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Originally published In Press as doi:10.1074/jbc.M502929200 on June 24, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29717-29727, August 19, 2005
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Differential Regulation of Dentin Sialophosphoprotein Expression by Runx2 during Odontoblast Cytodifferentiation*{boxs}

Shuo Chen{ddagger}§, Sheela Rani¶, Yimin Wu{ddagger}, Aaron Unterbrink{ddagger}, Ting Ting Gu{ddagger}, Jelica Gluhak-Heinrich||, Hui-Hsiu Chuang{ddagger}, and Mary MacDougall{ddagger}

From the Departments of {ddagger}Pediatric Dentistry, Pharmacology, and ||Orthodontics, The University of Texas Health Science Center, San Antonio, Texas 78229-3900

Received for publication, March 16, 2005 , and in revised form, June 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The spatial-temporal expression of DSPP is largely restricted during differentiational stages of dental cells. DSPP plays a vital role in tooth development. It is known that an osteoblast-specific transcription factor, Runx2, is essential for osteoblast differentiation. However, effects of Runx2 on DSPP transcription remain unknown. Here, we studied different roles of Runx2 in controlling DSPP expression in mouse preodontoblast (MD10-F2) and odontoblast (MO6-G3) cells. Two Runx2 isoforms were expressed in preodontoblast and odontoblast cells, and in situ hybridization assay showed that DSPP expression increased, whereas Runx2 was down-regulated during odontoblast differentiation and maturation. Three potential Runx2 sites are present in promoters of mouse and rat DSPP genes. Runx2 binds to these sites as demonstrated by electrophoretic mobility shift assay and supershift experiments. Mutations of Runx2 sites in mouse DSPP promoter resulted in a decline of promoter activity in MD10-F2 cells compared with an increase of its activity in MO6-G3 cells. Multiple Runx2 sites were more active than a single site in regulating the DSPP promoter. Furthermore, forced overexpression of Runx2 isoforms induced increases of endogenous DSPP protein levels in MD10-F2 cells but reduced its expression in MO6-G3 cells consistent with the DSPP promoter analysis. Thus, our results suggest that differential positive and negative regulation of DSPP by Runx2 is dependent on use of cytodifferentiation of dental ectomesenchymal-derived cells that may contribute to the spatial-temporal expression of DSPP during tooth development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tooth organogenesis is the result of reciprocal interactions between epithelial-mesenchymal cells leading to the terminal differentiation of matrix-producing cells (1-2). Dental papilla mesenchymal cells give rise to dental pulp cells, which maintain the homeostasis of dental mineralized tissues and support dentin, and odontoblasts, which synthesize dentin extracellular matrix. Recently, several in vitro and in vivo studies have demonstrated that dental pulp cells are capable of differentiating into odontoblasts and producing a mineralizing matrix, particularly during reparative dentinogenesis associated with injury and disease (3, 4).

Odontoblast and dental pulp cells synthesize and secrete several collagenous and non-collagenous proteins (NCPs)1 to form a unique dentin extracellular matrix. Dentin sialophosphoprotein (DSPP) is a phosphorylated parent protein that is cleaved post-translationally into two dentin NCPs: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) (5-6). DSPP gene is encoded by five exons and four introns (7) with the DSP sequences located at the NH2 terminus (exons 1-4 and the 5' region of exon 5) and the DPP domain found at the COOH region (remainder of exon 5). DSP and DPP contain high levels of carbohydrate and sialic acid as well as aspartic acid and phosphoserine, suggesting a function related to dentin mineralization and mineral nucleation (8). In situ hybridization and other experimental analyses have shown that DSPP is expressed predominantly in odontoblasts, transiently in preameloblasts, and at low levels in bone (9-11). This suggests that the functional role of DSPP is mainly involved in tooth formation and mineralization. Mutations of the DSPP gene cause dentinogenesis imperfecta type II and type III, and dentin dysplasia type II (12-16). These studies demonstrate that regulatory elements in the DSPP promoter control its temporal-spatial expression pattern in various cell types.

Mouse and rat DSPP gene promoters have been characterized and shown to contain an inverted TATA and CAATT box sequences, Sp1, Nrf1, and C/EBP sites, as well as several homeodomain (Dlx and Msx) motifs in the proximal regions (7, 17, 18). Further sequence analysis of the distal region of the mouse DSPP promoter reveals multiple potential Runx2 consensus motifs (19), but the biological function of these Runx2 binding sites in regulating DSPP expression has not been investigated.

Runx2 (also known as Cbfa1, Osf2, til-1, Pebp2aA, or AML-3) is a transcription factor belonging to the Drosophila runt family (20). It is essential for osteoblast differentiation and determines the lineage of osteoblasts from mesenchymal cells as well as also regulates many bone- and tooth-related genes (21-28). Runx2 mutations associated with the human syndrome cleidocranial dysplasia show that this gene has an essential role in bone and tooth formation (29-32). However, Runx2 transgenic mice inhibit osteoblast differentiation at the later stages (33), suggesting different roles during osteoblast differentiation. For example, Runx2 up- or down-regulates bone sialoprotein (BSP) gene expression in various cell types (22-23). DSPP, like BSP, is restrictively expressed in mineralizing tissues (9, 11) and belongs to the dentin/bone small integrin binding ligand N-linked glycoprotein (SIBLING) gene family (34). Regulation of BSP by Runx2 suggests that Runx2 may be a regulator of DSPP expression. In this study, we demonstrate that Runx2 regulates the mouse DSPP gene expression through its binding sites and sequences of the Runx2 binding sites are conserved with that of rat DSPP promoter. Runx2 up-regulates DSPP gene expression in mouse preodontoblasts and down-regulates its activity in mouse odontoblasts as shown by serial analyses of deletion and point mutations of Runx2 binding sites in the mouse DSPP promoter. Forced overexpression of Runx2 isoforms showed similar results with an increase of endogenous DSPP protein expression in preodontoblast cells with a corresponding decrease of DSPP levels in odontoblast cells. This study shows for the first time that Runx2 has a differential function on DSPP expression during odontoblast cytodifferentiation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Mouse odontoblast (MO6-G3) and preodontoblast (MD10-F2) cells were isolated from dental papilla mesenchymal cells of the first mandibular molars of Swiss Webster mice at embryonic day 18 (E-18) and established by immortalization using an SV-40 large T antigen vector and the neomycin gene for selection (35). MO6-G3 cells show characteristics of a differentiated odontoblast phenotype expressing tooth-related genes, whereas MD10-F2 cells have an undifferentiating dental pulp-like phenotype. These cells were grown at 33 °C under 5% CO2 in {alpha}-minimal essential medium supplemented with 10% fetal calf serum, 100 units/ml penicillin/streptomycin, 50 µg/ml ascorbic acid, and 10 mM sodium {beta}-glycerophosphate (Sigma).

