Overexpression of Smad2 inhibits proliferation of gingival epithelial cells
Overexpression of Smad2 inhibits proliferation of gingival epithelial cells
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DOI：
10.1111/jre.12106
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发表时间：
2014-06-01
影响因子：
3.5
通讯作者：
Takashiba, S.
中科院分区：
文献类型：
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作者：
Shimoe, M.;Yamamoto, T.;Takashiba, S.
牙周组织再生过程中，牙龈上皮细胞迁移和增殖的时空抑制是重要因素。转化生长因子β（TGF-β）在伤口愈合的多个方面是重要的，并且TGF-β的下游转录因子Smad2在牙龈伤口愈合期间对再上皮化具有抑制作用。因此，我们研究了迁移和增殖状态的影响，和内/胞外信号调节Smad2过表达在gingival epithelialcells. Material and MethodsGingival epithelial cells从腭牙龈组织的转基因小鼠过度表达Smad2角蛋白14启动子驱动分离。免疫印迹法和免疫荧光法检测Smad2的表达。划痕试验和5-溴-2 '-脱氧尿苷染色用于评估细胞迁移和增殖。为了抑制TGF-β I型受体，向培养物补充SB 431542。通过ELISA定量分泌的TGF-β。采用实时荧光定量PCR和免疫荧光法检测牙龈结合上皮细胞中Smad2基因的表达。划痕实验和5-溴-2 '-脱氧尿苷染色表明，Smad2过表达细胞的迁移能力与野生型细胞相比无显著差异，但增殖率降低。SB431542显著抑制Smad2磷酸化，这与Smad2过表达细胞的增殖率恢复一致。TGF-β释放的ELISA没有显示基因型之间的任何差异。细胞周期抑制剂，p15和p21，表现出显着上调Smad2过表达细胞相比，野生型对照。结论Smad2过表达激活牙龈上皮细胞的信号通路依赖于TGF-β I型受体，激活的Smad2可增加p15和p21的表达，抑制牙龈上皮细胞进入细胞周期，从而抑制牙龈上皮细胞的增殖。了解Smad2诱导的信号传导将有助于可能的临床应用，以调节牙龈上皮向下生长。
Background and ObjectiveSpatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor beta (TGF-beta) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-beta, 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 MethodsGingival 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-beta type I receptor, the cultures were supplemented with SB431542. Secreted TGF-beta was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium.ResultsSmad2-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-beta 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.ConclusionThe signaling activation triggered by overexpression of Smad2 was dependent on TGF-beta 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.