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Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation
Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Original Article
Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation Toshihiro Miyazaki a, Maaya Inoue a, Tomomi T. Baba b, Toshihisa Komori a,n a b
Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan Department of Oral Molecular Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 23 January 2017 Received in revised form 20 February 2017 Accepted 22 February 2017
Objective: Sp7 is required for cellular cementum formation and odontoblast differentiation in the root. Sp7 is expressed in preodontoblasts and is strongly expressed in odontoblasts. However, Sp7 expression was down-regulated in odontoblasts in the crown after root formation. We examined osteoblast/odontoblast-specific Sp7 transgenic mice to investigate the effects of sustained Sp7 expression in differentiated odontoblasts. Methods: Tooth development was examined by immunohistochemical analyses, using antibodies against Sp7, pro-collagen type1a1, osteocalcin, dentin sialoprotein (DSP), nestin, and osteopontin. Results: In 1-week-old Sp7 transgenic mice, the thickness of dentin was slightly reduced, the elongation of odontoblasts was inhibited, and odontoblasts in the cusp lost polarity. At 2 weeks of age, Sp7 was strongly expressed in odontoblasts in the root, but weakly expressed in the crown of wild-type mice. It was strongly expressed in odontoblasts in the crown and root and the thinning of dentin and impairments in elongation and polarization in odontoblasts were severe in the crown of Sp7 transgenic mice. Tooth fracture and pulp exposure occurred after tooth eruption. Expression of dentin matrix proteins (DSP and osteocalcin) and nestin were reduced, whereas the osteoblast marker gene, osteopontin, was not expressed in the odontoblasts of Sp7 transgenic mice. Conclusions: These results indicate that sustained Sp7 expression in differentiated odontoblasts inhibits odontoblast maturation and reduces the expression of dentin matrix proteins; however, it fails to induce the transdifferentiation of odontoblasts into osteoblasts, which occurs in Runx2 transgenic mice. Therefore, Sp7 expression needs to be down-regulated to an appropriate level for odontoblast maturation. & 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Sp7 Odontoblast differentiation Dentin matrix protein DSP Nestin
1. Introduction Mesenchymal cells, which are derived from neural crest cells, differentiate into odontoblasts through an interaction with oral epithelial cells and, in turn, these differentiate into ameloblasts [1– 3]. Runx2 is an essential transcription factor for osteoblast differentiation, bone formation, and tooth development [4–6]. Runx2deficient (Runx2–/–) mice have an arrest in tooth development at the late cap stage [4]. Runx2 is expressed in mesenchymal cells in tooth germs, preodontoblasts, and immature odontoblasts; its expression is down-regulated in fully differentiated odontoblasts [7–9]. Runx2 is also expressed in ameloblasts during the maturation phase of enamel formation. The overexpression of Runx2 in odontoblasts using a 2.3-kb pro-collagen type1a1 (Col1a1) promoter was shown to induce the transdifferentiation of odontoblasts into osteoblasts, forming a bone structure [9]. n
Corresponding author. E-mail address: [email protected] (T. Komori).
Sp7 is a target gene of Runx2 and is essential for osteoblast differentiation and bone formation [10]. Sp7 is expressed in odontoblasts and cementoblasts in addition to osteoblasts [11–16]. Although tooth morphogenesis progresses normally in Sp7 / mice, tooth organs are smaller in size and odontoblast maturation is disturbed [10,17]. The overexpression of Sp7 using a 3.6-kb Col1a1 promoter results in accelerated cementum formation. The conditional deletion of Sp7 by 2.3-kb Cola1 Cre or CreER leads to reductions in cellular cementum [15]. Furthermore, the conditional deletion of Sp7 by 2.3-kb Cola1 Cre, 3.6-kb Col1a1 Cre, or osteocalcin Cre impairs dentin formation in the root, but not the crown, via the disruption of odontoblast differentiation [18–20]. Therefore, Sp7 is required for cellular cementum formation and odontoblast differentiation in the root. In tooth development, Sp7 is detected in the dental papilla cells beneath the inner enamel epithelium in the future cusp area at E18.5 and in the differentiated odontoblasts of the whole crown [19]. Sp7 is also detected in newly differentiated odontoblasts in the root region [19]. However, we found that Sp7 expression is
http://dx.doi.org/10.1016/j.job.2017.03.003 1349-0079/& 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 1. Sp7 expression during tooth development. Expression pattern of the endogenous Sp7 protein in the lower first molars of 0-day-old (a), 3-day-old (b), 10-day-old (c), and 4-week-old (d) wild-type mice. Sp7 is localized in the nuclei of differentiated odontoblasts (arrows) and its expression decreases with the maturation of odontoblasts. It is also detected in osteoblasts. Eo, enamel organ; D, dentin; Dp, dental papilla (a, b) or dental pulp (c, d); E, enamel. Bars ¼ 100 μm.
down-regulated in the crown after root formation. Therefore, in order to investigate the effects of sustained Sp7 expression after dentin formation, we examined tooth development in Sp7 transgenic mice under the control of a 2.3-kb Col1a1 promoter.
