RESEARCH REPORTS

Biological

S.H. Oh1,2#, Y.C. Hwang1,2#, H. Yang1,2, J.H. Kang1,2, S.W. Hur1,2, N.R. Jung1,2, W.G. Jang1,2, K.N. Lee1,2, W.M. Oh1, J.C. Park3, S.H. Kim1,2, and J.T. Koh1,2*
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SHP is Involved in BMP2-induced Odontoblast Differentiation

Dental Science Research Institute and the BK21 Project; Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju 500-757, South Korea; 3Department of Oral Histology-Developmental Biology & Dental Research Institute, School of Dentistry, Seoul National University, Seoul, South Korea; and #authors contributing equally to this work as first authors; *corresponding author, jtkoh@chonnam.ac.kr J Dent Res 91(12):1124-1129, 2012

Abstract

Small Heterodimer Partner (SHP) interacts with diverse transcription factors such as Runx2 and regulates many cellular events including differentiation, proliferation, and energy metabolism. SHP is reported to be a positive regulator of BMP2-induced bone formation. This study aimed to clarify the role of SHP in odontoblast differentiation and matrix mineralization. Rat tooth germs were isolated, and gene expression was determined by RT-PCR and real-time PCR. Localization of SHP protein expression was identified by immunofluorescent analysis. Primary human dental pulp cells (HDPCs) were cultured with BMP2 and/or Ad-siSHP. Matrix mineralization was evaluated by Alizarin red staining. Transient transfection experiment was performed with the SHP or Dlx5 expressional plasmids and the DSPP gene. In tooth germs from post-natal days 3 to 9, BMP-2 and SHP expression increased with DSPP and DMP1 mRNA expression. In an immunostaining study, SHP was expressed in odontoblasts and surrounding osteoblasts. When HDPCs were cultured with BMP2 in mineralization-inducing medium, SHP expression also increased with an increase in DSPP expression. Down-regulation of SHP by Ad-siSHP inhibited matrix mineralization. In transient transfection experiments, overexpression of SHP was shown to enhance DSPP promoter activity through interactions between SHP and Dlx5. These results suggest that SHP may mediate BMP2 signaling to promote mineralization of the dentin matrix.

Introduction

KEY WORDS: orphan nuclear receptor, matrix mineralization, dentin matrix, dental pulp, DSPP, Dlx5.

DOI: 10.1177/0022034512461916 Received April 4, 2012; Last revision August 28, 2012; Accepted August 29, 2012 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. International & American Associations for Dental Research

evelopment of the tooth is initiated at the ectomesenchymal tissue and progresses via an initiation stage, bud stage, cap stage, and bell stage. During this process, the crown and root dentin mineralization occurs, and then the tooth erupts into the intra-oral space. During tooth germ development, diverse growth factors and cytokines, such as bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), hedgehog, and Wnt, are involved in the process (Thesleff, 2003). BMP2 belongs to the TGF- superfamily of proteins by virtue of its structure and function and was first identified as a protein that induces ectopic bone formation in rodents (Urist, 1965; Wozney and Rosen, 1998). Later, BMP2 was shown to be associated with osteogenesis and chondrogenesis (Chen et al., 2004; Rosen, 2009). During the process of tooth development in living organisms, BMP2 is initially expressed in embryonic ectomesenchymal tissue, and is strongly expressed in the tooth germs, which are involved in dentin formation after birth (Aberg et al., 1997; Thomadakis et al., 1999). In addition, BMP2 differentiates cultured pulp stem cells into odontoblast-like cells and induces expression of dentin matrix proteins and matrix mineralization (Nakashima, 2005; Chen et al., 2008; Yang et al., 2009). BMP2 induces the expression of the matrix proteins dentin sialophosphoprotein (DSPP) and dentin matrix protein (DMP)-1 by enhancing the activity of transcription factors, such as Msx, Dlx, Osx, NF-Y, and Runx2 (Yamashiro et al., 2003; Camilleri and McDonald, 2006; Chen et al., 2008, 2009; Cho et al., 2010). Nuclear receptors generally consist of a DNA-binding domain and ligandbinding domain and are activated by external stimulation to function as transcription factors (Mukherjee and Mani, 2010). The small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a DNA-binding domain, and its corresponding ligand has not yet been discovered. SHP has also been shown to regulate the expression of a diverse range of genes by binding to various nuclear receptors or transcription factors related to cell proliferation and differentiation (Chanda et al., 2008). Recently, we reported that SHP mediates BMP2-induced osteoblast differentiation and bone formation by interacting with the transcription factor Runx2 (Jeong et al., 2010). However, the role of SHP in odontoblast differentiation has not been well-studied. In this study, we investigated the role of SHP in BMP2-induced odontoblast differentiation and dentin matrix mineralization and demonstrated that SHP may be a stimulatory regulator of dentin formation.

