Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: implications for the induction of biomineralization.
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.
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.|Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, Suita, Osaka, Japan.
Kaori
K
Oya
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.|Department of Prosthodontics and Oral Rehabilitation, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
Ken
K
Ishida
Reserch Center for Ultra-High Voltage Electron Microscopy, Osaka University, Ibaraki, Osaka, Japan.
Tomoki
T
Nishida
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Sunao
S
Sato
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Mitsunobu
M
Kishino
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Katsutoshi
K
Hirose
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Yuzo
Y
Ogawa
Department of Prosthodontics and Oral Rehabilitation, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
Kazunori
K
Ikebe
Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, Suita, Osaka, Japan.
Fumio
F
Takeshige
Reserch Center for Ultra-High Voltage Electron Microscopy, Osaka University, Ibaraki, Osaka, Japan.
Hidehiro
H
Yasuda
Department of Cell Biology, Unit of Basic Medical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
Toshihisa
T
Komori
Department of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan. toyosawa@dent.osaka-u.ac.jp.
Satoru
S
Toyosawa
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002113:Calcification, Physiologic; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism*; D007150:Immunohistochemistry; D008297:Male; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
C081635:Dmp1 protein, rat; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; C000617556:Fam20C protein, rat; D017346:Protein Serine-Threonine Kinases
Dentin matrix protein 1 (Dmp1); Fam20C; Mineralization; Osteocyte; Phosphorylation