2. Materials and methods 2.1. Transgenic mice Osteoblast/odontoblast-specific Sp7 transgenic mice under the control of a 2.3-kb Co1a1 promoter were generated as previously described [21]. Transgenic lines were maintained in a B6C3H F1 background. 2.2. Histological analysis Histological examination and analyses were performed on 0-, 3-, 7-, 10-, 14-, 21-, and 28-day-old mice. After anesthetization by an intraperitoneal injection of sodium pentobarbital, the animals were perfused with 4% paraformaldehyde in a 0.05 M cacodylate buffer (pH 7.4). Dissected mandibles were further immersed in the same fixatives at 4 °C for 24 h, decalcified in 5% EDTA (pH7.4) at 4 °C for 1 day to 4 weeks, and then embedded in paraffin. Sections (4-μm-thick) were stained with hematoxylin and eosin, or
processed for immunohistochemical analyses, as described below. 2.3. Immunohistochemistry Immunohistochemistry was performed for Sp7, nestin, dentin sialoprotein (DSP), Col1a1, osteocalcin, and osteopontin. A monoclonal mouse anti-nestin antibody (Chemicon International Inc., CA, USA), polyclonal rabbit anti-Sp7 antibody (ab3742; Abcam, Cambridge, UK), polyclonal rabbit anti-DSP antibody (EMD Millipore Corp., CA, USA), polyclonal rabbit anti-Col1a1 antibody (EMD Millipore Corp., CA, USA), polyclonal rabbit anti-mouse osteocalcin antibody (Takara Bio Inc., Shiga, Japan), and polyclonal rabbit antimouse osteopontin antibody (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were used as primary antibodies at dilutions of 1:100, 1:200, 1:500, 1:250, 1:1000, and 1:50, respectively. As negative controls, normal mouse IgG (IBL) for nestin and normal rabbit IgG (IBL) for Sp7, DSP, Col1a1, osteocalcin, and osteopontin were used instead of the primary antibodies. After blocking endogenous peroxidase activity with 0.3% H2O2 in methanol, sections were processed with Histofine Simple Stain MAX-PO(M) (Nichirei, Tokyo, Japan) for nestin, and with Histofine Simple Stain MAX-PO (R) (Nichirei) for Sp7, DSP, Col1a1, osteocalcin, and osteopontin. Antibody-binding sites were colored brown by an incubation with Histofine Simple Stain DAB solution (Nichirei). Sections were then counterstained with methyl green.
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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3. Results 3.1. Sp7 expression during tooth development In newborn mice, Sp7 was detected in differentiating and differentiated odontoblasts in the cusp region of the lower first molar (Fig. 1a). Sp7 was strongly detected in differentiated odontoblasts in the whole crown at P3 (Fig. 1b). Sp7 was strongly expressed in differentiated odontoblasts in the root, while its expression in differentiated odontoblasts in the crown was weaker than that in the root at P10 (Fig. 1c). At 4 weeks of age, Sp7 expression in differentiated odontoblasts in the whole crown was barely detectable, while it was detected in differentiated odontoblasts in the root (Fig. 1d). Sp7 was strongly expressed in osteoblasts in the alveolar bone during tooth development (Fig. 1). We also compared the expression of Sp7, Col1a1, and osteocalcin at P9 (Fig. 2). Sp7 expression was detected at the preodontoblast stage (Fig. 2b), whereas Col1a1 expression was detected at the differentiated odontoblast stage and osteocalcin expression was detected at a more differentiated stage of odontoblasts (Figs. 2d, f). 3.2. Disturbed odontoblast differentiation in Sp7 transgenic mice
Fig. 2. Comparison of the expression of Sp7, Col1a1, and osteocalcin in odontoblasts. Comparison of the expression of Sp7 (a, b) with that of Col1a1 (c, d) and osteocalcin (OC) (e, f) in the tooth germs of first molars in 9-day-old wild-type mice. b, d, and f are high magnifications of the boxed areas in a, c, and e, respectively. Sp7 is expressed in preodontoblasts (thin arrows) as well as differentiated odontoblasts (b). On the other hand, Col1a1 and osteocalcin appeared in differentiated odontoblasts with the onset of dentinogenesis (d, f; thick arrows). Bars ¼ 200 μm (a, c, and e) and 20 μm (b, d, and f).
2.4. Real-time reverse-transcription-polymerase chain reaction (RTPCR) analysis Total RNA was extracted from the first and second molars in wild-type and Sp7 transgenic mice at 2 weeks of age by using ISOGEN (Wako, Osaka, Japan) and purified via the SV Total RNA Isolation System (Promega) according to the manufacturer's instructions. RNA samples were quantified with a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA) and the quality was confirmed with an Experion System (Bio-Rad Laboratories, Hercules, Calif., USA). Real-time RT-PCR was performed using the Mx3005P QPCR System (Agilent Technologies) for Sp7, dentin sialophosphoprotein (Dspp), nestin, and osteocalcin, as previously described [22]. The primer sequences used were as follows: Sp7, 5’-AGGCACAAAGAAGCCATAC-3’ and 5’-AATGAGTGAGGGAAGGGT-3’; Dspp, 5’-AACTCTGTGGCTGTGCCTCT-3’ and 5’TATTGACTCGGAGCCATTCC-3’; Nestin, 5’-AAGTGGCTACATACAGGA CTCT-3’ and 5’-GAATTCCTGTGTCTTCAGAAAG-3’; Osteocalcin, 5’GCTGCCCTAAAGCCAAACTCT-3′ and 5’-AGAGGACAGGGAGGATCA AGTTC-3’; and β-actin, 5’-CCACCCGCGAGCACAGCTTC-3’ and 5’TTGTCGACGACCAGCGCAGC-3’. We normalized the values to those of β-actin.
In 1-week-old wild-type mice when the molars were at the late bell stage, Sp7 was strongly expressed at the cervical loop regions and weakly at the coronal region, and odontoblasts exhibited a columnar shape, were polarized, and gradually elongated from the apical to coronal regions (Fig. 3a–f). Sp7 was strongly expressed in differentiated odontoblasts in Sp7 transgenic mice (Fig. 3j–l), and the shape of molars was similar between wild-type and Sp7 transgenic mice; however, the thickness of dentin was reduced in Sp7 transgenic mice (Fig. 3a, g). Odontoblasts in the cervical loop regions maintained their polarity and columnar shape, but were less elongated, while odontoblasts in the coronal region lost their polarity, columnar shape, and elongation in Sp7 transgenic mice (Fig. 3g–i). In 2-week-old wild-type mice, Sp7 was strongly expressed in odontoblasts in the root, but weakly in those in the crown (Fig. 4e– h); whereas Col1a1 was strongly expressed in odontoblasts in the root and crown (Fig. 4i–l). Odontoblasts were polarized and had a columnar shape and thick dentin developed in wild-type mice (Fig. 4a–d). In Sp7 transgenic mice, Sp7 was strongly expressed in odontoblasts in the root and crown, in which Col1a1 was also strongly expressed (Fig. 4q–x). Dentin was thin and less elongated odontoblasts were severely disturbed at the cusp (Fig. 4m–p). 3.3. Teeth had decayed in 3-week-old Sp7 transgenic mice In 3-week-old wild-type mice, teeth had erupted, thick dentin was formed by elongated and polarized odontoblasts, and dentinal tubules were observed in dentin (Fig. 5a–c). Although the shapes of the molars were similar between wild-type and Sp7 transgenic mice at 3 weeks of age, dentin was extremely thin and tooth fractures and exposure of the pulp were frequently observed in the erupted teeth of Sp7 transgenic mice (Fig. 5d–f). Inflammation or ectopic calcification was observed in the pulp of most molars (Fig. 5e). The ectopic calcification was likely formed as an inflammatory response following dental tissue damage [23]. Odontoblasts were observed in the root, but were barely detected in the crown (Fig. 5d–f). Furthermore, the elongation and polarity of odontoblasts were disturbed, the inner surface of dentin was notched in the root, and dentinal tubules were hardly observed throughout dentin (Fig. 5e, f).