Plasmid DNA Constructs—All plasmid constructs containing 5' deletions of the DSPP promoter were generated using standard cloning procedures (36). The 2698-bp XbaI-Hind III fragment of the mouse DSPP gene from nucleotides (nt) -2644 to +54 was cloned into the XbaI/Hind III sites of pGL3-basic vector (Promega, Madison, WI) and designated pDSPP2.6-luc. The 1501-bp EcoRI-HindIII fragment of the mouse DSPP gene between nt -1447 and +54 was cloned into the EcoRI/HindIII sites of the pGL3-basic vector and called pDSPP1.4-luc. The potential Runx2 binding sites in the mouse DSPP promoter were termed as site 1 (nt -2415 and -2410), site 2 (nt -2409 and -2404), and site 3 (nt -1796 and -1791) (Fig. 4A). For site-directed mutagenesis of the Runx2 sites, seven point mutation DNA constructs were generated by using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations in single, double, and triple Runx2 motifs were termed pDSPP2.6mut1-2-3-luc, pDSPP2.6mut1-2wt3-luc, pDSPP2.6mut1wt2mut3-luc, pDSPP2.6wt1mut2-3-luc, pDSPP2.6mut1wt2-3-luc, pDSPP2.6wt1mut2wt3-luc, and pDSPP2.6wt1-2mut3-luc (Table I and Fig. 5C). By using the same method, the above eight different DNA constructs of the mouse DSPP gene promoter were released from pDSPP-luc vectors with appropriate enzymes and subcloned into upstream of a chloramphenicol acetyltransferase (CAT) reporter gene (Promega) and termed pDSPP-CAT (Fig. 5D). Incorporation of the substitution mutations of the Runx2 sites in all constructs was confirmed by DNA sequencing.


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  		TABLE I
Oligonucleotides used in this study Substitution mutations are represented in lowercase letters. Core Runx2 binding sites are underlined.

 
The Runx2/Osf2 (type II) isoform expression construct was a kind gift from Dr. Gerard Karsenty (Baylor College of Medicine, Houston, TX). The cDNA of the Runx2/Pebp2{alpha}A (type I) isoform subcloned into the bluescript vector was generously provided by Dr. Yoshiaki Ito (Kyoto University, Kyoto, Japan). For the generation of the expression construct, cDNA of type I in the Bluescript vector was digested with XhoI and XbaI and then subcloned into the XhoI and XbaI sites of the pcDNA 3.1 vector (Invitrogen). Four tandem copies of the Runx2 binding site (OSE2) from the mouse osteocalcin gene promoter (22) containing the KpnI and XhoI sites at their 5'-ends was synthesized and subcloned into the KpnI and XhoI sites of the pGL-3 basic vector and named p4OSE2-luc. This p4OSE2-luc construct was kindly provided by Dr. Narayanasamy Elango (Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX).

Reverse Transcription-PCR—Total RNA was prepared from MO6-G3 and MD10-F2 cells. RNA extraction, cDNA synthesis, and PCR amplification were performed using standard protocols (36). The PCR conditions for the detection of each Runx2 isoform were described previously (19). For the detection of mouse DSPP expression, the following primer set was used (5): sense, 5'-CCAAAGAATCTGGGAAACTC-3'; antisense, 5'-AAGAAGCATCTCACGC-3'. The cycling parameters were: 94 °C for 4 min, followed by 25 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. As an internal control, a PCR analysis was performed with {beta}-actin-specific primers for 20 cycles using two primers: sense, 5'-CATCACTATTGGCAACGAGC-3'; antisense, 5'-ACTCATCGTACTCCTGCTTG-3' (37). For quantitative real-time RT-PCR (qRT-PCR), amplification reactions were analyzed in real-time on an ABI 7500 (Applied Biosystems, Foster City, CA) using SYBR Green chemistry, and the threshold values were calculated using SDS2 software (Applied Biosystems) as described earlier (38). The primer sequences used for qRT-PCR were as follows: cyclophilin (sense, 5'-GGTGACTTCACACGCCATAA-3'; antisense, 5'-CATGGCCTCCACAATATTCA-3'); dentin matrix protein 1 (DMP1) (sense, 5'-GGGAGCCAGAGAGGGTAGAG-3'; antisense, 5'-CCTCTGGGCTAGCTTGACTTT-3'); matrix extracellular phosphoglycoprotein (MEPE) (sense, 5'-GATGCAGGCTGTGTCTGTTG-3'; antisense, 5'-GCAGGCTCCTGTCTTCAT-3'); DSPP (sense, 5'-AACTCTGTGGCTGTGCCTCT-3'; antisense, 5'-TATTGACTCGGAGCCATTCC-3'); BSP (sense, 5'-AGTTAGCGGCACTCCAACTG-3'; antisense, 5'-TCCTCTGCTTCGGCTTCTT-3'); osteopontin (sense, 5'-CCCGGTGAAAGTGACGATT-3'; antisense, 5'-ATGGCTTTCATTGGAATTGC-3'); alkaline phosphatase (ALP) (5'-CGGGACTGGTACTCGGATAA-3'; antisense, 5'-TGAGATCCAGGCCATGTAGC-3'); collagen type 1{alpha}1 (sense, 5'-CCCCGGTCAGAGAGGAGAAA-3'; antisense, 5'-TCCAGAAGGACCTTGTTTGC-3'); osteocalcin (5'-CTTGGTGCACACCTAGCAGA-3'; antisense, 5'-ACCTTCTTGCCCTCCTGCTT-3'); and osteonectin (sense, 5'-AAACATGGCAAGGTGTGTGA-3'; antisense, 5'-TTGCATGGTCCGATGTAGTC-3').

In Situ Hybridization—Preparation of probes and details of the in situ hybridization procedures were performed as described earlier (19). The mouse Runx2 probe containing common coding region of the type I and II isoforms was a 234-bp fragment from nucleotide positions +17 to +251 (22).

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay—MO6-G3 cells were grown and harvested after confluency. Nuclear extracts were prepared using the methods of Dignam et al. (39). Protein concentrations were determined using the Bradford assay (40). For EMSA, oligonucleotides representing the wild type and mutant Runx2 binding sites in the mouse DSPP promoter as well as the OSE2 site shown in Table I were synthesized. The double-stranded oligonucleotides were labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase and purified on a 15% polyacrylamide gel. EMSA was performed as described before (22). For competition binding reactions, the unlabeled competitor in 100- and 200-fold molar excess of the labeled probe was used in the reaction. After incubation, the reaction mixtures were loaded onto a 5% native polyacrylamide gel in 0.5x Tris-boric acid-EDTA, electrophoresed, dried, and exposed to x-ray film. For antibody supershift experiments, 1 µl of anti-Runx2 antiserum (kindly provided by Dr. Gerard Karsenty) was added to the reaction mixture for 20 min prior to the addition of radiolabeled probe.

