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Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFβ1 in mouse gingival epithelial cells

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Abstract

Amelotin (AMTN) is expressed and secreted by ameloblasts in the maturation stage of amelogenesis and persist with low levels in the junctional epithelium (JE) of erupted teeth. The purpose of this study is to investigate the transcriptional regulation of the AMTN gene by transforming growth factor beta1 (TGFβ1) in gingival epithelial (GE1) cells in the apoptosis phase. Apoptosis was evaluated by the fragmentation of chromosomal DNA and TUNEL staining. A real-time PCR was carried out to examine the AMTN mRNA levels induced by TGFβ1 and Smad3 overexpression. Transient transfection analyses were completed using the various lengths of mouse AMTN gene promoter constructs with or without TGFβ1. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the Smad3 bindings to the AMTN gene promoter by TGFβ1. TGFβ1-induced apoptosis in GE1 cells were detected at 24 and 48 h by DNA fragmentation and TUNEL staining. AMTN mRNA levels increased at 6 h and reached maximum at 24 h in GE1 cells. Luciferase activities of the mouse AMTN gene promoter constructs were induced by TGFβ1. The results of the ChIP assays showed that there was an increase in Smad3 binding to Smad-binding element (SBE)#1 and SBE#2 after stimulation by TGFβ1. Immunohistochemical localization of AMTN was detected in the JE, and the AMTN protein levels in Smad3-deficient mice were decreased compared with wild-type mice. AMTN mRNA levels were induced at the initiation of apoptosis by TGFβ1, which mediated through the Smad3 bindings to SBEs in the mouse AMTN gene promoter.

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Acknowledgments

This work was supported in part by the Grant-in-Aid for Scientific Research (Young Scientists (B); No. 25862059, Scientific Research (C); No. 25463229), Nihon University Multidisciplinary Research Grant for 2014, Nihon University School of Dentistry at Matsudo Young Investigator Seed Funding Grant for 2014, and a Grant of Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT), 2010-2014 (S1001024), and an Operating Grant MOP-79449 (BG) from the Canadian Institutes of Health Research (CIHR).

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Authors and Affiliations

  1. Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, Japan

    Yohei Nakayama, Sari Matsui, Keisuke Noda, Mizuho Yamazaki, Yasunobu Iwai, Hiroyoshi Matsumura & Yorimasa Ogata

  2. Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Chiba, Japan

    Yohei Nakayama & Yorimasa Ogata

  3. Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan

    Takashi Izawa & Eiji Tanaka

  4. Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada

    Bernhard Ganss

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  1. Yohei Nakayama
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Correspondence to Yohei Nakayama or Yorimasa Ogata.

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Nakayama, Y., Matsui, S., Noda, K. et al. Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFβ1 in mouse gingival epithelial cells. Apoptosis 21, 1057–1070 (2016). https://doi.org/10.1007/s10495-016-1279-5

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