THE VITAMIN D RECEPTOR, RUNX2 AND THE NOTCH SIGNALING PATHWAY COOPERATE IN THE TRANSCRIPTIONAL REGULATION OF

OSTEOPONTIN Qi Shen and Sylvia Christakos+

From the Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School and Graduate School for Biomedical Sciences, Newark, New Jersey 07103

Running title: Transcriptional regulation of osteopontin Address correspondence to: Sylvia Christakos, Dept. of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103 Tel: 973-972 4033; FAX: 973-972 5594; Email: christak@umdnj.edu Summary Osteopontin (OPN), a glycosylated phosphoprotein that binds calcium, is present in bone extracellular matrix and has been reported to modulate both mineralization and bone resorption. Targeted disruption in mice of the vitamin D receptor (VDR) or Runx2 results in marked inhibition of OPN expression in osteoblasts. In this study we addressed possible cross talk between VDR and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] or Runx2 stimulated OPN transcription (mouse OPN promoter –777/+79) 2 –3 fold. However, coexpression of Runx2 and VDR in COS-7 cells and treatment with 1,25(OH)2D3 resulted in an 8 fold induction of OPN transcription, indicating for the first time functional cooperation between Runx2 and VDR in the regulation of OPN transcription. In ROS 17/2.8 and MC3T3-E1 cells, that contain endogenous Runx2, AML-1/ETO, that acts as a repressor of Runx2, significantly inhibited 1,25(OH)2D3 induction of OPN transcription, OPN mRNA and protein expression. Both a Runx2 site (–136/-130) and the vitamin

D response element (-757/-743) in the OPN promoter are needed for cooperative activation. ChIP analyses showed that 1,25(OH)2D3 can enhance VDR and Runx2 recruitment on the OPN promoter, further indicating cooperation between these two factors in the regulation of OPN. In osteoblastic cells Hes-1, a downstream factor of the Notch signaling pathway, was found to enhance basal and 1,25(OH)2D3 induced OPN transcription. This enhancement was inhibited by AML-1/ETO, an inhibitor of Runx2. Immunoprecipitation assays indicated that Hes-1 and Runx2 interact and that 1,25(OH)2D3 can enhance this interaction. Taken together these findings define novel mechanisms involving the intersection of three pathways, Runx2, 1,25(OH)2D3 and Notch signaling, that play a major role in the regulation of OPN in osteoblastic cells and therefore in the process of bone remodeling. Introduction Osteopontin (OPN) is a sialic acid rich glycosylated phosphoprotein, comprising about 2% of the non

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collagenous protein in bone (1, 2). OPN is produced by osteoblasts when they form bone matrix (1, 2). OPN is an extracellular matrix protein that contains arginine-glycine-aspartate (RGD) integrin binding motifs and promotes attachment of bone cells to the bone surface through binding to OPN receptors such as the αvβ3 integrin and CD44 (1-3). OPN has been suggested to be involved in the attachment of osteoclasts during bone resorption, to play a role in osteogenesis by attachment of osteoblasts when they form bone matrix and to act to regulate crystal size during bone mineralization (2). In addition OPN has been suggested to be a mediator of bone remodeling in response to mechanical strain (4). OPN null mice are resistant to mineral loss and bone resorption upon estrogen deprivation and have impaired activation of osteoclasts (3, 5-7). Also vascularization and resorption of bone discs have been reported to be significantly impaired in the absence of OPN (8). Although recent studies using OPN null mice have provided new insight on the role of OPN in vivo in bone metabolism, the factors that affect the regulation of OPN are not yet clearly defined. 1,25Dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, is a major calcitropic hormone involved in calcium homeostasis (9). One of its functions in bone is to regulate the synthesis of the bone calcium binding proteins osteocalcin (OC) and OPN (9). 1,25(OH)2D3 modulates the expression of these genes through transcriptional regulation. The actions of 1,25(OH)2D3 are mediated through the vitamin D receptor (VDR). Liganded VDR heterodimerizes with the retinoid X receptor (RXR) and interacts with a

