title: Smad2/3 is involved in growth inhibition of mouse embryonic palate mesenchymal cells induced by all-trans retinoic acid.
abstract: BACKGROUND
  Cleft palate is one of the major malformations induced by retinoic acid in both rodents and humans. The purpose of the present study was to elucidate the mechanism by which all-trans retinoic acid (atRA) induces the cleft palate.
  
  
  METHODS
  The cell cycle distribution of mouse embryonic palate mesenchymal (MEPM) cells under atRA (100 mg/kg) treatment on gestation day (GD) 10 or GD 12 were measured by immunohistochemistry and flow cytometry. The p21, phospho-Rb, Smad2/3, phospho-Smad2 and phospho-Smad3 protein expression levels were detected by western blot, respectively. Quantitative real-time PCR was performed for p21, Smad2, and Smad3 gene expression in each group under both conditions. Small interfering RNA (siRNA) was applied to inhibit Smad2/3 expression in MEPM cells and the effect was detected by western blot and flow cytometry.
  
  
  RESULTS
  The G(0)/G(1) arrest in MEPM cells in vivo was induced by atRA on GD 10. The protein expression levels of p21, Smad2/3, phospho-Smad2, and phospho-Smad3 were increased, while phospho-Rb was decreased in MEPM after atRA treatment on GD 10. These changes were not observed on the GD 12 group. Moreover, the mRNA expression levels of p21, Smad2, and Smad3 detected by quantitative real-time PCR were almost consistent with their protein expression trends. Furthermore, p21 was partially decreased and G(0)/G(1) arrest was partially released following Smad2/3 siRNA knockdown.
  
  
  CONCLUSIONS
  The induction of G(0)/G(1) block by atRA in MEPM cells varied with the development stage of exposure. Our study demonstrated that Smad2/3 regulation of p21 was partly required for atRA-induced cell cycle perturbations in MEPM cells.
authors.affiliation: Guanghua College of Stomatology, Sun Yat-sen University, Guangzhou, 510055, PR China.
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.forename: Hongzhang
authors.initials: H
authors.lastname: Huang
authors.forename: Yiyang
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D002972:Cleft Palate / Q000139:chemically induced* / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D020868:Gene Silencing; D008648:Mesoderm / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D051379:Mice; D010159:Palate / Q000002:abnormalities / Q000187:drug effects* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D013723:Teratogens / Q000633:toxicity*; D014212:Tretinoin / Q000633:toxicity*
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D013723:Teratogens; D014212:Tretinoin