Western Blot Analysis—Western blot analysis was performed on whole cell lysates from MO6-G3 and MD10-F2 cells with or without transiently transfected Runx2 isoform expression plasmids. For each construct, cells in 35-mm-diameter dishes were transfected with 2 µg of either empty pcDNA 3.1 or a Runx2 isoform expression plasmid. After 48 h, cells were washed with 1x PBS and lysed with RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenymethylsulfonyl fluoride, 30 µl/ml aprotinin, 100 mM sodium orthovanadate, Santa Cruz Biotechnology, Santa Cruz, CA). Proteins (50 µg/well) were resolved by 10% SDS-PAGE and transferred to a Trans-Blot membrane (Bio-Rad Laboratory, Inc., Hercules, CA). For the detection of mouse DSPP protein, Western blotting assay was performed as described earlier (41). As a control, goat anti-mouse actin antibody was used (Santa Cruz Biotechnology, Inc.).

Immunohistochemistry—Mouse MO6-G3 cells were cultured on glass slides for 3 days, rinsed twice with ice-cold PBS, and fixed for 10 min on ice with 4% formaldehyde. After washing once with 1x PBS, the cells were made permeable by incubation for 20 min on ice with 0.2% Triton X-100 in PBS. Slides were blocked with {alpha}-minimal essential medium containing 5% fetal calf serum, 10% normal goat serum for 45 min at 37 °C, and then washed 3x for 2 min with PBS-containing serum. The cells were incubated at 37 °C for 1 h with the appropriate dilution (1:250) of primary anti-Runx2 antibody and washed 3x for 5 min with PBS containing 0.1% goat serum, followed by an incubation with a 1:500 dilution of the secondary antibody (goat anti-rabbit secondary antibody) for 1 h at 37 °C. Excess secondary antibody was removed by washing the cells three times with PBS. The cells were stained with 4',6-diamidino-2-phenylindole (0.1% 4',6-diamidino-2-pheylindole, 0.1% Triton X-100 in PBS) for 5 min on ice followed by a single rinse with 0.1% Triton X-100 in PBS and mounted using Vectashied (Vector Laboratory, Inc., Burlingame, CA). The cells were viewed with a Zeiss Axioplan2 microscope (Carl Zeiss, Inc., Germany) equipped with a Sony video capturing system and photographed with a 35-mm camera using a TMS-F inverted microscope. For quantitative detection of expression of collagenous and non-collagenous proteins in both MO6-G3 and MD10-F2 cells, fluorescent immunohistochemistry was performed using antibodies directed against mouse DSP and DMP1 (Alpha Diagnostic International, San Antonio, TX), MEPE (kindly provided by Dr. Peter Rowe, The University of Texas Health Science Center at San Antonio, TX), BSP (a gift from Dr. Larry Fisher, NIDCR), osteopontin, ALP, osteonectin, and collagen type 1{alpha}1 (Santa Cruz Biotechnology, Inc.). Negative control of mouse IgG 1 was purchased from Dakocytomation (Carpinteria, CA). The samples were incubated for overnight at 4 °C with a 1:100 dilution of primary antibodies and then washed with 1x PBS, followed by the secondary antibodies with Alexa Fluo® 488 (Molecular Probes, Eugene, OR). Images of Alexa Fluo® 488 staining of the various proteins in cultures were obtained at the Core Optical Imaging Facility at University of Texas Health Science Center under the same parameters in an Olympus wide field microscope and quantitated by means of MetaMorph software (Universal Imaging Corp., West Chester, PA). For each experiment, all sides were simultaneously processed for a specific antibody, so that homogeneity in the staining procedure was ensured between the samples. After capture of the images at the same magnification, the threshold was set and maintained for each slide in the experiment. The optical density was calculated by use of the morphometric analysis within the software package.

DNA Transfection and Promoter Activity—For each transfection experiment, MO6-G3 and MD10-F2 cells were seeded at 2 x 105 cells/35-mm diameter dishes and underwent transfection 18 h later with reporter plasmid DNA, the effector plasmid (pCMV-Runx2 isoform or pcDNA 3.1 (mock)) and pRL-TK (Promega) using Lipofectamine Plus reagent (Invitrogen) as specified by the manufacturer. Three hours after the start of transfection, serum-free DNA containing medium was replaced by fresh growth medium with 20% serum. Measurement of luciferase activity was performed as described earlier (7). CAT activity was determined using a CAT enzyme assay system (Promega). Briefly, pDSPP-CAT constructs were transiently transfected into MO6-G3 and MD10-F2 cells, respectively, using LipofectMINE Plus reagent (Invitrogen). Three hours after the start of transfection, serum-free DNA-containing medium was replaced by fresh growth medium with 20% serum. For CAT assay, the cells were harvested after a 48-h transfection and washed twice with ice-cold PBS and lysed with 300 µl of 1x reporter lysis buffer (Promega) for 15 min at room temperature. Cell lysate (50 µl) was incubated with the reaction mixture for 6 h at 37 °C. The products were separated by thin-layer chromatography (Sigma). After autoradiography, each of the butyrylated 14C-labeled chloramphenicol products was cut from plates and counted in a scintillation counter for quantitation of CAT activity. The data were normalized protein concentrations of the cell extracts using Bradford reagent (BioRad). Transfection results were computed as CAT activities per milligram of total protein. All experiments were repeated three times using at least three different DNA preparations.

Mouse DSP Expression in Bacteria—The DNA sequence corresponding to amino acid residues between +18 and +389 of the mouse DSP gene was amplified by PCR from a mouse DSPP cDNA using the following primers (5): forward with added Ndel site (lowercase), 5'-catatgATTCCGGTTCCCCAG-3', and reverse with BamH I (lowercase), 5'-ggatcctaTTTACTTCCACTGAGTTTCCC-3'. The 1127-bp PCR product was digested with Ndel I and BamH I, subcloned into the corresponding sites of the pET-11a expression vector (Novagen, Madison, WI), and confirmed by DNA sequencing. This vector containing the mouse DSP gene was transformed into the Escherichia coli strain BL21 (Novagen). A positive clone was grown in 2x YT medium until the culture reached an A600 = 0.4-0.6, at which point protein production was induced by the addition of 1 mM isoproyl-1-thio-{beta}-D-galactopyanoside (Sigma). The cells were harvested 6 h post-induction, centrifuged, and lysed. The recombinant DSP protein was purified by high-performance liquid chromatography. The yield of the purification and the relative amount of the recombinant protein were estimated by SDS-PAGE and Coomassie Blue staining.