vitamin D response element (VDRE). The VDRE in the mouse OPN promoter (at -757/-743) is a perfect direct repeat of the motif GGTTCA spaced by three nucleotides (10). Transcription proceeds through the interaction of VDR with coactivators and coregulators including SRC-1/NcoA1, SRC-2/GR-interacting protein(GRIP-1)/NcoA2, SRC-3/ACTR and the multisubunit DRIP (vitamin D receptor interacting protein) complex (11). Although a VDRE has been identified in the mouse OPN promoter (10) and VDR null mice show marked inhibition of OPN expression in osteoblasts (12), the exact mechanisms, including protein-protein and protein DNA interactions, involved in 1,25(OH)2D3 regulated OPN transcription are not well understood. Runx2/Cbfa1 is a member of the runt/Cbfa family of transcription factors that was first identified as an osteoblast specific transcription factor and a regulator of osteoblast differentiation (13, 14). Runx2 -/- mice die shortly after birth and show a complete lack of mineralized bone tissue (13,14). Marked decreases in the expression of osteopontin and osteocalcin are observed in Runx2 -/- mice, indicating the regulation of these genes by Runx2 (13). Three Runx2 binding motifs have been identified in the rat OC promoter (15). In addition, Runx2 has been shown to play a key role in the 1,25(OH)2D3 regulation of rat OC (15, 16). However it is not yet known whether a similar cooperation occurs between VDR and Runx2 in the regulation of OPN. Hes-1 (Hairy and Enhancer of Split homologue-1), a downstream target of the Notch signaling pathway, is a helix-loop-helix transcription factor that has been reported to play a role in developmental processes including

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myogenesis and neurogenesis (17). The expression of the Hes-1 gene is widely detected in embryos as well as adults (17). Hes-1 is also expressed in osteoblastic cells (18). Hes-1 is coexpressed with Runx2 in osteoblastic cells and Runx2 and Hes-1 physically interact (19, 20). In addition, studies in Drosophila indicate that runt and hairy contribute to common transcriptional regulatory events (21, 22). Due to the relationship between Hes-1 and Runx2 and the suggested role of Runx2 in OPN regulation (13, 14, 19, 20, 23), we tested the possibility that Hes-1 may cooperate with Runx2 in the regulation of OPN. Our finding define, for the first time, novel mechanisms involving the intersection of Runx2, 1,25(OH)2D3 and Notch signaling that are involved in the regulation of the OPN gene. Material and Methods Materials. [γ-32P]-ATP [3,000 Ci (111 TBq)/mmol], nylon membrane and the enhanced chemiluminescent detection system (ECL) were purchased from NEN Life Science Products (Boston, MA). DMEM plus Ham’s F12 nutrient mixture (DMEM-F12), DMEM, fetal bovine serum (FBS), PSN Antibiotic Mixture were purchased from GIBCO Invitrogen Corporation (Carlsbad, CA). α-MEM medium was purchased from Sigma (St. Louis, MO). VDR antiserum (C-20), mouse OPN antiserum (P-18) and histone deacetylase-1 (HDAC1) antiserum (H-51) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Runx2 antiserum was purchased from Oncogene Research Products (San Diego, CA). The antiserum reacting to Hes-1 (a gift from T. Sudo, Kamakura, Japan) was produced by immunizing

rabbits with a fusion protein consisting of C-terminal 19 aa (SPSSGSSLTSDSMWRPWRN) of mouse Hes-1 coupled to keyhole limpet haemocyanin. 1,25-Dihydroxyvitamin D3 was a generous gift from Dr. Milan Uskokovic, Hoffmann-LaRoche (Nutley, NJ) Cell culture. COS-7 African green monkey kidney cells were obtained from American Type Culture Collection (Manassas, Va.) and were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). ROS17/2.8 cells (a gift of S. Rodan and G. Rodan, Merck Research Labs, West Point, PA) were maintained in DMEM/F-12 medium supplemented with 5% FBS, 1% PSN. MC3T3-E1 cells (Riken Cell Bank, Tsukuba, Japan) were cultured in α-MEM medium supplemented with 10% FBS, 1% PSN. All cells were cultured in a humidified atmosphere of 95% air-5% CO2 at 37 C. Cells were seeded at 70-80% confluence 24 hours before experiments. Treatments with 1, 25(OH)2D3 were performed in medium supplemented with 2% charcoal-stripped serum. Transient transfection and Dual Luciferase assay. The mouse osteopontin promoter (-777/+79) firefly luciferase reporter construct was kindly provided by D. Denhardt (Rutgers University, Piscataway, NJ). pCMV-Runx2 was a gift of G. Karsenty, Baylor College of Medicine, Houston, TX and pCMV-AML-1/ETO expression vector was from S.W. Hiebert (Vanderbilt University School of Medicine, Nashville, TN). pcDNA3-Hes1 expression vector was a gift from Dr. S. Stifani (McGill University, Montreal, Quebec, Canada). Cells were seeded in a