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 3. Molars in 1-week-old Sp7 transgenic mice. Hematoxylin-eosin (H-E) staining (a-c, g-i) and an immunohistochemical analysis of Sp7 (d-f, j-l) in sections of the lower first molars of 1-week-old wild-type (wt) (a-f) and Sp7 transgenic (tg) (g-l) mice. b-c, e-f, h-i, and k-l are high magnifications of the boxed areas in a, d, g, and j, respectively. Odontoblasts in Sp7 transgenic mice are morphologically similar to those in wild-type mice at an early stage of dentinogenesis (b and h, long arrows); thereafter, they lose their columnar shape (h and i, short arrows) as dentinogenesis advances (compare h and i with b and c, respectively). Sp7 is strongly expressed in odontoblasts at an early stage of dentinogenesis in wild-type (e) and Sp7 transgenic (k) mice. Its expression in odontoblasts is decreased in the coronal region as dentinogenesis advances in wildtype mice (f), but not in Sp7 transgenic mice (l). Bars ¼ 500 μm (a, d, g, and j) and 20 μm (b, c, e, f, h, i, k, and l).
3.4. Reduced dentin matrix protein production in Sp7 transgenic mice
3.5. Reduced expression of nestin and absence of osteopontin expression in odontoblasts of Sp7 transgenic mice
In order to investigate the characteristics of odontoblasts in Sp7 transgenic mice, we examined the expression of the two following dentin matrix proteins at 3 days, 1 week, and 2 weeks of age using immunohistochemistry: DSP, which is formed by the cleavage of DSPP, and osteocalcin (Fig. 6). DSP was detected in differentiated odontoblasts and dentinal tubules in wild-type mice, whereas DSP protein levels were markedly reduced in odontoblasts in Sp7 transgenic mice (Fig. 6a–f). The osteocalcin protein was also detected in the differentiated odontoblasts of wild-type mice, whereas its levels were reduced in the odontoblasts of Sp7 transgenic mice, particularly at the cusp (Fig. 6g–l). The mRNA expression of Dspp and osteocalcin were also weaker in Sp7 transgenic mice than in wild-type mice (Fig. 7).
We also examined the expression of nestin, an intermediate filament and marker of odontoblasts (Fig. 8a–f). Nestin was localized in the cytoplasm close to dentin and dentinal tubules in differentiated odontoblasts; its expression increased during odontoblast differentiation and this strong expression was sustained in fully differentiated odontoblasts at the cusp in wild-type mice (Fig. 8a, c, d). Nestin was also localized in the cytoplasm close to dentin, but in limited quantities in dentin, which reflects the poor formation of dentinal tubules in Sp7 transgenic mice (Fig. 8b, e, f). Analysis by real-time RT-PCR revealed that the expression of nestin was weaker in Sp7 transgenic mice than in wild-type mice (Fig. 7). However, the expression of osteopontin, which is a marker of osteoblasts, was not observed in the odontoblasts of wild-type or Sp7 transgenic mice (Fig. 8g, h).
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 4. Molars in 2-week-old Sp7 transgenic mice. Hematoxylin-eosin (H-E) staining (a-d, m-p) and immunohistochemical analysis of Sp7 (e-h, q-t) and Col1a1 (i-l, u-x) in sections of the lower first molars of 2-week-old wild-type (wt) (a-l) and Sp7 transgenic (tg) (m-x) mice. b-d, f-h, j-l, n-p, r-t, and v-x are high magnifications of the boxed areas in a, e, i, m, q, and u, respectively. Odontoblasts are polarized, have a columnar shape, and thick dentin develops in wild-type mice (b-d), whereas those of Sp7 transgenic mice have a low columnar shape in the root, their polarity is disturbed in the crown, and dentin is thinner than that of wild-type mice (n-p). Sp7 is strongly expressed in odontoblasts in the root, but decreases in the coronal region in wild-type mice (f-h). Sp7 is strongly detected in odontoblasts in the root and crown regions of Sp7 transgenic mice (r-t). Col1a1 is strongly expressed in the differentiated odontoblasts of the entire region of the tooth germ in wild-type and Sp7 transgenic mice (j-l, v-x). Od, odontoblasts. Bars ¼ 200 μm (a, e, i, m, q, and u) and 20 μm (b-d, f-h, j-l, n-p, r-t, and v-x).
4. Discussion The overexpression of Sp7 by the 2.3-kb Col1a1 promoter disturbed the polarity and elongation of odontoblasts during their maturation and resulted in the reduction of dentin matrix
proteins, including DSP and osteocalcin. The expression of DSP and nestin, which are markers for odontoblasts, increased during odontoblast maturation in wild-type mice, but not in Sp7 transgenic mice. Furthermore, dentin formation was severely impaired and tooth fractures and the exposure of pulp occurred after tooth
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 5. Molars in 3-week-old Sp7 transgenic mice. Light micrographs of the lower first molars of 3-week-old wild-type (wt) (a-c) and Sp7 transgenic (tg) (d-f) mice stained with hematoxylin and eosin. b-c and e-f are high magnifications of the boxed areas in a and d, respectively. Dentin in Sp7 transgenic mice is thinner than that in wild-type mice, and dentinal tubules and predentin are barely detectable. In the coronal pulp regions of Sp7 transgenic mice, inflammation (d, asterisks) or ectopic calcifications (e, long arrow) are observed, and odontoblasts are barely detectable (e). In the root regions in Sp7 transgenic mice, odontoblasts lose their columnar structure, and the inner surface of dentin shows undulations (f, short arrows). D, dentin; Dp, dental pulp; Pl, periodontal ligament. Bars ¼ 200 μm (a and d) and 50 μm (b, c, e, and f).