Von Kossa and Alkaline Phosphatase Analyses—Cells were washed with PBS and then fixed with 10% formalin for 30 min. Von Kossa assay was performed as previously described (42). For in situ alkaline phosphatase (ALP) analysis, cells were fixed and stained according to the instructions provided by the Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad).

Data Analysis—Expression of CollA1 and NCP mRNAs in MD10-F2 and MO6-G3 cells was quantitative by qRT-PCR. The data were analyzed using Student's t test. p values < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Runx2 and DSPP in Mouse Dental Cell lines—It has been documented that MO6-G3 cells show a differentiated odontoblast-like phenotype and MD10-F2 cells represent a less differentiated preodontoblast-like phenotype (35, 43). The cell morphology of MD10-F2 cells is spindly and fibroblastic-like, whereas MO6-G3 cells appear polygonal with short cellular branches (data not shown). To determine expression of Runx2 types I and II in MD10-F2 and MO6-G3 cells, RT-PCR analysis was performed using four specific primers for each isoform (19). Fig. 1A shows that expression of two Runx2 isoforms was detected in both MD10-F2 and MO6-G3 cells. Furthermore, Runx2 protein expression was seen in the two dental cells using Western blot analysis (Fig. 1B). Molecular mass (65 kDa) of Runx2 identified in these cells was the same size as that reported in osteoblastic cells (22, 44). In addition, the cellular localization of Runx2 in the dental cells was specifically found in the nucleus by immunohistochemistry (Fig. 1C). Taken together, we conclude that the two Runx2 isoforms were expressed in MD10-F2 and MO6-G3 cells consistent with previous in situ hybridization studies in mouse dental tissues (19).

Because DSPP is one of the major markers of odontoblast differentiation and dentin formation (5, 9), analysis of DSPP expression in the dental cells was performed using specific mouse DSPP primers by RT-PCR analysis (5). The results show that DSPP expression was detected in these two dental cells with higher levels in MO6-G3 cells (Fig. 1A, lanes 7 and 8). In agreement with other reporters (9, 45), DSPP was preferentially expressed at high levels in odontoblasts. Cloning and sequencing of the resulting PCR products demonstrated that they were specific for Runx2 types I and II and DSPP (data not shown).

Quantitative Gene Expression of Collagenous and Non-collagenous Proteins and Mineralization in MD10-F2 and MO6-G3 Cells—Because odontoblast cells express collagens (collagen type 1 accounting for 95%) and various NCPs, we then measured expression levels of collagen type 1 and various NCP genes in both MD10-F2 and MO6-G3 cells using qRT-PCR and immunohistochemistry assays. The results in Fig. 2 (A and B) show that both MD10-F2 and MO6-G3 cells synthesized collagen type 1 and a member of SIBLING gene family, including DSPP, DMP1, osteopontin, MEPE, and BSP, as well as other extracellular matrix proteins osteocalcin, osteonectin, and ALP. Quantitative RT-PCR assays demonstrated that expression of these NCPs genes was higher in MO6-G3 cells than that of MD10-F2 cells, but there was no significant difference of collagen type 1{alpha}1gene expression between both the MD10-F2 and MO6-G3 cells. These results were further confirmed by quantitative immunohistochemical analyses (Supplemental Fig. S1). Increasing expression of NCP genes during osteoblast and dental cell differentiation was observed in the in vitro and in vivo by other laboratories (9, 45-52). Furthermore, ALP activity in MD10-F2 and MO6-G3 cells was measured at days 5 and 20 post-confluency. These results show higher ALP activity in MO6-G3 cells at days 5 and 20 compared with that of MD10-F2 cells (Fig. 2C). We next examined cell mineralization using von Kossa assays and higher density, and larger sized mineralized nodules were seen in MO6-G3 cells at the two time points of cell confluency tested (Fig. 2D). Hence, these studies indicate that MD10-F2 and MO6-G3 cells express distinct stages of odontoblast cytodifferentiation.



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  		FIG. 1.
Gene expression of Runx2 and DSPP in mouse cell lines. A, gene expression of Runx2 isoforms and DSPP in mouse cell lines shown by RT-PCR. 1 µg of total RNA was reverse-transcribed, and PCR was performed for 20-35 cycles with primers specific to mouse Runx2 isoforms and DSPP as described under "Materials and Methods." PCR products were resolved in 1.5% agarose gels and visualized with ethidium bromide. M is the molecular marker (pGEM3). Lane 2 is the negative control. The PCR products from Runx2 are as follows: Type I, 356 bp (lanes 3 and 4); Type II, 348 bp (lanes 5 and 6); DSPP, 314 bp (lanes 7 and 8); and {beta}-actin gene, 352 bp (lanes 9 and 10). Neg, negative; 10-F2 and 6-G3 indicate MD10-F2 and MO6-G3. B, expression of Runx2 protein in mouse dental cell lines demonstrated by Western blot analysis. Whole cell lysates were separated on 10% SDS-polyacrylamide gels and electroblotted onto Trans-Blot membranes. The blots were probed with anti-Runx2 antibody, and an arrow indicates a detected signal. Lane 1, the gel containing whole cell lysates was silver-stained; lanes 2 and 3; whole cell proteins from MD10-F2 and MO6-G3 cells were detected by anti-Runx2 antibody. C, immunohistochemical analysis of endogenous Runx2 in MO6-G3 cells. Representation of Runx2 was seen in the nuclear matrix.

 
Expression of DSPP and Runx2 in during Tooth Development by in Situ Hybridization—In situ hybridization study showed that DSPP mRNA was strongly expressed in odontoblast and preameloblast cells at E-16 (Fig. 3B). In comparison, expression of Runx2 was also evident in odontoblasts, preamloblast, pulp cells, and osteoblast cells within alveolar bone (Fig. 3H). At postnatal day 1, DSPP expression was apparent in odontoblast cells but dramatically decreased in ameloblast cells (Fig. 3D). In contrast, Runx2 expression was relatively increased in ameloblast and osteoblast cells, whereas it was down-regulated in odontoblast and pulp cells (Fig. 3J). At postnatal day 3, DSPP signal was barely detected both in ameloblast and osteoblast cells but continued intensely in odontoblast cells (Fig. 3F). Conversely, Runx2 expression became more intense in osteoblast and ameloblast cells but hardly seen in odontoblast and pulp cells (Fig. 3L). In addition, Runx2 signal was highly observed in osteoblast cells in developing alveolar bones of all stages. The results were similar to previous observations (32). Thus, the data indicate that the level of Runx2 expression is likely relevant to the DSPP expression at the late stages during odontoblast differentiation and maturation.