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24-well culture dish 24 hours prior transfection at 70% confluence. Cells in each well were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacture’s instructions. Empty vectors were used to keep the total DNA concentration the same. Efficiency of transfection, as assessed by green fluorescent protein cotransfection and subsequent visualization was estimated at 60 -70%. 1,25(OH)2D3 (10-8M) or TSA (15 nM) was added to cells 24hr post-transfection for another 24 hours. Cells were washed twice with phosphate buffered saline (PBS) and harvested by incubating with 1X Passive Lysis Buffer, supplied by the Dual-Luciferase Reporter Assay kit (Promega). The luciferase activity assay was performed according to the protocol of the manufacturer and normalized to values for pRL-TK-Renilla luciferase. For all transcription studies OPN promoter activity (firefly/Renilla luciferase) is represented as fold induction by comparison to basal levels (basal levels refer to levels of OPN promoter activity in cells transfected with vector alone (vec) and treated with vehicle). Site Directed Mutagenesis. Mutant mouse OPN promoter (-777/+79) luciferase reporter constructs were generated by site directed mutagenesis using the Quick Change site directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotides used to generate the Runx2 mutated site (shown in lowercase) were as follows: 5’-CCT TTT TTT TTT TTT AAg aAC AAA ACC AGA GGA GG-3’ (top strand) and 5’-CCT CCT CTG GTT TTG Ttc TTA AAA AAA AAA AAA GG-3’ (lower strand). The oligonucleotides used to generate the VDRE mutated site (shown

in lowercase) were as follows: 5’-CAG AGC AAC AAG Gcc CAC GAG GTT CAC GTC-3’ (top strand) and 5’-GAC GTG AAC CTC GTG ggC CTT GTT GCT CTG-3’ (bottom strand). Northern Blot analysis. ROS17/2.8 cells or MC3T3-E1 cells, plated at 70% confluence in 100 mm tissue culture dishes, were transfected using Lipofectamine 2000 reagent, with AML-1/ETO or Hes-1 expression vector or vector alone. Twenty four hours after transfection cells were treated for 24 h with 1,25(OH)2D3 (10-8M) or vehicle control. The treated cells were then harvested by trypsinization, pelleted and washed with PBS. Total RNA was isolated by RNA-bee RNA extraction solution (Tel-Test, Friendswood, TX) and precipitated by chloroform and isopropanol. 20 μg of total RNA from each sample was used for Northern Blot analysis as previously described (24). 32P-labeled cDNA was prepared using Random Primers DNA Labeling System (Life Technologies, Inc.) according to the random primer method (25). The mouse osteopontin cDNA was generated by HindIII digestion and was a gift from D. Denhardt (Rutgers University, Piscataway, NJ). The β-actin cDNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The blots were hybridized with the 32P-labeled mouse OPN cDNA probes for 16 h at 42°C, washed, air-dried and exposed to Kodak BIOMAX MR film at -80°C for one day. The same blots were stripped and probed with 32P-labeled β-actin cDNA. Autoradiograms were analyzed by densitometric scanning using the Dual-Wavelength Flying Spot Scanner. The relative optical density obtained using the OPN probe was divided by the relative optical density

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obtained after probing with β-actin to normalize for sample variation. OPN Western Blot Analysis. MC3T3-E1 cells, plated at 70% confluence in 100 mm tissue culture dishes, were transfected with vector alone or pCMV-AML1/ETO and treated with vehicle or 1,25(OH)2D3 (10-8M) for 24 h and harvested by trypsinization. For Western blot analysis 50 μg protein from total cell lysates was loaded onto a 10% SDS polyacrylamide gel and separated by electrophoresis. Protein was transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Membranes were incubated overnight at 4 oC with mouse OPN polyclonal antibody (P-18, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 dilution in phosphate buffered saline (PBS) containing 5% nonfat milk. The membrane was washed with PBS and incubated for 1 h with the corresponding secondary antibody conjugated with horseradish peroxidase. The enhanced chemiluminescent Western blotting detection system (NEN Life Science Products) was used to detect the antigen/antibody complex. Chromatin Immunoprecipitation (ChIP) assay. MC3T3-E1 cells were cultured in α-MEM supplemented with 10% FBS to 95% confluency prior to the experiment and then treated in α-MEM supplemented with 2% charcoal stripped serum under the conditions and for the times indicated. Treated cells were used for the ChIP assay (26, 27). Briefly, cells were first washed with PBS and subjected to a cross-link reaction with 1% formaldehyde for 15 min. The cross-link reaction was stopped by adding glycine to a final concentration of 0.125 M. Cells were washed with ice-cold PBS

twice. The cells were collected by scraping and lysed sequentially in 5 mM Pipes pH8.0, 85 mM KCl, 0.5% NP-40 and then in 1% SDS, 10 mM EDTA, 50 mMTris-HCl pH 8.1 for 20 min individually. The chromatin pellets were sonicated to an average DNA size of 500 bp DNA (assessed by 1% agarose gel electrophoresis) using a Fisher Model 100 sonic Dismembranator at a power setting of 1. The sonicated extract was centrifuged for 10 min at maximum speed and then diluted into ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 150 mM NaCl, 0.01%SDS, 1.1% Triton X-100, 1.2 mM EDTA). Immunoprecipitations were performed at 4 °C overnight with the indicated antibody overnight. After a 1 h incubation with salmon sperm DNA and bovine serum albumin (BSA) pretreated Zysorbin (Zymed, San Francisco, CA), the precipitates were collected by centrifugation. Precipitates were washed sequentially in buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and TE buffer (10 mM Tris, 1mM EDTA) twice. The protein-DNA was then eluted by using 1% SDS and 0.1 M NaHCO3 for 15 min twice. Cross-links were reversed by incubating at 65°C overnight in elution buffer with 0.2 M NaCl. DNA fragments were purified using Qiagen QIAquick PCR purification Kits (Valencia, CA) and subjected to PCR using the primers designed to amplify fragments of murine osteopontin promoter VDRE motif (upper: 5’- ACC ACC TCT TCT GCT CTA TAT GGC –3’; lower: 5’- TGA CAC TTG AAC TAT GCA GCC GC-