Fig. 6. Immunohistochemical analysis of dentin sialoprotein (DSP) and osteocalcin in Sp7 transgenic mice. The expression of DSP (a-f) and osteocalcin (OC) (g-l) proteins was examined in the tooth germs of first molars in wild-type (wt) (a, c, e, g, i, k) and Sp7 transgenic (tg) (b, d, f, h, j, l) mice at 3 days (a, b, g, h), 1 week (c, d, i, j), and 2 weeks (e, f, k, l) of age. DSP expression was exclusively detected in differentiated odontoblasts and dentin in wild-type mice, whereas it was markedly decreased in Sp7 transgenic mice. Osteocalcin was also exclusively expressed in differentiated odontoblasts in wild-type mice and its expression in odontoblasts decreased with the maturation of odontoblasts in Sp7 transgenic mice (arrows). Bars ¼ 200 μm.
eruption in Sp7 transgenic mice. These results indicate that sustained Sp7 expression in differentiated odontoblasts impairs the production of dentin matrix proteins due to the inhibition of odontoblast maturation.
Odontoblasts in Runx2 transgenic mice using the same 2.3-kb Col1a1 promoter lose their polarity and columnar shape and dentin formation is severely impaired. This was also observed in Sp7 transgenic mice[9]. However, dentin is deposited around
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 7. Real-time RT–PCR analysis. A real-time RT–PCR analysis of nestin, osteocalcin, Dspp, and Sp7 in the tooth germs at 2 weeks of age. Levels in wild-type (wt) mice were set as one and relative levels in Sp7 transgenic (tg) mice are shown. Data are the mean 7 SD of eight wild-type and seven Sp7 transgenic mice. * vs. wild-type mice. *P o 0.05, **Po 0.01.
odontoblasts and it possesses lacunae and a canaliculi-like structure in Runx2 transgenic mice. Odontoblasts in Runx2 transgenic mice also lose the expression of odontoblast marker genes, such as Dspp and nestin, and increase the expression of osteoblast marker genes, including osteocalcin, osteopontin, and Dmp1. Therefore, the overexpression of Runx2 in odontoblasts induces the transdifferentiation of odontoblasts into osteoblasts. Although the overexpression of Sp7 in odontoblasts reduced the expression of DSP and nestin, it failed to induce bone-like structures in dentinogenesis or increase the expression of osteocalcin and osteopontin. Therefore, Sp7 has the
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ability to inhibit odontoblast maturation, but not induce the transdifferentiation of odontoblasts into osteoblasts. Tooth morphogenesis progresses normally until the mid-bell stage in Sp7 / mice[10,17]. However, odontoblasts are disorganized in Sp7 / newborn mice, indicating that Sp7 is involved in odontoblast maturation[17]. Since the expression of Sp7 preceded that of Col1a1 and osteocalcin, the involvement of Sp7 in the differentiation of preodontoblasts into odontoblasts needs to be investigated in more detail. In conditional Sp7 knockout mice, using 3.6-kb Col1a1 Cre, 2.3-kb Col1a1 Cre, or osteocalcin Cre mice, the formation of root dentin, but not crown dentin is impaired due to the disruption of odontoblast differentiation in the root [13,18– 20]. In these studies, the expression of Sp7 in odontoblasts in the crown after root formation was controversial. Sp7 expression is maintained or down-regulated in the crown after root formation. In our analysis, Sp7 expression was down-regulated in the crown after root formation. Since the sustained expression of Sp7 in differentiated odontoblasts inhibited odontoblast maturation and dentin matrix formation, Sp7 expression may need to be downregulated after odontoblasts are fully differentiated. Although the overexpression of Sp7 has been shown to promote the expression of Dspp in odontoblast-like cells in vitro [11], the overexpression of Sp7 in odontoblasts reduced the expression of Dspp in vivo. These findings indicate that the tuning of Sp7 expression in odontoblasts is important for proper odontoblast maturation as well as the production of dentin matrix proteins.
Fig. 8. Immunohistochemical analysis of nestin and osteopontin. An immunohistochemical analysis of nestin (Nes) (a-f) and osteopontin (OP) [23] (g, h) in tooth germs of the first molars in wild-type (wt) (a, c, d, g) and Sp7 transgenic (tg) (b, e, f, h) mice at one week of age. c-d and e-f are high magnifications of the boxed areas in a and b, respectively. In wild-type mice, nestin expression is exclusively and strongly detected in the apical cytoplasm and process of differentiated odontoblasts (Ob) in wild-type mice (a, c, d), while it decreases with the maturation of odontoblasts (Ob) in Sp7 transgenic mice (b, e, f). Osteopontin expression is detected in osteoblasts of bone tissues (arrows), but not in tooth germs of wild-type and Sp7 transgenic mice. Bars ¼ 200 μm (a, b, g, and h) and 20 μm (c-f).
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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5. Conclusions This study shows the physiological importance of the regulation of Sp7 expression during odontoblast maturation. Overexpression of Sp7 in odontoblasts inhibited odontoblast maturation and Sp7 expression in odontoblasts in the crown was downregulated after root formation in wild-type mice. Therefore, our findings indicate that Sp7 expression needs to be down-regulated to an appropriate level for full odontoblast maturation and dentin formation.
Ethical approval Prior to this study, all experiments were reviewed and approved by the Animal Care and Use Committee of Nagasaki University Graduate School of Biomedical Sciences.
Conflict of interest The authors have no conflict of interest.