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Materials & Methods

Isolation of Tooth Germs
Forty Sprague-Dawley rats (Damool Science, Daejeon, Korea) were used according to the guidelines of the Chonnam National University Animal Care and Use Committee. The rat pups at 3, 6, and 9 days post partum were sacrificed, and maxillae including developing tooth germs were removed. The 2nd and 3rd molar tooth germs were surgically isolated under a stereomicroscope (Leica, Wetzlar, Germany). Ten tooth germs per group were pooled for analysis of gene expression.

Cell Culture
Primary human dental pulp cells (HDPCs) were isolated from sound third molars (Hwang et al., 2008). HDPCs were maintained in -MEM containing 10% heatinactivated FBS, 100 U/mL penicillin, and 100 g/mL streptomycin. To induce differentiation and mineralization, we added 50 g/mL ascorbic acid, 5 mM -glycerophosphate (-GP), and/or 100 ng/ mL rhBMP2 (R&D Systems, Minneapolis, Figure 1.Expressional profile of BMP2, DSPP, DMP1, and SHP in rat tooth germs from MN, USA) to the culture medium. For the post-natal days 3 to 9. (A,B) RT-PCR analysis. Total RNA was isolated from tooth germs of matrix mineralization assay, the cells were the 2nd molars at post-natal days 3 (bell stage), 6 (crown formation stage), and 9 (root plated at a density of 50,000 cells/cm2 in formation stage) and of 3rd molar tooth germs at post-natal day 9 (cap stage). During the -MEM containing 10% FBS for 24 hrs, developmental process, BMP2, DSPP, DMP1, and SHP expression was determined by and then the culture medium was changed RT-PCR. -actin was used as an internal control. (C) Real-time PCR analysis for quantifying to a serum-free medium with or without the BMP2, DSPP, and SHP mRNA expression. SHP mRNA expression at days 6 and 9 adenoviral vector encoding siSHP increased by 2- and 3-fold, respectively, when compared with the expression at day 3. *p < 0.05; **p < 0.01. (Ad-siSHP). Four hrs later, the same volume of medium containing 10% FBS was added to the cultures. After 20 hrs, the culture medium was replaced with the mineralization-inducing Data were expressed as a relative value over the SHP expression medium. The medium was replaced every other day. at day 3.

RNA Isolation and RT-PCR Analysis

Total RNA was extracted from the isolated tooth germs of rats or HDPCs with the Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized with random primers and the AccessQuick RT-PCR system (Promega, Madison, WI, USA). Gene expression was assessed by PCR with the synthesized cDNA and selective primers. PCR primers and reaction conditions are shown in Appendix Table 1. The level of gene expression was evaluated by ethidium bromide staining. For quantitative analysis, real-time PCR was performed with a Rotor-Gene RG-3000 (Corbett Research, Mortlake, Australia) and SYBR Green PCR Master Mix Reagent kit (Qiagen, Valencia, CA, USA). Primers and reaction conditions are shown in Appendix Table 2. Corbett Robotics Rotor-Gene software (Rotor-Gene 6 version 6.1, Build 90) was used for data analysis.

Immunofluorescent Staining
Immunofluorescent staining was performed with a TSATM Kit (Invitrogen, Carlsbad, CA, USA). The maxillae containing the tooth germ were fixed in 4% paraformaldehyde and decalcified in 10% EDTA solution (pH 7.4) for 6 to 8 wks. The tissues were treated with ethanol dehydration, embedded in paraffin, cut into 4-m sections, and reacted with goat anti-SHP (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 48 hrs and HRP-conjugated secondary antibody (1:200, Santa Cruz Biotechnology) for 1 hr after the endogenous peroxidase was blocked with 1% H2O2. The sections were counter-stained with propidium iodide for nuclear morphology and photographed by LSM confocal microscopy (Carl Zeiss, Oberkochen, Germany). We tested immunological specificity by substituting the primary antibody with normal serum.