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3’) and the primers designed to amplify the Runx2 motif (upper: 5’- TTC CGG GAT TCT AAA TGC AGT CTA –3’; lower: 5’- CTC CCA GAA TTT AAA TGC TGG TCC –3’). PCR analysis was carried out in the linear range of DNA amplification. PCR products were resolved in 5% TBE acrylamide gel and visualized using ethidium bromide staining. DNA acquired prior to precipitation was collected and used as the input. 10% of input was used for PCR evaluation. In re-chromatin immunoprecipitation (re-ChIP) experiments complexes were eluted by incubation for 30 min at 37oC in 60 μl of elution buffer containing 10 mM dithiothreitol. The eluted samples were diluted 50 times with ChIP dilution buffer and subjected again to the ChIP procedure with specific antibodies. Nuclear Extracts. Cells were washed with cold PBS twice, harvested by scraping, pelleted by centrifuging at 4,000 rpm for 4 min. The pellets were washed and lysed in hypotonic buffer containing 10 mM HEPES [pH 7.4], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, phosphatase inhibitors (0.5 mM phenylmethyl sulfonylfluoride, 1 mg/ml pepstatin A, 2 mg/ml leupeptin, 2 mg/ml aprotinin) and 1% Triton X-100. Nuclei were pelleted at 4,000 rpm for 4 min, and cytoplasmic supernatants were separated. Nuclei were resuspended in hypertonic buffer containing 0.42 mM NaCl, 0.2 mM EDTA, 25% glycerol, and the phosphatase and protease inhibitors indicated above. After 2 h incubation at 4°C nuclear soluble proteins were collected by centrifuging at 13,000 rpm for 10 min. Protein concentration of the supernatant was measured by the method of Bradford (28) and aliquots were stored at -80°C.

Immunoprecipitation. To examine the association of Runx2 and Hes-1 in the presence or absence of 1,25(OH)2D3 coimmunoprecipitation experiments were done. Nuclear extracts were prepared as indicated above from ROS17/2.8 cells or MC3T3-E1 cells and protein concentration was detected by the Bradford method (28). 500 μg of each preparation was used for immunoprecipitation with the addition of 4 μg of Hes-1 antiserum or 4 μg of Runx2 antiserum in the presence or absence of 1,25(OH)2D3 (10-8M) for 24 h at 4°C. Then, 30 μl protein A Sepharose 4 Fast Flow Beads (Amersham Biosciences) were added to each sample and, after further incubation by rotating at 4 °C for 1 h, the immunoprecipitated complex was collected by centrifuging at 3,000 rpm for 5 min. The complex was separated by 12% SDS-PAGE and probed with Runx2 antibody or Hes-1 antibody. Immunoprecipitation experiments were also done as described above using COS-7 cells transfected with VDR and treated with 1,25(OH)2D3 (10-8M for 24h) and cotransfected with vector alone (pCMV) or 2 μg pCMV-Runx2 to examine the association of Hes-1 with HDAC1 in the presence or absence of Runx2. For these studies 500 μg of nuclear extract was used for immunoprecipitation with the addition of 4 μg HDAC1 antiserum followed by the addition of protein A Sepharose 4 Fast Flow Beads incubated and collected by centrifugation as described above. The complex was separated by 12% SDS-PAGE and probed with Hes-1 antibody. Statistical analysis. Results are expressed as the mean ± SE and significance was determined by analysis