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Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Original Article
Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation Toshihiro Miyazaki a, Maaya Inoue a, Tomomi T. Baba b, Toshihisa Komori a,n a b
Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan Department of Oral Molecular Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 23 January 2017 Received in revised form 20 February 2017 Accepted 22 February 2017
Objective: Sp7 is required for cellular cementum formation and odontoblast differentiation in the root. Sp7 is expressed in preodontoblasts and is strongly expressed in odontoblasts. However, Sp7 expression was down-regulated in odontoblasts in the crown after root formation. We examined osteoblast/odontoblast-specific Sp7 transgenic mice to investigate the effects of sustained Sp7 expression in differentiated odontoblasts. Methods: Tooth development was examined by immunohistochemical analyses, using antibodies against Sp7, pro-collagen type1a1, osteocalcin, dentin sialoprotein (DSP), nestin, and osteopontin. Results: In 1-week-old Sp7 transgenic mice, the thickness of dentin was slightly reduced, the elongation of odontoblasts was inhibited, and odontoblasts in the cusp lost polarity. At 2 weeks of age, Sp7 was strongly expressed in odontoblasts in the root, but weakly expressed in the crown of wild-type mice. It was strongly expressed in odontoblasts in the crown and root and the thinning of dentin and impairments in elongation and polarization in odontoblasts were severe in the crown of Sp7 transgenic mice. Tooth fracture and pulp exposure occurred after tooth eruption. Expression of dentin matrix proteins (DSP and osteocalcin) and nestin were reduced, whereas the osteoblast marker gene, osteopontin, was not expressed in the odontoblasts of Sp7 transgenic mice. Conclusions: These results indicate that sustained Sp7 expression in differentiated odontoblasts inhibits odontoblast maturation and reduces the expression of dentin matrix proteins; however, it fails to induce the transdifferentiation of odontoblasts into osteoblasts, which occurs in Runx2 transgenic mice. Therefore, Sp7 expression needs to be down-regulated to an appropriate level for odontoblast maturation. & 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Sp7 Odontoblast differentiation Dentin matrix protein DSP Nestin
1. Introduction Mesenchymal cells, which are derived from neural crest cells, differentiate into odontoblasts through an interaction with oral epithelial cells and, in turn, these differentiate into ameloblasts [1– 3]. Runx2 is an essential transcription factor for osteoblast differentiation, bone formation, and tooth development [4–6]. Runx2deficient (Runx2–/–) mice have an arrest in tooth development at the late cap stage [4]. Runx2 is expressed in mesenchymal cells in tooth germs, preodontoblasts, and immature odontoblasts; its expression is down-regulated in fully differentiated odontoblasts [7–9]. Runx2 is also expressed in ameloblasts during the maturation phase of enamel formation. The overexpression of Runx2 in odontoblasts using a 2.3-kb pro-collagen type1a1 (Col1a1) promoter was shown to induce the transdifferentiation of odontoblasts into osteoblasts, forming a bone structure [9]. n
Corresponding author. E-mail address: [email protected] (T. Komori).
Sp7 is a target gene of Runx2 and is essential for osteoblast differentiation and bone formation [10]. Sp7 is expressed in odontoblasts and cementoblasts in addition to osteoblasts [11–16]. Although tooth morphogenesis progresses normally in Sp7 / mice, tooth organs are smaller in size and odontoblast maturation is disturbed [10,17]. The overexpression of Sp7 using a 3.6-kb Col1a1 promoter results in accelerated cementum formation. The conditional deletion of Sp7 by 2.3-kb Cola1 Cre or CreER leads to reductions in cellular cementum [15]. Furthermore, the conditional deletion of Sp7 by 2.3-kb Cola1 Cre, 3.6-kb Col1a1 Cre, or osteocalcin Cre impairs dentin formation in the root, but not the crown, via the disruption of odontoblast differentiation [18–20]. Therefore, Sp7 is required for cellular cementum formation and odontoblast differentiation in the root. In tooth development, Sp7 is detected in the dental papilla cells beneath the inner enamel epithelium in the future cusp area at E18.5 and in the differentiated odontoblasts of the whole crown [19]. Sp7 is also detected in newly differentiated odontoblasts in the root region [19]. However, we found that Sp7 expression is
http://dx.doi.org/10.1016/j.job.2017.03.003 1349-0079/& 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 1. Sp7 expression during tooth development. Expression pattern of the endogenous Sp7 protein in the lower first molars of 0-day-old (a), 3-day-old (b), 10-day-old (c), and 4-week-old (d) wild-type mice. Sp7 is localized in the nuclei of differentiated odontoblasts (arrows) and its expression decreases with the maturation of odontoblasts. It is also detected in osteoblasts. Eo, enamel organ; D, dentin; Dp, dental papilla (a, b) or dental pulp (c, d); E, enamel. Bars ¼ 100 μm.
down-regulated in the crown after root formation. Therefore, in order to investigate the effects of sustained Sp7 expression after dentin formation, we examined tooth development in Sp7 transgenic mice under the control of a 2.3-kb Col1a1 promoter.