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  		FIG. 2.
Expression of collagenous and non-collagenous protein genes and mineralization in MD10-F2 and MO6-G3 cells. A, quantitative RT-PCR analysis of mRNA expression of cyclophilin A, DMP1, DSPP, osteopontin, BSP, osteonectin, ALP, osteocalcin, MEPE, and Col1Al genes from MD10-F2 and MO6-G3 cells. Expression of those mRNAs in MD10-F2 cells acts as a 1.0-fold increase. Asterisks show significant differences between the two cells (p < 0.05). B, qRT-PCR products from one of three experiments were run onto 1.5% agarose gels and stained with ethidium bromide. M, DNA marker; Neg, negative control; Cyclo, cyclophilin as internal control. 10-F2 and 6-G3 indicate MD10-F2 and MO6-G3. C and D, ALP and von Kossa analyses in MD10-F2 and MO6-G3 cells at days 5 and 20 after cell confluency.

 



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  		FIG. 3.
Expression of DSPP and Runx2 genes in tooth organs during tooth development. A-L, hematoxylin and in situ hybridization of mouse tooth developmental stages from E-16 to a postnatal day 3 with DSPP (B, D, and F) and Runx2 (H, J, and L) antisense probes. Incisors at E-16 (A), molars at PN 1 (C), and at PN 3 (E) were stained with hematoxylin; incisors at E-16 (B), molars at PN1 (D), and at PN3 (F) were hybridized with antisense DSPP probe. B',D', and F' were higher magnifications of B, D, and F. Incisors at E-16 (G), at PN 1 (I), and molars (K) at PN3 were stained with hematoxylin; incisors at E-16 (H), at PN1 (J), and molars at PN3 (L) were hybridized with antisense Runx2 probe. H', J', and L' were higher magnifications of H, J, and K. od, odontoblasts; am, ameloblasts; dp, dental pulp cells; b, alveolar bone; e, enamel; d, dentin.

 
Identification of Runx2 Binding Sites in Mouse DSPP Gene Promoter—Previously, a 1501-bp segment of mouse DSPP gene promoter from nt -1447 to +54 was cloned and characterized (7). Computer analysis demonstrated that this 5'-flanking region contains many potential binding sites of known and unknown transcription factors, such as CBF/NF-Y, C/EBP, SP-1, AP-1, and Msx, but lacks potential Runx2 binding sites. Further analysis of a larger 2644-bp segment of the mouse DSPP gene found that the region beyond nt -1447 contains three potential Runx2 response elements in addition to a number of known putative binding sites of other transcription factors (19). These elements have a consensus Runx2 sequence as described in several other bone and dentin matrix genes (21-28). Two potential Runx2 binding sites are proximal at positions nt -2415 to -2410 and nt -2409 to -2404 in the mouse DSPP gene promoter (Fig. 4, A and B). The third site is located at nt -1796 to -1791. These sites are termed as sites 1, 2, and 3, respectively. As rat DSPP gene promoter has been cloned and sequenced (17), the sequences of the three sites and their positions in the mouse DSPP promoter were compared with that of the rat DSPP promoter. All three sites were present in the rat DSPP promoter and sites 1 and 3 at approximately the same location except site 2 that is located about 200 bp downstream of the mouse position.

To determine whether Runx2 binds to these potential sites in the mouse DSPP gene promoter, double-stranded oligonucleotides were synthesized and used for EMSA (Table I). The first, named wt1-2, has the wild-type sites 1 and 2 and their surrounding sequences. The second, termed wt1mut2, contains the wild-type site 1 and a mutant site 2. The third, called mut1wt2, carries a mutant site 1 and wild-type site 2. The fourth, termed mut1-2, has mutations in both sites 1 and 2. The other two oligonucleotides, termed wt3 and mut3, were generated to test the binding of site 3. These double-stranded oligonucleotides and an OSE2 site from the mouse osteocalcin gene promoter (22), used as a standard control, were labeled as probes in EMSA using nuclear extracts from MO6-G3 cells. The results revealed that DNA-protein complexes were formed with the wt1-2, wt3, and OSE2 probes (Fig. 4C and Ref. 19). The complexes from the wt1-2 and wt3 probes show the same migration as those from the OSE2 probe (Fig. 4C, lanes 1, 8, and 15). For cross-competition experiments, the complexes from these probes were competed away with 100- and 200-fold molar excess of unlabeled wt1-2, wt3, and OSE2 oligonucleotides (Fig. 4C, lanes 2-5, 9-12, and 16-19), but not with the unlabeled mut1-2 and mut3 oligonucleotides, respectively (Fig. 4C, lanes 6-7, 13-14, and 20-21). To assess binding to individual Runx2 site within the wt1-2, wt1mut2 and mut1wt2 probes were used in EMSA. The results show that both the wt1mut2 and mut1wt2 probes interacted with the nuclear extracts (Fig. 4D). These complexes were abolished by the unlabeled homologous and OSE2 oligonucleotides (lanes 2-3, 4-5, 9-10, and 11-12), but not by the mut1-2 oligonucleotide (lanes 6-7 and 13-14). However, the mut1wt2 probe produced one additional retarded band (marked with an asterisk) and binding was competed away with the unlabeled homologous oligonucleotide (Fig. 4D, lanes 9 and 10), but not with the unlabeled OSE2 and mut1-2 oligonucleotides (lanes 11-12 and 13-14). We speculate that a new binding site was generated with the mutated clustered Runx2 binding sites. Therefore, we have not explored it further.