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with Student’s t test for two group comparison or ANOVA for multiple group comparison. Results Runx2 cooperates with VDR in regulating OPN. Targeted disruption in mice of VDR or Runx2 results in a marked inhibition of OPN expression in osteoblasts (12, 13). In order to address possible cross talk between VDR and Runx2 in regulating OPN transcription, studies were done using COS-7 cells (that lack endogenous VDR and Runx2) transfected with the mouse OPN promoter (-777/+79; VDRE –757/-743) and hVDR and/or Runx2 expression vectors. In 1,25(OH)2D3 treated (10-8M for 24 h) VDR transfected COS-7 cells, OPN transcription was induced 3.0 ± 0.4 fold. OPN transcription was induced 2.4 ± 0.1 fold by cotransfection of Runx2 expression vector in the absence of 1,25(OH)2D3. Coexpression of Runx2 and VDR and treatment with 1,25(OH)2D3 (10-8M 24 h) resulted in an 8.3 ± 0.8 fold induction of OPN transcription, suggesting functional cooperation between Runx2 and VDR in the regulation of OPN. The chimeric protein AML-1/ETO can efficiently block Runx2 mediated transcriptional activation (29). In COS-7 cells the enhancement of the inductive action by 1,25(OH)2D3 and Runx2 was inhibited by AML-1/ETO in a dose dependent manner (Fig. 2A). In ROS17/2.8 cells and MC3T3-E1 cells, that contain endogenous Runx2, AML-1/ETO significantly diminished the 1,25(OH)2D3 induction of OPN transcription (Fig. 2B, C) further indicating cooperation between Runx2 and VDR in the regulation of OPN transcription.

Northern blot analysis was also performed to assess the effect of AML-1/ETO on endogenous 1,25(OH)2D3 induced OPN mRNA expression. Expression of AML-1/ETO in ROS17/2.8 osteoblastic cells resulted in a significant inhibition of the levels of basal and 1,25(OH)2D3 induced OPN mRNA (Fig. 3A). Note (Fig. 3A last two bars) although there is a 50% decrease in basal OPN mRNA, there is a 75% decrease in 1,25(OH)2D3 induced OPN mRNA. Similar results were observed using MC3T3-E1 cells (Fig. 3B). In addition, inhibition of 1,25(OH)2D3 OPN protein expression was also observed in the presence of AML-1/ETO (Fig. 3C). These findings suggest that VDR and Runx2 cooperate in vivo to regulate the expression of OPN. Both the VDRE and the Runx2 site are needed for cooperative activation of OPN transcription. A Runx2 site was noted in the mouse osteopontin promoter (AACCACA at –136/-130; 23). Gel shift assays using synthetic oligonucleotides corresponding to the wild type (WT) (-136/-130) or mutated (AAgaACA) Runx2 binding sequences and nuclear extracts from Runx2 transfected COS-7 cells indicated that Runx2 interacted with the WT oligonucleotides in a dose dependent manner (not shown). No protein-DNA interaction was detected using the mutant oligonucleotide and preincubation with cold WT oligonucleotide but not mutant oligonucleotide resulted in a dose dependent depletion of the binding of Runx2 to the labeled probe (not shown). These electrophoretic mobility shift assays indicated, similar to previous studies (23), that –136/-130 in the mOPN promoter is a binding site for Runx2. To investigate the specific

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contribution of the VDRE and the Runx2 site to the cooperative activation of OPN transcription, mutant OPN promoter constructs were generated with either the Runx2 site (-136/-130) mutated or the VDRE (-757/-743) mutated (Fig. 4A). Mutation of the Runx2 site did not affect the induction by 1,25(OH)2D3 of OPN transcription in VDR transfected COS-7 cells (Fig. 4B, vector transfected, vehicle and 1,25(OH)2D3 treated) and resulted in a decreased (but not abolished) 1,25(OH)2D3 response in ROS 17/2.8 cells (not shown). However, in COS-7 cells Runx2 could no longer activate OPN transcription [Fig. 4B, Runx2 transfected, vehicle treated; p> 0.4 compared to vector transfected vehicle treated (lane 3 vs. lane 1)] indicating that Runx2 acts through this site in the mOPN promoter –777/+79 and not through additional sites (unlike the regulation of OC by Runx2; 15). Also mutation of the Runx2 site resulted in a loss of the cooperative response (Fig. 4B, lane 4; compare to Fig. 1, lane 4). The decrease in the response to 1,25(OH)2D3 in the presence of Runx2 using the OPN promoter with the mutated Runx2 site may be due to the reported binding of Runx2 to VDR (16). Runx2, in the presence of a mutated Runx2 site in the OPN promoter, may bind to VDR and thus less VDR would be available for 1,25(OH)2D3 induced transcription. Using the OPN promoter construct bearing a mutation in the VDRE, 1,25(OH)2D3 was unable to activate the OPN promoter in VDR transfected COS-7 cells (Fig. 4C, vector transfected (Vec), 1,25(OH)2D3 treated) or in ROS 17/2.8 cells (not shown). However, transfection of COS-7 cells with Runx2 could still result in enhanced OPN transcription and, similar to the mutation of the Runx2 site, the