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Luc promoter, pcDNA3.1-SHP, pcDNA 3.1-Dlx5, and pCMV--galactosidase (gal) constructs as described previously (Cho et al., 2010; Jeong et al., 2010). We adjusted the total amount of DNA in each transfection by adding the appropriate amount of pcDNA3 plasmid. After transfection, cells underwent lysis and were then assayed with the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA). The luciferase activity was normalized by -galactosidase activity.

Protein-Protein Interaction Assay

MDPC-23 cells were transfected with 500 ng of the indicated plasmids with Lipofectamine LTX and PLUS reagents (Invitrogen). Whole-cell extracts underwent lysis with buffer containing 1% Triton X-100, 1% deoxycholate, 50 mM Tris (pH 7.4), 100 mM NaCl, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, and 2 mM EDTA. The lysates were precleared with 50 L of protein A/G-agarose beads (Invitrogen) for 2 hrs, which were removed by centrifugation. A total of 2 g of polyclonal antibody against Dlx5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the pre-cleared lysates. After centrifugation, the immunoprecipitated complexes were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted with specific antibodies.

Figure 2. Expression of SHP protein in rat tooth germs at post-natal day 9. (A) H&E staining of 2nd and 3rd molar tooth germs at post-natal day 9. (B-D) Immunofluorescent staining was performed with the TSATM kit and DSP- or SHP-specific antibody, and imaged by LSM confocal microscopy. The antibody of dentin sialoprotein (DSP) was used for identifying odontoblast differentiation, and the nuclear staining with propidium iodide was performed to distinguish the cells from the matrices. (B) The negative control stained with IgG was immunofluorescentnegative. (C) Immunoreactivity to the anti-DSP antibody (green) was observed in the odontoblasts of the 2nd molar germ. (D) Immunoreactivity to the anti-SHP antibody (green) was observed in the dentin-forming odontoblasts of the 2nd molar germ as well as in boneforming osteoblasts surrounding tooth germs. Notably, SHP immunoreactivity in 2nd molar germs was stronger than that in 3rd molar germs, being consistent with DSP reactivity. Od, odontoblast; Ob, osteoblast; DP, dental papilla; Am, ameloblast; D, dentin; E, enamel; 2nd, 2nd molar; 3rd, 3rd molar.

Statistical Analysis

Alizarin Red Staining

HDPCs were fixed with 70% ethanol, washed 5 times with distilled water, and then stained with 40 mM Alizarin red (AR-S, pH 4.2, Sigma, St. Louis, MO, USA) solution. After being washed with de-ionized water, the stained cell culture plate was moved to a scanner (Epson Perfection V700 Photo Scanner, Epson Korea Co., Seoul, Korea), and the stained image was acquired. For quantitative evaluation, the sample was reacted with 10% cetylpyridinium chloride (pH 7.0) solution at room temperature for 15 min to dissolve the stain, and absorbance was measured at a wavelength of 540 nm with a standard solution (Stanford et al., 1995; Heng et al., 2007).

All experiments were performed in triplicate on the same sample, and the experiments were repeated twice in another cell culture or tissue. Experimental results were expressed as means standard deviation. Differences between groups were evaluated by analysis of variance (ANOVA) and the Duncan test with the SPSS 17.0 software program (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.

Results
SHP mRNA Expression is Increased during Tooth Development
To determine whether SHP has a role in dentin mineralization, we first examined SHP mRNA expression in the different developmental stages of tooth germs: bell stage (day 3), crown formation stage (day 6), and root formation stage (day 9) of maxillary 2nd molar germs, and cap stage (day 9) of maxillary 3rd molar germs.

Transient Transfection Assay

Transient transfections with Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA) were carried out with pGL3-basic DSPP-

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RT-PCR results showed that mRNA expression of BMP2, DSPP, DMP1, and SHP gradually increased depending on the developmental stage (Figs. 1A, 1B). The results of real-time PCR analysis demonstrated that SHP mRNA expression at the crown and root formation stages increased by approximately 2 or 3 times, respectively, compared with that at the bell stage. BMP2 expression increased by 2.6 or 4.2 times, and DSPP increased by 3.3 or 6.0 times (Fig. 1C).