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  		FIG. 4.
Determination of Runx2 binding sites in mouse DSPP promoter by electrophoretic mobility shift assay. A, three Runx2 sites are present in mouse and rat DSPP gene promoters. Runx2 consensus sequences of site 1 between nt -2415 and -2410, site 2 nt -2409 and -2404, and site 3 nt -1796 and -1791 are underlined. B, diagram of the mouse DSPP promoter and each of the Runx2 binding sites. The arrow indicates the start site of transcription (+1). The position indicated for each Runx2 site is relative to the transcriptional start site. C, double-stranded DNA sequences of wt1-2 and wt3 as well as OSE2 oligonucleotides were used in EMSA (Table I). EMSA was performed with MO6-G3 cell nuclear extracts and either end-labeled with OSE2 (lanes 1-7) or wt1-2 (lanes 8-14) or wt3 probes (lanes 15-21) in the presence or absence of 100- and 200-fold molar excess of the unlabeled DNA competitors. DNA-protein complexes from the wt1-2, wt3, and OSE2 probes were each competed (lanes 2-5, 9-12, and 16-19), respectively, but not with the mut1-2 and mut3 oligonucleotides (lanes 6-7, 13-14, and 20-21). Lanes 2-3, 11-12, and 18-19 are OSE2 oligonucleotide; lanes 4-5 and 9-10, wt1-2 oligonucleotide; lanes 16-17, wt3 oligonucleotide; lanes 6-7 and 13-14, mut1-2 oligonucleotide; lanes 20-21, mut3 oligonucleotide. Lanes 1, 8, and 15, DNA-protein complexes without DNA competitors. D, Wt1mut2 and mut1wt2 probes were used in EMSA. The end-labeled double-stranded wt1mut2 and mut1wt2 probes interacted with nuclear extracts from MO6-G3 cells. Competitions with 100-fold and 200-fold molar excess of the unlabeled oligonucleotides are shown in lanes 2-7 (wt1mut2) and lanes 9-14 (mut1wt2). Lanes 1 and 8, DNA-protein complex only. The asterisk shows a nonspecific DNA-protein complex. E, antibody supershift assay. Nuclear extracts were incubated with end-labeled OSE2 (lane 1), wt1-2 (lane 3), and wt3 (lane 5) oligonucleotides with nonspecific serum. Anti-Runx2 antibody was incubated with nuclear extracts and end-labeled OSE2 (lane 2), wt1-2 (lane 4), and wt 3 (lane 6) probes. The arrowhead indicates the supershift band. The arrow shows the DNA-protein complex.

 
Binding of Runx2 to these sites was further verified with a supershift assay using an anti-Runx2 antibody. Incubation of MO6-G3 cell nuclear extracts with the antibody prior to addition of the wt1-2, wt3, and OSE2 probes led to the formation of a slower migrating band (Fig. 4E, lanes 2, 4, and 6), whereas nonspecific serum had no effect (lanes 1, 3, and 5). Together, these data demonstrate that these binding sites are bound by Runx2.

Regulation of DSPP Gene Promoter Activity by Runx2—To assess the biological function of mouse DSPP promoter mediated through these Runx2 sites, two DSPP-promoter-luciferase reporter gene constructs, pDSPP 2.6-luc containing three Runx2 sites, and pDSPP 1.4-luc lacking the Runx2 sites, were transiently transfected into MD10-F2 and MO6-G3 cells, respectively. The results show that promoter activity in pDSPP 2.6-luc resulted in a 2.4-fold increase in MD10-F2 cells, but a 3-fold decrease in MO6-G3 cells as compared with that of the pDSPP1.4-luc (Fig. 5A). To explore these observations further, the p4OSE2-luc, containing four tandem copies of the OSE2 site ligated into pGL-3 basic vector, was transfected into both cell lines. Compared with the empty expression vector, luciferase activity of the p4OSE2-luc was 4.8-fold higher in MD10-F2 and 5-fold lower in MO6-G3 cells (Fig. 5B) consistent with the results obtained from the native mouse DSPP promoter.

To understand the effect of each of the Runx2 sites in the mouse DSPP gene promoter, seven different mutant constructs were generated within the three sites (Fig. 5C) and transfected into both cell lines, respectively. Fig. 5C shows that promoter activity of these constructs was similar to that obtained from Fig. 5 (A and B). Even a single site was capable of influencing the transcriptional activity of the mouse DSPP gene (Fig. 5C, rows C-E), however multiple sites were more effective (Fig. 5C, rows F and G). Compared with promoter activity of pDSPP2.6mut1wt2-3-luc and pDSPP2.6wt1mut2wt3-luc constructs, pDSPP2.6wt1-2mut3-luc caused a lesser change in promoter activity (Fig. 5C, row H). This difference is probably due to the overlap between both the sites 1 and 2 (Table I), because there is limited space for Runx2 to simultaneously bind to both the sites; suggesting only one Runx2 occupies either site 1 or site 2. The space between these two binding sites may be critical for mouse DSPP promoter activity (53). Furthermore, substitution mutations in the three Runx2 sites in the 2.6-kb DSPP gene promoter did not completely abolish or stimulate transactivation in both dental cell lines, suggesting that additional factors besides Runx2 may be involved in regulating DSPP gene expression (7, 17, 18, 41, 54). Using the CAT gene as a reporter, similar results for the above DNA constructs of the mouse DSPP gene promoter were observed (Fig. 5D). These studies indicate a differential role of Runx2 on the regulation of mouse DSPP in MD10-F2 and MO6-G3 cells.

Effects of Overexpression of Runx2 Isoforms on the Mouse DSPP Gene—Runx2 isoforms are shown to have different actions on bone matrix genes in osteoblastic and other cell lines (23, 37, 55-57). To determine the effect of Runx2 isoforms on mouse DSPP expression, we examined forced expression of Runx2 isoforms using pDSPP2.6-luc and pDSPP1.4-luc as reporter gene constructs in MD10-F2 and MO6-G3 cells. As shown in Fig. 6A, overexpression of both isoforms caused a 3.4- to 3.7-fold increases of promoter activity in MD10-F2 cells, but a 3.5- to 4.3-fold decline in MO6-G3 cells when using pDSPP2.6-luc as compared with that of pDSPP1.4-luc. Moreover, when cotransfected with either p4OSE2-luc or empty vector and Runx2 isoform expression constructs into these dental cells, luciferase activity in p4OSE2-luc was 5- to 7-fold higher in MD10-F2 cells, whereas 6- to 7-fold lower in MO6-G3 cells than that of the empty vector (Fig. 6B). Finally, we ascertained whether overexpression of Runx2 isoforms could change expression levels of endogenous DSPP in the two dental cell lines. MO6-G3 and MD10-F2 cells were transiently transfected with either empty or each Runx2 isoform expression vector and Western blot assay shows that each Runx2 isoform induced a 3-fold increase of endogenous DSPP levels in MD10-F2 cells but reduced DSPP levels by 3- to 4-fold in MO6-G3 cells (Fig. 6C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Runx2 is necessary for osteoblast differentiation, bone formation, and tooth development (20, 22, 29, 31). In addition to the skeletal defects in Runx2 null mice (30), developing tooth germs are arrested at cap/early bell stage development (32). However, transgenic mice of Runx2 overexpression show that osteoblast maturation is disrupted at the later stage of osteoblast differentiation, and the expression of genes related to bone matrix formation is reduced in terminally differentiated osteoblasts (33). Thus, the function of Runx2 is likely cell type-specific or dependent on the stage of cytodifferentiation. For instance, Runx2 induces BSP expression in C3H10T1/2 and mouse skin fibroblast cells (22) but represses BSP activity in chicken calvarial, rat osteosarcoma (ROS) 17/2.8, and HeLa cells (23). As both BSP and DSPP are members of the SIBLING gene family (34), we investigated the role of Runx2 in regulating the mouse DSPP promoter during odontoblast cytodifferentiation.



