Shimoe M, Yamamoto T, Shiomi N, Tomikawa K, Hongo S, Yamashiro K, Yamaguchi T, Maeda H, Takashiba S
Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan.
J Periodontal Res. 2014 Jun;49(3):290-8. doi: 10.1111/jre.12106. Epub 2013 Jun 6.
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.
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.
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.
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.
牙龈上皮顶端迁移和增殖的时空抑制是牙周再生的重要因素。转化生长因子β（TGF-β）在伤口愈合的多个方面发挥重要作用，而Smad2作为TGF-β的下游转录因子，在牙龈伤口愈合过程中对再上皮化具有抑制作用。因此，我们研究了Smad2过表达对牙龈上皮细胞迁移和增殖状态以及细胞内/外信号调节的影响。
从由角蛋白14启动子驱动的Smad2过表达转基因小鼠的腭部牙龈组织中分离牙龈上皮细胞。通过蛋白质免疫印迹和免疫荧光分析鉴定Smad2的表达。进行划痕试验和5-溴-2'-脱氧尿苷染色以评估细胞迁移和增殖。为使TGF-β I型受体失活，在培养物中添加SB431542。通过酶联免疫吸附测定（ELISA）对分泌的TGF-β进行定量。通过实时逆转录聚合酶链反应（RT-PCR）和牙龈结合上皮的体内免疫荧光分析检测Smad2靶基因的表达。
证实过表达Smad2的细胞在细胞核中有显著磷酸化的Smad2。划痕试验和5-溴-2'-脱氧尿苷染色表明，过表达Smad2的细胞迁移无显著差异，但与野生型对照相比增殖率降低。SB431542显著抑制Smad2磷酸化，这与过表达Smad2的细胞增殖率恢复一致。TGF-β释放的ELISA检测未显示不同基因型之间存在任何差异。与野生型对照相比，细胞周期抑制剂p15和p21在过表达Smad2的细胞中显著上调。此外，转基因小鼠的结合上皮显示P-Smad2、p15和p21的表达增加。
Smad2过表达引发的信号激活依赖于TGF-β I型受体，激活的Smad2增加p15和p21的表达，从而抑制细胞周期进入，对牙龈上皮细胞产生抗增殖作用。了解Smad2诱导信号对于调节牙龈上皮向下生长的可能临床应用具有重要意义。