2. Materials and methods 2.1. Transgenic mice Osteoblast/odontoblast-specific Sp7 transgenic mice under the control of a 2.3-kb Co1a1 promoter were generated as previously described [21]. Transgenic lines were maintained in a B6C3H F1 background. 2.2. Histological analysis Histological examination and analyses were performed on 0-, 3-, 7-, 10-, 14-, 21-, and 28-day-old mice. After anesthetization by an intraperitoneal injection of sodium pentobarbital, the animals were perfused with 4% paraformaldehyde in a 0.05 M cacodylate buffer (pH 7.4). Dissected mandibles were further immersed in the same fixatives at 4 °C for 24 h, decalcified in 5% EDTA (pH7.4) at 4 °C for 1 day to 4 weeks, and then embedded in paraffin. Sections (4-μm-thick) were stained with hematoxylin and eosin, or
processed for immunohistochemical analyses, as described below. 2.3. Immunohistochemistry Immunohistochemistry was performed for Sp7, nestin, dentin sialoprotein (DSP), Col1a1, osteocalcin, and osteopontin. A monoclonal mouse anti-nestin antibody (Chemicon International Inc., CA, USA), polyclonal rabbit anti-Sp7 antibody (ab3742; Abcam, Cambridge, UK), polyclonal rabbit anti-DSP antibody (EMD Millipore Corp., CA, USA), polyclonal rabbit anti-Col1a1 antibody (EMD Millipore Corp., CA, USA), polyclonal rabbit anti-mouse osteocalcin antibody (Takara Bio Inc., Shiga, Japan), and polyclonal rabbit antimouse osteopontin antibody (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were used as primary antibodies at dilutions of 1:100, 1:200, 1:500, 1:250, 1:1000, and 1:50, respectively. As negative controls, normal mouse IgG (IBL) for nestin and normal rabbit IgG (IBL) for Sp7, DSP, Col1a1, osteocalcin, and osteopontin were used instead of the primary antibodies. After blocking endogenous peroxidase activity with 0.3% H2O2 in methanol, sections were processed with Histofine Simple Stain MAX-PO(M) (Nichirei, Tokyo, Japan) for nestin, and with Histofine Simple Stain MAX-PO (R) (Nichirei) for Sp7, DSP, Col1a1, osteocalcin, and osteopontin. Antibody-binding sites were colored brown by an incubation with Histofine Simple Stain DAB solution (Nichirei). Sections were then counterstained with methyl green.
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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3. Results 3.1. Sp7 expression during tooth development In newborn mice, Sp7 was detected in differentiating and differentiated odontoblasts in the cusp region of the lower first molar (Fig. 1a). Sp7 was strongly detected in differentiated odontoblasts in the whole crown at P3 (Fig. 1b). Sp7 was strongly expressed in differentiated odontoblasts in the root, while its expression in differentiated odontoblasts in the crown was weaker than that in the root at P10 (Fig. 1c). At 4 weeks of age, Sp7 expression in differentiated odontoblasts in the whole crown was barely detectable, while it was detected in differentiated odontoblasts in the root (Fig. 1d). Sp7 was strongly expressed in osteoblasts in the alveolar bone during tooth development (Fig. 1). We also compared the expression of Sp7, Col1a1, and osteocalcin at P9 (Fig. 2). Sp7 expression was detected at the preodontoblast stage (Fig. 2b), whereas Col1a1 expression was detected at the differentiated odontoblast stage and osteocalcin expression was detected at a more differentiated stage of odontoblasts (Figs. 2d, f). 3.2. Disturbed odontoblast differentiation in Sp7 transgenic mice
Fig. 2. Comparison of the expression of Sp7, Col1a1, and osteocalcin in odontoblasts. Comparison of the expression of Sp7 (a, b) with that of Col1a1 (c, d) and osteocalcin (OC) (e, f) in the tooth germs of first molars in 9-day-old wild-type mice. b, d, and f are high magnifications of the boxed areas in a, c, and e, respectively. Sp7 is expressed in preodontoblasts (thin arrows) as well as differentiated odontoblasts (b). On the other hand, Col1a1 and osteocalcin appeared in differentiated odontoblasts with the onset of dentinogenesis (d, f; thick arrows). Bars ¼ 200 μm (a, c, and e) and 20 μm (b, d, and f).
2.4. Real-time reverse-transcription-polymerase chain reaction (RTPCR) analysis Total RNA was extracted from the first and second molars in wild-type and Sp7 transgenic mice at 2 weeks of age by using ISOGEN (Wako, Osaka, Japan) and purified via the SV Total RNA Isolation System (Promega) according to the manufacturer's instructions. RNA samples were quantified with a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA) and the quality was confirmed with an Experion System (Bio-Rad Laboratories, Hercules, Calif., USA). Real-time RT-PCR was performed using the Mx3005P QPCR System (Agilent Technologies) for Sp7, dentin sialophosphoprotein (Dspp), nestin, and osteocalcin, as previously described [22]. The primer sequences used were as follows: Sp7, 5’-AGGCACAAAGAAGCCATAC-3’ and 5’-AATGAGTGAGGGAAGGGT-3’; Dspp, 5’-AACTCTGTGGCTGTGCCTCT-3’ and 5’TATTGACTCGGAGCCATTCC-3’; Nestin, 5’-AAGTGGCTACATACAGGA CTCT-3’ and 5’-GAATTCCTGTGTCTTCAGAAAG-3’; Osteocalcin, 5’GCTGCCCTAAAGCCAAACTCT-3′ and 5’-AGAGGACAGGGAGGATCA AGTTC-3’; and β-actin, 5’-CCACCCGCGAGCACAGCTTC-3’ and 5’TTGTCGACGACCAGCGCAGC-3’. We normalized the values to those of β-actin.
In 1-week-old wild-type mice when the molars were at the late bell stage, Sp7 was strongly expressed at the cervical loop regions and weakly at the coronal region, and odontoblasts exhibited a columnar shape, were polarized, and gradually elongated from the apical to coronal regions (Fig. 3a–f). Sp7 was strongly expressed in differentiated odontoblasts in Sp7 transgenic mice (Fig. 3j–l), and the shape of molars was similar between wild-type and Sp7 transgenic mice; however, the thickness of dentin was reduced in Sp7 transgenic mice (Fig. 3a, g). Odontoblasts in the cervical loop regions maintained their polarity and columnar shape, but were less elongated, while odontoblasts in the coronal region lost their polarity, columnar shape, and elongation in Sp7 transgenic mice (Fig. 3g–i). In 2-week-old wild-type mice, Sp7 was strongly expressed in odontoblasts in the root, but weakly in those in the crown (Fig. 4e– h); whereas Col1a1 was strongly expressed in odontoblasts in the root and crown (Fig. 4i–l). Odontoblasts were polarized and had a columnar shape and thick dentin developed in wild-type mice (Fig. 4a–d). In Sp7 transgenic mice, Sp7 was strongly expressed in odontoblasts in the root and crown, in which Col1a1 was also strongly expressed (Fig. 4q–x). Dentin was thin and less elongated odontoblasts were severely disturbed at the cusp (Fig. 4m–p). 3.3. Teeth had decayed in 3-week-old Sp7 transgenic mice In 3-week-old wild-type mice, teeth had erupted, thick dentin was formed by elongated and polarized odontoblasts, and dentinal tubules were observed in dentin (Fig. 5a–c). Although the shapes of the molars were similar between wild-type and Sp7 transgenic mice at 3 weeks of age, dentin was extremely thin and tooth fractures and exposure of the pulp were frequently observed in the erupted teeth of Sp7 transgenic mice (Fig. 5d–f). Inflammation or ectopic calcification was observed in the pulp of most molars (Fig. 5e). The ectopic calcification was likely formed as an inflammatory response following dental tissue damage [23]. Odontoblasts were observed in the root, but were barely detected in the crown (Fig. 5d–f). Furthermore, the elongation and polarity of odontoblasts were disturbed, the inner surface of dentin was notched in the root, and dentinal tubules were hardly observed throughout dentin (Fig. 5e, f).