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  		FIG. 5.
Biological activities of Runx2 in mouse DSPP promoter. A, DSPP2.6-luc, DSPP1.4-luc, or pGL3-basic vector (control) as well as pRL-TK plasmid were transfected into MD10-F2 and MO6-G3 cells. B, p4OSE2-luc or a mock vector (pGL3-basic vector only) was transfected in MD10-F2 and MO6-G3 cells. C, constructs containing 2.6 kb of the DSPP promoter with or without Runx2 site mutations were cloned upstream of a luciferase gene and transfected into MD10-F2 and MO6-G3 cells. 48 h after the start of transfection, luciferase activity was measured with the Promega dual-Luciferase reporter assay system as indicated by the manufacturer. The value (ratio between firefly and Renilla luciferase) was obtained from the control group (pGL3-basic vector only) and taken as 1-fold. The -fold luciferase activity was calculated by dividing the individual value by the control group value. The data show the mean ± S.E. from at least five separate experiments performed in triplicate. D, representative CAT autoradiogram of the mouse DSPP gene promoter. The same DNA constructs from C containing 2.6 kb of the DSPP promoter with or without Runx2 site mutations were released from pDSPP-luc vectors and subcloned into CAT-basic vector. The pDSPP-CAT constructs were transiently transfected into MO6-G3 and MD10-F2 cells, respectively. After a 48-h transfection, CAT assay was measured as described under "Experimental Procedures." The data were representative from one of three experiments each yielding similar results. The Runx2 sites are between nt -2415 and -2410 (1), nt -2409 and -2404 (2), and nt -1796 and -1791 (3). Mutant Runx2 sites are shown by an oval with cross lines.

 
In this study, we identified three Runx2 sites in the mouse DSPP gene promoter region. The sequences of these Runx2 binding sites share conservation with those identified in promoters of other bone- and tooth-related genes as well as Runx2 (Fig. 7). Using EMSA and antibody supershift analyses, we showed that these three sites bind Runx2. Moreover, we studied the effects of these Runx2 sites on the mouse DSPP gene promoter. Surprisingly, the Runx2 sites in the mouse DSPP gene promoter express contrasting regulatory activities in MD10-F2 preodontoblast versus MO6-G3 odontoblast cells. Deletion and point mutations of the Runx2 motifs resulted in decrease of promoter activity in MD10-F2 cells while increasing activity in MO6-G3 cells. To further evaluate these observations, the introduction of a reporter gene construct containing four tandem copies of OSE2 site in the two dental cell lines led to similar results. Each Runx2 site in the DSPP promoter was functional, however the multiple sites were found to be more active. Enhanced effects of multiple Runx2 sites within the osteoprotegerin gene promoter have also been demonstrated (28).

Two Runx2 isoforms have been identified in alternative promoter usages (61, 63). The Runx2 type I (pebp2{alpha}A) is transcribed by a proximal promoter (P2), whereas the type II (til-1/Osf2) uses a distal promoter (P1). Recent studies have shown that these isoforms have functional differences in regulating bone matrix genes in osteoblastic and non-osteoblastic cell lines (23, 37, 55-57). However, the precise function of each isoform has not been clarified in the regulation of tooth matrix genes in dental-derived cells. Therefore, we initially examined the expression of the Runx2 isoforms in mouse preodontoblast and odontoblast cells by RT-PCR and found that both isoforms were expressed (Fig. 1) supporting our previous in situ hybridization studies in developing teeth (19). Next, we studied the effects of each isoform on DSPP activity in MD10-F2 and MO6-G3 cells. Transient transfection studies showed each isoform stimulated DSPP promoter activity in MD10-F2 cells while inhibited its transcription in mature MO6-G3 odontoblast cells (Fig. 6, A and B). These results further ascertained the same regulation of endogenous DSPP protein expression in the two cell lines (Fig. 6C). Our studies did not distinguish different biological roles for the two Runx2 isoforms in the transcriptional control of DSPP in the two dental cells. Other in vitro and in vivo experiments have shown similar results (23, 37, 55, 56).

Although Runx2 has been implicated in both positive and negative regulation of gene expression, the mechanisms of DSPP transcriptional repression and activation in response to Runx2 are as yet unclear. Javed et al. (23) reported that BSP gene suppression by Runx2 is cell type-independent and does not involve the interaction of the repressor protein, groucho/TLE, whereas others have found that the effects of Runx2 on its target genes are dependent on certain cell types (22, 55). During early stages of osteoblast differentiation, mesenchymal cells activate Runx2 expression, and ectopic Runx2 induces expression of osteoblast-specific genes in mesenchymal cells (22). Targeted inactivation of the Runx2 gene in mice results in the absence of osteoblast differentiation (30, 31). The Runx2 knock-out mice also exhibited impaired odontoblast and ameloblast differentiation during early tooth development (32). However, Runx2 transgenic mice show that osteoblast maturation is disrupted at the later stages of osteoblast differentiation, expression of ALP and osteocalcin genes is diminished in differentiated osteoblasts, and these mice exhibit bone osteoponia and tooth fragility (33).



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  		FIG. 6.
Effects of Runx2 isoforms on DSPP expression in dental cell lines. The cells were transfected with either pDSPP1.4-luc or pDSPP 2.6-luc or empty vector (pGL3-basic only) and type I or type II expression plasmid as well as pRL-TK (Renilla luciferase) as an internal control (A); p4OSE2-luc or empty (pGL3-basic only) and type I or type II expression vector with pRL-TK (B). After 48 h, cells were harvested and luciferase activity was tested. The value (ratio between firefly and Renilla luciferase) was obtained from the control group (empty vector only) as 1-fold. The -fold luciferase activity was calculated by dividing the individual value by the control group value. The data show the mean ± S.E. from at least five separate experiments performed in triplicate. C, endogenous DSPP protein expression by Runx2 isoforms. MD10-F2 and MO6-G3 cells were transfected with 2 µg of either empty (pcDNA 3.1) or Runx2 type I or type II expression vector. Total cellular proteins were isolated 48 h post-transfection. 50 µg of total cellular proteins was run at 10% SDS-PAGE gels and subsequently electroblotted. Membranes were probed with affinity-purified anti-mouse DSP antibody. Lane 1 is recombinant mouse DSP protein as a positive control. Lanes 2 and 5, pcDNA 3.1; lanes 3 and 6, type I; lanes 4 and 7, type II. The arrow indicates detected DSP. The arrowhead shows actin protein as a control.

 



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  		FIG. 7.
Comparison of Runx2 binding sites in promoters of bone- and tooth-related genes as well as Runx2. Runx2 consensus sequences were underlined. W = A or T; Y = C or T; R = A or G; D = G, A, or T. S and As indicate sense- and antisense-stranded DNA.