SHP Protein is Localized in Odontoblasts and Osteoblasts

In the hematoxylin and eosin staining of post-natal day 9 maxilla, the 2nd molar germ showed the root-forming stage, and the 3rd molar germ showed the cap stage Figure 3.SHP mediates BMP2-induced odontoblast differentiation in primary human dental pulp cells. (A) HDPCs were maintained in -MEM containing ascorbic acid (AA; 50 g/mL)/ (Fig. 2A). Immunofluorescent staining -glycerophosphate (-GP; 5 mM) and BMP2 (100 ng/mL). At designated time-points, cells were showed that positive immunoreactivity harvested for total RNA extraction, and RT-PCR (upper panel) or real-time PCR (lower panel) was to the anti-DSP was observed in odontoperformed with primers specific for SHP and odontoblast differentiation marker DSPP and DMP1. blasts of the 2nd molar germs at the root-actin was used as an internal control. (B) The cells were cultured with Ad-siSHP in mineralizationforming stage (Fig. 2C). Positive inducing medium for 14 days. After being fixed with 70% ethanol, the cells were stained with AR-S immunoreactivity to the anti-SHP was solution and imaged (upper panel). For quantitative analysis of matrix mineralization, the stained also observed in odontoblasts of the 2nd cell cultures were treated with cetylpyridinium chloride, and the AR-S concentration was measured by spectrophotometry as described in Materials & Methods (lower panel). Data are expressed as molar germ and osteoblasts (Fig. 2D). the mean SD of triplicate samples. **p < 0.01. DM, differentiation medium. However, there was less anti-SHP and anti-DSP reactivity in the 3rd molar germ at the cap stage than in the 2nd molar tooth germs. Discussion

SHP is Mediated in Dentinal Matrix Mineralization

When HDPCs were cultured with mineralization-inducing medium, SHP expression was increased in a time-dependent manner, accompanied by an increase in DSPP (Fig. 3A). To identify the role of SHP in matrix mineralization, we cultured HDPCs in differentiation medium with or without Ad-siSHP for 5 days, and performed Alizarin red staining. Ad-siSHP, an adenoviral vector that encodes siSHP, which inhibits SHP expression, blocked matrix mineralization (Fig. 3B).

SHP Stimulates the Transcription of DSPP Genes through Interaction with Dlx5
A transient transfection experiment was performed with DSPP promoter genes, Dlx5, and SHP expression plasmids to determine a putative regulatory mechanism by which SHP regulates dentin matrix mineralization. When 0.7-, 1.5-, or 2.6-Kb DSPP-Luc promoter gene was transfected with pcDNA3.1-SHP, luciferase activity increased in proportion to the dose of pcDNA3.1-SHP (Fig. 4A). In addition, SHP enhanced Dlx5-induced DSPP promoter luciferase activity (Fig. 4B). The immunoprecipitation assay revealed that SHP directly interacted with transcriptional factor Dlx5 (Fig. 4C).

During the process of tooth development, BMP2 is initially found in the embryo of the oral epithelial cell and is strongly expressed in mesenchymal dental papilla cells after the bell stage, where dentin formation progresses (Aberg et al., 1997; Thomadakis et al., 1999). The dentinal matrix protein DSPP is strongly expressed in odontoblasts during the bell stage (Chen et al., 2009). Previously, we identified the developmental stages of the rat tooth germ with post-natal days, based on histological analysis. Briefly, the 2nd molar germ at post-natal days 3, 6, and 9 was shown to be in the bell stage, crown formation stage, and root formation stage. The third molar germ at post-natal day 9 was in the cap stage (Kim et al., 2009). From the isolated germs, we also found that BMP2, DSPP, and DMP1 expression increased gradually as development progressed from the cap stage to the root formation stage, consistent with our previous findings. BMPs are observed in relatively high amounts in the cap stage, but expression of the matrix proteins DSPP and DMP1, rarely observed in the cap stage, markedly increases after the bell stage (DSouza et al., 1997; Butler, 1998; Chen et al., 2005). The present study also showed that SHP expression was strongly increased after the crown formation stage. These findings suggest that the SHP may be related to dentin formation by BMP2 in the late phase of tooth development, but other factors, such as cytokines and transcription factors, also may play a major role in the early stages of development.

