Essential role of osterix for tooth root but not crown dentin formation - PubMed (original) (raw)

Essential role of osterix for tooth root but not crown dentin formation

Hua Zhang et al. J Bone Miner Res. 2015 Apr.

Abstract

Tooth is made of crown and root. It is widely believed that dentin formation in crown and root uses the same regulatory mechanism. However, identification of nuclear factor 1 C (NFIC)'s unique function in determining root but not crown dentin formation challenges the old thinking. In searching for the target molecules downstream of NFIC, we unexpectedly found a sharp reduction of osterix (OSX), the key transcription factor in skeleton formation, in the Nfic knockout (Nfic-KO) tooth root. We then demonstrated a dose-dependent increase of Osx in the odontoblast cell line due to a transient transfection of Nfic expression plasmid. Studies of global and conditional Osx-KO mice revealed no apparent changes in the crown dentin tubules and dentin matrix. However, the OSX conditional KO (cKO) mice (crossed to the 2.3-kb collagen type 1 [Col1]-Cre) displayed an increase in cell proliferation but great decreases in expressions of root dentin matrix proteins (dentin matrix protein 1 [DMP1] and dentin sialophosphoprotein [DSPP]), leading to an inhibition in odontoblast differentiation, and short, thin root dentin with few dentin tubules. Compared to the Nfic-KO tooth, which contains essentially no dentin tubules and remains in a "root-less" status at adult stages, the Osx-cKO root phenotype had partially improved at the late stage, indicating that other factors can compensate for OSX function. Thus, we conclude that OSX, one of the key downstream molecules of NFIC, plays a critical role in root, but not crown, formation.

Keywords: DENTIN; NFIC; ODONTOBLAST; OSTERIX; TOOTH ROOT.

© 2014 American Society for Bone and Mineral Research.

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Figures

Fig 1

Fig 1

Osx, downstream of Nfic, is not required for crown formation but is needed for root dentin formation during postnatal development. (a) The 2-week-old Nfic KO displayed short molar roots as viewed by uCT (lower panel); (b) Immunohistochemistry stains showed few positive OSX signals in the KO odontoblasts (lower panel); (c) Osx cKO molar roots (lower panel) were short and thin according to uCT findings; (d) Backscattered SEM images revealed large and immature osteocytes in the Osx cKO alveolar bone (lower panel); (e) FITC-confocal images displayed no apparent differences in the cKO (lower panel) and control crown dentin tubules; (f–g) backscattered SEM (f) and acid-etched SEM (g) showed that the cKO dentin tubules were poorly formed in structure and sharply reduced in number with ~1/3 region without dentin tubules (right panels).

Fig 2

Fig 2

Defects in dentin mineralization in Osx cKO mice were caused by an increase in cell proliferation and a decrease in odontoblast differentiation (right panels). (a) The double-labeling images unveiled a great reduction in the Osx cKO root at the age of 4 weeks, which is significantly different from the age-matched control (n = 4, **, P < 0.01); (b) The BrdU assay showed a sharp increase in the cKO odontoblast cell proliferation with a statistical significance from the control (n=4, * P<0.01); (c) The H&E stain documented an immature odontoblast cell layer with no sign of polarization morphology in the cKO root; (d–f) The in situ hybridization assays displayed a great decrease in type 1 collagen (d), Dmp1 (e), and Dspp (f) in the cKO root; (g) The immunostain images confirmed a great reduction in tubulin expressions in the immature flat odontoblast in cKO; (h) overexpression of PEX3-Osx plasmids in the odontoblast cell line led to an increase in mineral nodules and expressions of Dmp1 and Dspp; and (i) The working hypothesis: OSX is the key downstream molecule of NFIC, which controls root but not crown dentin via its inhibitory role in cell proliferation and stimulatory function in the cell differentiation/dentin tubule formation (right panel). Deletion of Osx will lead to root but not crown defects (left panel). In addition, there must be unknown factors, which partly compensate for OSX function during late root formation.

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