 
These findings raise several significant questions related to the regulation of DSPP during odontoblast differentiation by Runx2. First, other and our studies have shown converse expression patterns between Runx2 and DSPP during tooth development (32) (Fig. 3). In situ hybridization analysis reveals that at the late bell stage Runx2 expression is down-regulated throughout the differentiating odontoblasts, whereas DSPP expression increases with odontoblast differentiation and is highest in fully differentiated mature odontoblasts. Additionally, DSPP is transiently expressed in preameloblasts, and its expression is dramatically decreased with ameloblast differentiation at the secretory stage while mature ameloblasts appear strongly positive for Runx2. In contrast to odontoblasts, high levels of Runx2 are expressed in osteoblasts throughout craniofacial ossification where levels of DSPP expression are extremely low. These observations suggest that Runx2 inhibits DSPP gene expression in odontoblasts, ameloblasts, and osteoblasts during the later stages of cell differentiation. Gailkwad et al. (64) reported that forced overexpression of the Osf2/Runx2 gene (type II) in another mouse odontoblast cell line (MDPC-23) resulted in the selective down-regulation of DSPP expression. As compared with MO6-G3 and MDPC-23 cells, MD10-F2 cells are less differentiated, thus representing preodontoblasts as shown by ALP and von Kossa staining analyses, as well as lower levels of DSPP and other NCP gene expression (Figs. 1 and 2). Therefore, it is hypothesized that the effects of Runx2 on DSPP expression depend on stages of cell differentiation with Runx2 activating DSPP expression in preodontoblast cells and repressing this gene at later stages of maturation.

The question remains why Runx2 plays a differential role in DSPP regulation during odontoblast differentiation. Studies have shown that Runx2 is necessary but not sufficient by itself for osteogenic differentiation and that coordinated action of Runx2 with other factors is required (65). For instance, it has been documented that Runx factors interact with Smads, a family of signaling proteins that regulate a diverse array of developmental and biological processes in response to transforming growth factor (TGF)-{beta}/bone morphogenetic proteins (BMPs) in osteoblast differentiation (66-67). Positive and negative regulation of BMPs/TGF-{beta} on Runx2 and its target genes is cell type-dependent (68-70). Expression of Smads and TGF-{beta}/BMPs during tooth development has been observed (71-74). Although bone and teeth have common characteristics, the physical function between odontoblasts and osteoblasts reveals some differences. For example, BMPs/TGF-{beta} stimulate expression of Runx2 and its target genes in osteoblastic cells (55, 70). However, expression of Runx2 in dental mesenchyme is induced by the fibroblast growth factor family members, but not by BMPs at cap/early bell stages (E-13/E-14) (32). Thyagarajan et al. (75) reported that at later stages (starting at E-17), transgenic mice with TGF-{beta} overexpression under the control of the mouse DSPP gene promoter result in a significant reduction in tooth mineralization and defective dentin formation. Expression of DSPP is down-regulated in these transgenic mice, but expression levels of collagens I and III are increased. Recently, He et al. (76) have found that TGF-{beta} inhibits DSPP gene expression in a mouse odontoblast cell line mediated through the Smad 3 signal pathway. Thus, it is implied that Runx2 may be inducing or interacting with a known or an unknown factor in response to fibroblast growth factors that function as an activator at the early stages of dental cell differentiation. Runx2 may also interact with signaling molecules related to TGF-{beta}, which acts as a repressor such as Smad 3 at the later stages of tooth development, either directly as a DNA-binding protein or indirectly as a Runx2 partner protein. This suggests that the cytodifferentiation-dependent changes in DSPP gene expression during tooth formation appear to be coordinated by more than one trans-regulator. In addition to Runx2, we have recently described two transcription factors, core binding factor (CBF/NF-Y) and a novel repressor (DSPP Factor 2, DF2) (41). CBF up-regulates DSPP gene expression in MO6-G3 cells and mutations of CBF binding sites on the mouse DSPP promoter cause a 5-fold decline in promoter activity in transfected cells. In contrast, DF2 is a conserved transcription factor whose activity in the mouse odontoblast nuclear extract declines ~3-fold during odontoblast differentiation. Deletion and point mutations of the DF2 binding site result in a 4.6-fold increase of DSPP promoter activity in MO6-G3 cells. Both of the proteins have functional interactions on the mouse DSPP promoter activity.

Regulation of DSPP expression during dental cell differentiation may require the coordinated action of Runx2 with other factors (7, 17, 18, 41) such as CBF, DF2, C/EBP, Nrf1, and even yet unidentified contributing factors, because substitution mutations of all three Runx2 sites in the DSPP promoter did not completely abolish or stimulate Runx2 transactivation in the two dental cell lines. Our results suggest that activation and suppression of DSPP gene expression by Runx2 during dental cell differentiation is a significant physiological consequence and may contribute to spatial-temporal expression pattern of DSPP during dentinogenesis. Investigations into potential cooperative interactions among these sites should help in further understanding mechanisms of the DSPP transcriptional regulation.


   FOOTNOTES
 
* This work was supported by NIDCR, National Institutes of Health Grants PO1-DE113221 and RO3-DE01448401A2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. S1. Back

§ To whom correspondence should be addressed: Dept. of Pediatric Dentistry, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-6642; Fax: 210-567-6603; E-mail address: chens0{at}uthscsa.edu.

1 The abbreviations used are: NCP, non-collagenous protein; DSPP, dentin sialophosphoprotein; DSP, dentin sialoprotein; DPP, dentin phosphoprotein; BSP, bone sialoprotein; nt, nucleotide(s); RT, reverse transcription; qRT, quantitative real-time RT; MEPE, matrix extracellular phosphoglycoprotein; ALP, alkaline phosphatase; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; SIBLING, dentin/bone small integrin binding ligand N-linked glycoprotein gene family; TGF, transforming growth factor; E, embryonic day. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Howard Dang and Julie Dickson for critical reading of the manuscript. We are grateful to Drs. Gerard Karsenty (Baylor College of Medicine, Houston, TX), Yoshiaki Ito (Kyoto University, Kyoto, Japan), and Narayanasamy Elango (Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX) for providing Runx2/Osf2, Runx2/Pebp2{alpha}A, and 4OSE-2 vectors. We thank Drs. Larry Fisher (NIDCR, National Institutes of Health) and Peter Rowe (The University of Texas Health Science Center, San Antonio, TX) for providing BSP and MEPE antibodies. We also thank Dr. Victoria C. Frohlich and Mark W. Blaylock in the Core Optical Imaging Facility, University of Texas Health Science Center, for fluorescent immunohistochemistry.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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