengli
Z
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects* / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014212:Tretinoin / Q000494:pharmacology*
D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D014212:Tretinoin