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Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: implications for the induction of biomineralization

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Abstract

Dmp1 is an acidic phosphoprotein that is specifically expressed in osteocytes. During the secretory process, the full-length, precursor Dmp1 is cleaved into N- and C-terminal fragments. C-terminal Dmp1 is phosphorylated, becoming a highly negatively charged domain that may assist in bone mineralization by recruiting calcium ions and influencing subsequent mineral deposition. It has been recently reported that the Golgi-localized protein kinase Fam20C phosphorylates Dmp1 in vitro. To investigate this phosphorylation in situ, we determined the locations of phosphorylated Dmp1 and Fam20C in rat bones using immunohistochemistry. During osteocytogenesis, osteoblastic, osteoid, and young osteocytes (but not old osteocytes) express Dmp1 mRNA and contain Dmp1 protein in the Golgi apparatus. These Dmp1-producing cells were distributed across the surface layer of cortical bone. Using immunofluorescence, we found that N- and C-terminal Dmp1 fragments were predominantly distributed along the lacunar walls and canaliculi of mineralized bone, respectively, but were not present in the osteoid matrix. We also found that Fam20C and its substrate, C-terminal Dmp1, colocalized in the Golgi of osteoblastic, osteoid, and young osteocytes. Furthermore, phosphorylated C-terminal Dmp1 was present in the Golgi of young osteocytes. Double-labeling immunoelectron microscopy revealed that phosphorylated C-terminal Dmp1 localized to the canalicular wall in mineralized bone. These findings suggest that C-terminal Dmp1 is phosphorylated within osteocytes and then secreted into the pericanalicular matrix of mineralized bone. Phosphorylated, negatively charged C-terminal Dmp1 in the pericanalicular matrix may play an important role in bone mineralization by recruiting calcium ions.

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Acknowledgments

This work was partially supported by JSPS KAKENHI Grant Number 24390409 and 26670801, and was partially supported by “Nanotechnology Platform” (Project No. 12024046) of MEXT. We acknowledge Emeritus Prof. Yoshiro Takano and Dr. Yukiko Nakano who belonged to Tokyo Medical and Dental University for their support with immunohistochemistry, and Dr. Yu Usami of Osaka University for advice regarding the confocal microscope images.

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Authors and Affiliations

  1. Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Kaori Oya, Ken Ishida, Sunao Sato, Mitsunobu Kishino, Katsutoshi Hirose, Yuzo Ogawa & Satoru Toyosawa

  2. Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, Suita, Osaka, Japan

    Kaori Oya & Fumio Takeshige

  3. Reserch Center for Ultra-High Voltage Electron Microscopy, Osaka University, Ibaraki, Osaka, Japan

    Tomoki Nishida & Hidehiro Yasuda

  4. Department of Prosthodontics and Oral Rehabilitation, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

    Ken Ishida & Kazunori Ikebe

  5. Department of Cell Biology, Unit of Basic Medical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

    Toshihisa Komori

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  1. Kaori Oya
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  2. Ken Ishida
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  3. Tomoki Nishida
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Correspondence to Satoru Toyosawa.

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Oya, K., Ishida, K., Nishida, T. et al. Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: implications for the induction of biomineralization. Histochem Cell Biol 147, 341–351 (2017). https://doi.org/10.1007/s00418-016-1490-z

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  • DOI: https://doi.org/10.1007/s00418-016-1490-z

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