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 3. Molars in 1-week-old Sp7 transgenic mice. Hematoxylin-eosin (H-E) staining (a-c, g-i) and an immunohistochemical analysis of Sp7 (d-f, j-l) in sections of the lower first molars of 1-week-old wild-type (wt) (a-f) and Sp7 transgenic (tg) (g-l) mice. b-c, e-f, h-i, and k-l are high magnifications of the boxed areas in a, d, g, and j, respectively. Odontoblasts in Sp7 transgenic mice are morphologically similar to those in wild-type mice at an early stage of dentinogenesis (b and h, long arrows); thereafter, they lose their columnar shape (h and i, short arrows) as dentinogenesis advances (compare h and i with b and c, respectively). Sp7 is strongly expressed in odontoblasts at an early stage of dentinogenesis in wild-type (e) and Sp7 transgenic (k) mice. Its expression in odontoblasts is decreased in the coronal region as dentinogenesis advances in wildtype mice (f), but not in Sp7 transgenic mice (l). Bars ¼ 500 μm (a, d, g, and j) and 20 μm (b, c, e, f, h, i, k, and l).
3.4. Reduced dentin matrix protein production in Sp7 transgenic mice
3.5. Reduced expression of nestin and absence of osteopontin expression in odontoblasts of Sp7 transgenic mice
In order to investigate the characteristics of odontoblasts in Sp7 transgenic mice, we examined the expression of the two following dentin matrix proteins at 3 days, 1 week, and 2 weeks of age using immunohistochemistry: DSP, which is formed by the cleavage of DSPP, and osteocalcin (Fig. 6). DSP was detected in differentiated odontoblasts and dentinal tubules in wild-type mice, whereas DSP protein levels were markedly reduced in odontoblasts in Sp7 transgenic mice (Fig. 6a–f). The osteocalcin protein was also detected in the differentiated odontoblasts of wild-type mice, whereas its levels were reduced in the odontoblasts of Sp7 transgenic mice, particularly at the cusp (Fig. 6g–l). The mRNA expression of Dspp and osteocalcin were also weaker in Sp7 transgenic mice than in wild-type mice (Fig. 7).
We also examined the expression of nestin, an intermediate filament and marker of odontoblasts (Fig. 8a–f). Nestin was localized in the cytoplasm close to dentin and dentinal tubules in differentiated odontoblasts; its expression increased during odontoblast differentiation and this strong expression was sustained in fully differentiated odontoblasts at the cusp in wild-type mice (Fig. 8a, c, d). Nestin was also localized in the cytoplasm close to dentin, but in limited quantities in dentin, which reflects the poor formation of dentinal tubules in Sp7 transgenic mice (Fig. 8b, e, f). Analysis by real-time RT-PCR revealed that the expression of nestin was weaker in Sp7 transgenic mice than in wild-type mice (Fig. 7). However, the expression of osteopontin, which is a marker of osteoblasts, was not observed in the odontoblasts of wild-type or Sp7 transgenic mice (Fig. 8g, h).
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 4. Molars in 2-week-old Sp7 transgenic mice. Hematoxylin-eosin (H-E) staining (a-d, m-p) and immunohistochemical analysis of Sp7 (e-h, q-t) and Col1a1 (i-l, u-x) in sections of the lower first molars of 2-week-old wild-type (wt) (a-l) and Sp7 transgenic (tg) (m-x) mice. b-d, f-h, j-l, n-p, r-t, and v-x are high magnifications of the boxed areas in a, e, i, m, q, and u, respectively. Odontoblasts are polarized, have a columnar shape, and thick dentin develops in wild-type mice (b-d), whereas those of Sp7 transgenic mice have a low columnar shape in the root, their polarity is disturbed in the crown, and dentin is thinner than that of wild-type mice (n-p). Sp7 is strongly expressed in odontoblasts in the root, but decreases in the coronal region in wild-type mice (f-h). Sp7 is strongly detected in odontoblasts in the root and crown regions of Sp7 transgenic mice (r-t). Col1a1 is strongly expressed in the differentiated odontoblasts of the entire region of the tooth germ in wild-type and Sp7 transgenic mice (j-l, v-x). Od, odontoblasts. Bars ¼ 200 μm (a, e, i, m, q, and u) and 20 μm (b-d, f-h, j-l, n-p, r-t, and v-x).
4. Discussion The overexpression of Sp7 by the 2.3-kb Col1a1 promoter disturbed the polarity and elongation of odontoblasts during their maturation and resulted in the reduction of dentin matrix
proteins, including DSP and osteocalcin. The expression of DSP and nestin, which are markers for odontoblasts, increased during odontoblast maturation in wild-type mice, but not in Sp7 transgenic mice. Furthermore, dentin formation was severely impaired and tooth fractures and the exposure of pulp occurred after tooth
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 5. Molars in 3-week-old Sp7 transgenic mice. Light micrographs of the lower first molars of 3-week-old wild-type (wt) (a-c) and Sp7 transgenic (tg) (d-f) mice stained with hematoxylin and eosin. b-c and e-f are high magnifications of the boxed areas in a and d, respectively. Dentin in Sp7 transgenic mice is thinner than that in wild-type mice, and dentinal tubules and predentin are barely detectable. In the coronal pulp regions of Sp7 transgenic mice, inflammation (d, asterisks) or ectopic calcifications (e, long arrow) are observed, and odontoblasts are barely detectable (e). In the root regions in Sp7 transgenic mice, odontoblasts lose their columnar structure, and the inner surface of dentin shows undulations (f, short arrows). D, dentin; Dp, dental pulp; Pl, periodontal ligament. Bars ¼ 200 μm (a and d) and 50 μm (b, c, e, and f).
