Overexpression of Smad2 inhibits proliferation of gingival epithelial cells

M. Shimoe, Tadashi Yamamoto, N. Shiomi, K. Tomikawa, S. Hongo, Keisuke Yamashiro, T. Yamaguchi, H. Maeda, Shogo Takashiba

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Background and 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. Material and 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. Conclusion: 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.

Original languageEnglish
Pages (from-to)290-298
Number of pages9
JournalJournal of Periodontal Research
Volume49
Issue number3
DOIs
Publication statusPublished - 2014

Fingerprint

Transforming Growth Factors
Epithelial Cells
Epithelial Attachment
Growth Factor Receptors
Bromodeoxyuridine
Wound Healing
Transgenic Mice
Fluorescent Antibody Technique
Cell Cycle
Enzyme-Linked Immunosorbent Assay
Staining and Labeling
Re-Epithelialization
Cell Movement
Real-Time Polymerase Chain Reaction
Regeneration
Transcription Factors
Up-Regulation
Epithelium
Western Blotting
Genotype

Keywords

  • Gingival epithelial cells
  • Proliferation
  • SB431542
  • Smad2

ASJC Scopus subject areas

  • Periodontics
  • Medicine(all)

Cite this

Overexpression of Smad2 inhibits proliferation of gingival epithelial cells. / Shimoe, M.; Yamamoto, Tadashi; Shiomi, N.; Tomikawa, K.; Hongo, S.; Yamashiro, Keisuke; Yamaguchi, T.; Maeda, H.; Takashiba, Shogo.

In: Journal of Periodontal Research, Vol. 49, No. 3, 2014, p. 290-298.

Research output: Contribution to journalArticle

Shimoe, M. ; Yamamoto, Tadashi ; Shiomi, N. ; Tomikawa, K. ; Hongo, S. ; Yamashiro, Keisuke ; Yamaguchi, T. ; Maeda, H. ; Takashiba, Shogo. / Overexpression of Smad2 inhibits proliferation of gingival epithelial cells. In: Journal of Periodontal Research. 2014 ; Vol. 49, No. 3. pp. 290-298.
@article{af9e28bc01d445b882163264f4721eb3,
title = "Overexpression of Smad2 inhibits proliferation of gingival epithelial cells",
abstract = "Background and 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. Material and 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. Conclusion: 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.",
keywords = "Gingival epithelial cells, Proliferation, SB431542, Smad2",
author = "M. Shimoe and Tadashi Yamamoto and N. Shiomi and K. Tomikawa and S. Hongo and Keisuke Yamashiro and T. Yamaguchi and H. Maeda and Shogo Takashiba",
year = "2014",
doi = "10.1111/jre.12106",
language = "English",
volume = "49",
pages = "290--298",
journal = "Journal of Periodontal Research",
issn = "0022-3484",
publisher = "Blackwell Munksgaard",
number = "3",

}

TY - JOUR

T1 - Overexpression of Smad2 inhibits proliferation of gingival epithelial cells

AU - Shimoe, M.

AU - Yamamoto, Tadashi

AU - Shiomi, N.

AU - Tomikawa, K.

AU - Hongo, S.

AU - Yamashiro, Keisuke

AU - Yamaguchi, T.

AU - Maeda, H.

AU - Takashiba, Shogo

PY - 2014

Y1 - 2014

N2 - Background and 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. Material and 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. Conclusion: 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.

AB - Background and 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. Material and 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. Conclusion: 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.

KW - Gingival epithelial cells

KW - Proliferation

KW - SB431542

KW - Smad2

UR - http://www.scopus.com/inward/record.url?scp=84899633888&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84899633888&partnerID=8YFLogxK

U2 - 10.1111/jre.12106

DO - 10.1111/jre.12106

M3 - Article

VL - 49

SP - 290

EP - 298

JO - Journal of Periodontal Research

JF - Journal of Periodontal Research

SN - 0022-3484

IS - 3

ER -