title: Overexpression of Smad2 inhibits proliferation of gingival epithelial cells.
abstract: OBJECTIVE
  Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor β (TGF-β) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-β, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells.
  
  
  METHODS
  Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-β type I receptor, the cultures were supplemented with SB431542. Secreted TGF-β was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium.
  
  
  RESULTS
  Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-β release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21.
  
  
  CONCLUSIONS
  The signaling activation triggered by overexpression of Smad2 was dependent on TGF-β type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.
authors.affiliation: Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Shimoe
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: N
authors.initials: N
authors.lastname: Shiomi
authors.forename: K
authors.initials: K
authors.lastname: Tomikawa
authors.forename: S
authors.initials: S
authors.lastname: Hongo
authors.forename: K
authors.initials: K
authors.lastname: Yamashiro
authors.forename: T
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: H
authors.initials: H
authors.lastname: Maeda
authors.forename: S
authors.initials: S
authors.lastname: Takashiba
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001973:Bromodeoxyuridine; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000032:analysis; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis; D004149:Dioxoles / Q000494:pharmacology; D004846:Epithelial Attachment / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005881:Gingiva / Q000166:cytology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000032:analysis; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000502:physiology; D051899:Smad2 Protein / Q000032:analysis / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; C493956:CDKN1A protein, human; D050762:Cyclin-Dependent Kinase Inhibitor p15; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004149:Dioxoles; D047428:Protein Kinase Inhibitors; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D016212:Transforming Growth Factor beta; D001973:Bromodeoxyuridine
keywords: SB431542; Smad2; gingival epithelial cells; proliferation