Fig. 6. Immunohistochemical analysis of dentin sialoprotein (DSP) and osteocalcin in Sp7 transgenic mice. The expression of DSP (a-f) and osteocalcin (OC) (g-l) proteins was examined in the tooth germs of first molars in wild-type (wt) (a, c, e, g, i, k) and Sp7 transgenic (tg) (b, d, f, h, j, l) mice at 3 days (a, b, g, h), 1 week (c, d, i, j), and 2 weeks (e, f, k, l) of age. DSP expression was exclusively detected in differentiated odontoblasts and dentin in wild-type mice, whereas it was markedly decreased in Sp7 transgenic mice. Osteocalcin was also exclusively expressed in differentiated odontoblasts in wild-type mice and its expression in odontoblasts decreased with the maturation of odontoblasts in Sp7 transgenic mice (arrows). Bars ¼ 200 μm.
eruption in Sp7 transgenic mice. These results indicate that sustained Sp7 expression in differentiated odontoblasts impairs the production of dentin matrix proteins due to the inhibition of odontoblast maturation.
Odontoblasts in Runx2 transgenic mice using the same 2.3-kb Col1a1 promoter lose their polarity and columnar shape and dentin formation is severely impaired. This was also observed in Sp7 transgenic mice[9]. However, dentin is deposited around
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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Fig. 7. Real-time RT–PCR analysis. A real-time RT–PCR analysis of nestin, osteocalcin, Dspp, and Sp7 in the tooth germs at 2 weeks of age. Levels in wild-type (wt) mice were set as one and relative levels in Sp7 transgenic (tg) mice are shown. Data are the mean 7 SD of eight wild-type and seven Sp7 transgenic mice. * vs. wild-type mice. *P o 0.05, **Po 0.01.
odontoblasts and it possesses lacunae and a canaliculi-like structure in Runx2 transgenic mice. Odontoblasts in Runx2 transgenic mice also lose the expression of odontoblast marker genes, such as Dspp and nestin, and increase the expression of osteoblast marker genes, including osteocalcin, osteopontin, and Dmp1. Therefore, the overexpression of Runx2 in odontoblasts induces the transdifferentiation of odontoblasts into osteoblasts. Although the overexpression of Sp7 in odontoblasts reduced the expression of DSP and nestin, it failed to induce bone-like structures in dentinogenesis or increase the expression of osteocalcin and osteopontin. Therefore, Sp7 has the
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ability to inhibit odontoblast maturation, but not induce the transdifferentiation of odontoblasts into osteoblasts. Tooth morphogenesis progresses normally until the mid-bell stage in Sp7 / mice[10,17]. However, odontoblasts are disorganized in Sp7 / newborn mice, indicating that Sp7 is involved in odontoblast maturation[17]. Since the expression of Sp7 preceded that of Col1a1 and osteocalcin, the involvement of Sp7 in the differentiation of preodontoblasts into odontoblasts needs to be investigated in more detail. In conditional Sp7 knockout mice, using 3.6-kb Col1a1 Cre, 2.3-kb Col1a1 Cre, or osteocalcin Cre mice, the formation of root dentin, but not crown dentin is impaired due to the disruption of odontoblast differentiation in the root [13,18– 20]. In these studies, the expression of Sp7 in odontoblasts in the crown after root formation was controversial. Sp7 expression is maintained or down-regulated in the crown after root formation. In our analysis, Sp7 expression was down-regulated in the crown after root formation. Since the sustained expression of Sp7 in differentiated odontoblasts inhibited odontoblast maturation and dentin matrix formation, Sp7 expression may need to be downregulated after odontoblasts are fully differentiated. Although the overexpression of Sp7 has been shown to promote the expression of Dspp in odontoblast-like cells in vitro [11], the overexpression of Sp7 in odontoblasts reduced the expression of Dspp in vivo. These findings indicate that the tuning of Sp7 expression in odontoblasts is important for proper odontoblast maturation as well as the production of dentin matrix proteins.
Fig. 8. Immunohistochemical analysis of nestin and osteopontin. An immunohistochemical analysis of nestin (Nes) (a-f) and osteopontin (OP) [23] (g, h) in tooth germs of the first molars in wild-type (wt) (a, c, d, g) and Sp7 transgenic (tg) (b, e, f, h) mice at one week of age. c-d and e-f are high magnifications of the boxed areas in a and b, respectively. In wild-type mice, nestin expression is exclusively and strongly detected in the apical cytoplasm and process of differentiated odontoblasts (Ob) in wild-type mice (a, c, d), while it decreases with the maturation of odontoblasts (Ob) in Sp7 transgenic mice (b, e, f). Osteopontin expression is detected in osteoblasts of bone tissues (arrows), but not in tooth germs of wild-type and Sp7 transgenic mice. Bars ¼ 200 μm (a, b, g, and h) and 20 μm (c-f).
Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i
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5. Conclusions This study shows the physiological importance of the regulation of Sp7 expression during odontoblast maturation. Overexpression of Sp7 in odontoblasts inhibited odontoblast maturation and Sp7 expression in odontoblasts in the crown was downregulated after root formation in wild-type mice. Therefore, our findings indicate that Sp7 expression needs to be down-regulated to an appropriate level for full odontoblast maturation and dentin formation.
Ethical approval Prior to this study, all experiments were reviewed and approved by the Animal Care and Use Committee of Nagasaki University Graduate School of Biomedical Sciences.
Conflict of interest The authors have no conflict of interest.
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Please cite this article as: Miyazaki T, et al. Overexpression of Sp7 in odontoblasts results in dentinogenesis imperfecta due to the inhibition of odontoblast maturation. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.03.003i