cooperative response was not observed (Fig. 4C). These findings suggest that the Runx2 site at –136/-130 and the VDRE are essential for cooperative effects of Runx2 and VDR in activating mOPN transcription. Runx2 and VDR interact with the OPN promoter in intact osteoblastic cells. In order to further understand mechanisms involved in activation of OPN transcription, we examined VDR and Runx2 complex formation on the OPN promoter in MC3T3-E1 cells using the ChIP assay and specific antibodies against Runx2 and VDR. The antibodies were used to precipitate sonicated chromatin cross links from whole cells lysates after formaldehyde cross linking of DNA to transcription factors. DNA was amplified using specific primers directed against the VDRE or the Runx2 binding region of the OPN promoter. In the PCR procedure the number of cycles was chosen so that the amplification was conducted in the linear range of amplification efficiency. No signal was detected in the presence of IgG (Fig. 5A). The ChIP analysis showed that 1,25(OH)2D3 can enhance both VDR and Runx2 recruitment to the OPN promoter (Fig. 5A). Note that transfection of MC3T3-E1 cells with AML-1/ETO resulted in decreased recruitment of Runx2 to the OPN promoter (Fig. 5B). The 1,25(OH)2D3 enhancement of Runx2 as well as VDR DNA binding affinity could be one possible mechanism involved in the cooperative activation. Hes-1 can potentiate the Runx2 mediated transactivation of OPN transcription. Hes-1, a downstream target of the Notch signaling pathway, is coexpressed with Runx2 in osteoblastic cells and Hes-1 and Runx2 have been

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reported to contribute to common transcriptional regulatory events (19, 20). We therefore tested the possibility that Hes-1 may be involved in 1,25(OH)2D3 and Runx2 mediated regulation of OPN transcription. In ROS17/2.8 cells and MC3T3-E1 cells, that contain endogenous Runx2, tranfection of Hes-1 (0.1 – 1 μg) resulted in an enhancement of both basal and 1,25(OH)2D3 induced OPN transcription (Fig. 6A, 6B). Expression of Hes-1 also resulted in an enhancement of basal and 1,25(OH)2D3 induced OPN mRNA expression (Fig. 6C). The enhancement of the induction of OPN transcription by Hes-1 in ROS17/2.8 cells was inhibited by AML-1/ETO, a repressor of Runx2 (Fig. 7). In COS-7 cells, in the absence of tranfected Runx2, expression of Hes-1 resulted in a repression of 1,25(OH)2D3 dependent induction of OPN transcription and co-tranfection of Runx2 in COS-7 cells reversed the inhibition by Hes-1 (not shown), further suggesting functional cooperation between Hes-1 and Runx2. Immunoprecipitation assays using ROS17/2.8 cells indicated that Hes-1 and Runx2 interact and that 1,25(OH)2D3 can increase this interaction (Fig. 8A), suggesting that 1,25(OH)2D3 may enhance functional cooperation between Hes-1 and Runx2 by enhancing Hes-1/Runx2 interaction. Similar results were observed using MC3T3-E1 cells (not shown). Further, reChIP analysis shows that Runx2 and Hes-1 bind simultaneously to the OPN promoter (Fig. 8B). Since both Runx2 and Hes-1 can interact with TLE (transducin-like Enhancer of split) proteins, which can recruit histone deacetylases (20, 30), we asked whether inhibition of histone deacetylation may be involved in the

activation by Hes-1. In ROS17/2.8 cells TSA, a histone deacetylase inhibitor, was able to rescue the inhibition by AML-1/ETO of Hes-1 enhanced 1,25(OH)2D3 induced OPN transcription (Fig. 9A). In addition, in COS-7 cells, in the absence of transfected Runx2, inhibition of 1,25(OH)2D3 induced OPN transcription by Hes-1 was reversed in the presence of TSA (not shown). These findings suggest that Hes-1/Runx2 binding may interfere with Runx2-TLE and Hes-1-TLE interactions thus preventing repression which may be mediated, at least in part, by histone decacetylation. Coimmunoprecipitation studies showed the association of Hes-1 and HDAC1 and a decrease in this association in the presence of Runx2 (Fig. 9B). Taken together, these findings show that Hes-1 can potentiate VDR mediated OPN transcription in the presence of Runx2 and define new mechanisms and functional interactions that are involved in the regulation of OPN and may therefore affect the process of bone remodeling. Discussion This study describes for the first time cooperative effects between Runx2, VDR and Hes-1 in the transcriptional regulation of OPN. Functional cooperation was demonstrated between Runx2 and VDR in the regulation of OPN transcription, OPN mRNA and protein expression. 1,25(OH)2D3 was found to enhance both VDR and Runx2 recruitment on the OPN promoter in vivo, further indicating cooperation between these two factors in the regulation of OPN. Hes-1, a downstream target of the Notch signaling pathway, was found to act as an enhancer of basal and 1,25(OH)2D3 induced OPN transcription and OPN