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Because SHP lacks a DNA-binding domain, unlike other nuclear receptors, the interpretation is that it functions as a transcriptional co-regulator by binding to other transcription factors, such as Runx2. In the present study, BMP2 was shown to induce DSPP and SHP mRNA expression in HDPCs, and overexpression of SHP increased DSPP promoter activity, indicating that SHP may regulate the expression of odontoblast matrix proteins at the transcriptional level. The DSPP gene contains some transcriptional factor binding motifs, such as Msx, Dlx, Osx, NF-Y, and Runx2 (Yamashiro et al., 2003; Camilleri and McDonald, 2006; Chen et al., 2008, 2009; Cho et al., 2010). Recently, Dlx5 and Runx2 were reported to play a critical role in BMP2-induced DSPP expression, which recognizes the -433- ~ -430- and -2415- ~ -2409-bp regions on the DSPP promoter gene, respectively (Cho et al., 2010). To predict which transcriptional Figure 4. SHP stimulates DSPP gene expression via physically interacting with transcriptional factors are related to SHP-induced DSPP factor Dlx5. (A) Cells were co-transfected with 200 ng of the DSPP-luciferase reporter transcription, we performed a transient constructs (0.7-, 1.5-, 2.6-kb) and pcDNA3.1-SHP constructs (+, 100 ng; ++, 200 ng). The transfection study using 0.7, 1.5, and 2.6 cells underwent lysis 48 hrs after transfection, and luciferase activity was measured. (B) Cells kb of the DSPP promoter and SHP expreswere co-transfected with DSPP-luciferase reporter (200 ng), pcDNA3.1-Dlx5 (+, 100 ng; ++, 200 ng), and pcDNA3.1-SHP constructs (100 ng). The cells underwent lysis 48 hrs after sional constructs. The results of these transfection, and luciferase activity was measured. (C) Physical interaction between SHP and experiments revealed that SHP similarly Dlx5. Cells were transfected with 100 ng of pcDNA3.1-Dlx5, and 100 ng of GST-SHP increased all of the promoter activities, constructs (100 ng). As a control, GST vector was also transfected. After 48 hrs, the GST pullsuggesting that transcription factors that down assay was performed. All data are expressed as the mean SD of triplicate samples. interact within the -0.7 kb of the DSPP *p < 0.05; **p < 0.01. (D) Schematic diagram describes the role of SHP in BMP2-induced gene and SHP play a major role in SHPodontoblast differentiation. When BMP2 signal is activated, SHP expression is increased in induced DSPP expression. When we odontoblasts, and it enhances the transcriptional activity of Dlx5 to induce DSPP expression via direct interaction between SHP and Dlx5. Finally, SHP is a stimulatory regulator of transduced Dlx5 and SHP expression vecodontoblast differentiation and dentinal matrix mineralization. tors simultaneously with the DSPP-Luc promoter, the luciferase activity was In the present study, the immunofluorescence assay demonshown to be dependent on Dlx5 concentration. Furthermore, the strated that the SHP protein was expressed at low levels in the cap immunoprecipitation assay showed that SHP directly interacted stage germs but was highly expressed in odontoblasts during the with Dlx5. These results support that SHP mediates dentin matrix late stage. In addition, alveolar bone tissue surrounding tooth mineralization through interacting with the transcriptional factor germs also showed anti-SHP reactivity, which is consistent with Dlx5. our previous results that SHP stimulates osteoblast differentiation Overall, our study showed that SHP is a downstream factor (Jeong et al., 2010). These findings indicate that nuclear receptor of BMP2 signaling in odontoblasts and may stimulate dentin SHP plays an important role in hard tissue mineralization. matrix mineralization via enhancing the transcriptional activity In this study, HDPCs were cultured in mineralization-inducing of Dlx5. Orphan nuclear receptors have become molecular tarmedium, and DSPP expression increased in a time-dependent mangets for drug development due to their ligand-binding activity ner, consistent with results reported previously (Iohara et al., 2004). and modulation of gene expression. Chemical ligands for moduUnder the same culture conditions, SHP expression was also gradulating SHP need to be studied to better examine the potential ally increased. This expression pattern was similar to that observed uses of SHP in therapeutic applications for regenerating dentin. in rat tooth germs. We also observed that down-regulation of SHP expression inhibited matrix mineralization in cell cultures. From Acknowledgments these findings, we understand that SHP may function as a signal The authors thank Dr. H.M. Ryoo for a gift of Dlx5 construct and mediator of BMP2 in dentin matrix mineralization. Dr. J.E. Nr for supplying MDPC-23 cells. This study was Our previous study demonstrated that SHP stimulates the transupported by a grant from the National Research Foundation of scription of osteocalcin and osteoblastic bone formation by alterKorea (NRF), funded by the Korea government (MEST) (No. ing the transcriptional activity of Runx2 (Jeong et al., 2010).

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2011-0030757). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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