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mRNA in the presence of Runx2. Coimmunoprecipitation analysis indicated that Hes-1 and Runx2 interact and 1,25(OH)2D3 enhances this interaction. We propose that these three major pathways, Runx2, 1,25(OH)2D3 and Notch signaling, intersect and play a major role in the regulation of OPN in osteoblastic cells and therefore in the process of bone remodeling. Runx2 was found not only to upregulate OPN basal promoter activity but also to enhance 1,25(OH)2D3 induced OPN transcription. Runx2 has been reported to be essential for osteogenic differentiation (31, 32). 1,25(OH)2D3 promotes osteoblastic differentiation and directly stimulates the production of OC and OPN (33, 34). OPN has been reported to be present in preosteoblasts and is present in high concentrations in the osteoblast (35, 36). Bone sialoprotein (BSP), another calcium binding protein present in bone matrix that shares structural features with OPN, is expressed after OPN but earlier than OC in the development of the osteoblast phenotype (35, 36). OC is the latest of the differentiation markers to be expressed. OC is abundantly expressed in mature osteoblasts (35, 36). These calcium binding proteins may function in regulating the ordered deposition of mineral (2, 37). Although much work has been done concerning the regulation of OC, we are only beginning to understand mechanisms involved in the regulation of OPN and BSP. Two Runx2 sites had previously been suggested in the OPN promoter (at –136/-130) (23) and on the reverse strand at –695/-690 (14). Mutation of the Runx2 site at –136/-130 resulted in a complete block of the activation of OPN transcription by Runx2 (Fig. 4A), indicating that Runx2 can act through

this single site in the OPN promoter. This is unlike the regulation of rat OC. The rat OC promoter contains two distal Runx2 sites (A, B) and a proximal Runx2 site (C). All three sites are required for maximal OC promoter activity. Mutation of the proximal site C has the least effect on basal OC promoter activity (15, 16). Three Runx sites have also been noted in the BSP promoter (38). The Runx sites in the BSP promoter mediate repression of BSP (38). 1,25(OH)2D3 also represses BSP expression (39). It has been suggested that the context of the multiple Runx2 motifs within a promoter may contribute to the formation of Runx2 regulatory complexes and secondary interactions that mediate either repression or activation (38). However, previous studies have also indicated, similar to our study, that multiple Runx2 sites are not always required for regulation by Runx2. For example, Drissi et al. (40) reported that, although more than one Runx2 site is present in the Runx2 promoter, a single site within the proximal promoter is sufficient to confer negative autoregulation. It is of interest that although the function of all three calcium binding proteins, BSP, OC and OPN, is associated with ordered deposition of mineral, OC and OPN are induced by 1,25(OH)2D3 and are positively regulated by Runx2 and BSP is inhibited by 1,25(OH)2D3 and Runx mediates its repression. Thus differential regulation by Runx2 and 1,25(OH)2D3 may be needed to regulate the timing of the expression of these proteins and to accommodate their functional role at various stages of osteoblast differentiation. Although Runx2 enhanced VDR mediated OPN transcription, the Runx2 site was not required for 1,25(OH)2D3

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induction of OPN transcription. Mutation of the Runx2 site in the OPN promoter did not affect the 1,25(OH)2D3 response in COS-7 cells (Fig. 4B) and resulted in a decreased (but not abolished) 1,25(OH)2D3 response in ROS 17/2.8 cells, indicating the involvement of tissue specific factors and a cooperative effect of VDR and Runx2 in bone cells. However, in osteoblastic cells mutation of the Runx2 sites in the rat OC promoter blocks 1,25(OH)2D3 OC transcription (15, 16). The 1,25(OH)2D3 regulation of rat OC requires a functional Runx2 site B which is adjacent to the OC VDRE (15, 16). In addition, both Runx2 and AP1 binding sites are required for PTH stimulation of collagenase 3 transcription (41, 42). In the collagenase 3 promoter there is an overlapping AP1 and Runx2 site and Runx2 has been reported to interact with c-fos and c-jun (43, 44). It has been suggested that PTH dependent collagenase 3 expression involves cooperation between Runx2 and AP1 transcription factors and the composite Runx2/TRE element as well as a distal Runx2 site (43). For the regulation of OPN, the Runx2 site is not adjacent or overlapping the VDRE (VDRE –757/-743; Runx2 site –136/-130). It is possible that the Runx2 site in the OPN promoter is not critical for 1,25(OH)2D3 regulation of OPN since the Runx2 site is not adjacent or overlapping the VDRE and that it is the organization of the Runx2 motifs that is important in the regulation of gene expression and the responsiveness to physiological regulation. In our study we also examined the effect on OPN transcription of the transcription factor Hes-1, a downstream target of the Notch signaling pathway, which is known to bind and modulate

the transactivating function of Runx2 (20). We found that Hes-1 is able to enhance basal and VDR mediated OPN transcription and OPN protein expression in the presence of Runx2. Although Hes-1 null mice die during late gestation, we found that OPN mRNA (determined by RT-PCR analysis) is not significantly different in Hes-1-/- and wild type (WT) 15 day old embryo littermates [n = 3 WT and 3 Hes-1-/-

embryos, p > 0.5 (unpublished observation); heterozygote mating pairs were obtained from Dr. Q. Al-Awqati at the College of Physicians and Surgeons of Columbia University and the Hes-1-/- mice were originally generated in the laboratory R. Kageyama, Kyoto University, Japan; 45]. It is possible that cell type specific differences in OPN mRNA expression may be observed which were undetectable using whole embryos or that there may be compensation by Hes-5 in the regulation of OPN. Hes-5 has been reported to compensate for the lack of Hes-1 in studies examining neuronal differentiation (46). Hes-1 is generally thought to act as a negative regulator (47-50). In Drosophila Hes proteins are known to interact with the transcriptional corepressor Groucho (51, 52) and mutations that inhibit the Groucho/Hes interaction interfere with the ability of the Hes proteins to repress transcription (50). The mammalian homolog of Groucho, TLE, associates with Hes-1. It has been suggested that TLE can mediate transcriptional repression by Hes-1 by recruiting histone deacetylases (20, 30). However, Runx2 also interacts with Hes-1 and Hes-1 and Runx2 were reported to colocalize to the nuclear matrix in osteoblastic cells (19). TLEs also associate with the nuclear matrix (53).

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Our transcription assay results as well as the coimmunoprecipitation studies in the presence or absence of Runx2 suggest that Runx2/Hes-1 binding may inhibit the Hes-1/TLE interaction and therefore the association with histone deacetylases thereby acting as a negative regulator of the inhibitory activity of Hes1. Runx2 also interacts with TLE (20) and Hes-1 binding to Runx2 may also interfere with this interaction. It has been reported that the binding of Hes-1 to Runx2 potentiates Runx2 mediated transactivation of OC by interfering with TLE mediated transcriptional repression (20). In our study we found that 1,25(OH)2D3 enhances Runx2/Hes-1 binding. Thus the enhancement of the

interaction of Runx2 with Hes-1 may be an additional mechanism involved in the activation of OPN transcription by 1,25(OH)2D3 in osteoblastic cells. These findings define novel mechanisms involving the intersection of Runx2, 1,25(OH)2D3 and Notch signaling that are involved in the regulation of OPN. Our findings suggest that VDR mediated transcriptional regulation of OPN is modulated by both Runx2 and Hes-1 and that 1,25(OH)2D3 may have a role in osteoblast differentiation by altering the balance of transcription factors affecting their interaction as well as their recruitment to the OPN promoter.

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Figure Legends Figure 1. Functional cooperation between VDR and Runx2 in the regulation of OPN transcription. COS-7 cells were plated in a 24 well culture dish and cells in each well were cotransfected with 0.3 μg mouse OPN promoter firefly luciferase construct (–777/+79) and 0.02 μg hVDR expression plasmid in the absence or presence of 0.1 μg pCMV-Runx2. Empty vectors were used to keep the total DNA concentration the same. Cells were treated with vehicle (-D) or 10-8 M 1,25(OH)2D3 (+D) for 24 h, harvested and luciferase activity was determined. The data were normalized to values for pRL-TK-Renilla luciferase as an internal control. OPN promoter activity (firefly/Renilla luciferase activity) is represented as fold induction (mean ± SE; n= 3-10 observations) and quantitated by comparison to basal levels. 1,25(OH)2D3 treatment or expression of Runx2 led to a significant increase in OPN promoter activity compared to basal levels (p<0.05). 1,25(OH)2D3 treatment in the presence of Runx2 led to a significant enhancement of OPN promoter activity compared to treatment with 1,25(OH)2D3 [vector (vec) +D] or expression of Runx2 (Runx2 –D) (p< 0.05). Figure 2. AML1/ETO inhibits cooperative effects between VDR and Runx2 in COS-7 cells and 1,25(OH)2D3 induced OPN transcription in osteoblastic cells. (A) COS-7 cells were plated in a 24 well culture dish and cells in each well were co-transfected with 0.3 μg mouse OPN promoter luciferase construct (–777/+79) and 0.02 μg hVDR expression vector in the absence or presence of pCMV-Runx2 (0.1 μg) and increasing concentrations of pCMV-AML1/ETO expression plasmid. In COS-7 cells there was no effect of AML-1/ETO on basal or 1,25(OH)2D3 induced levels of OPN transcription even at high concentrations (0.5 μg) of pCMV-AML-1/ETO [open bar vector transfected (vec) vs. vec +AML-1 ETO ; p> 0.5 and +1,25(OH)2D3 and +1, 25(OH)2D3 + AML-1 ETO ; p > 0.5] (B) ROS17/2.8 cells, containing endogenous VDR and Runx2, were transfected with 0.3 μg mouse OPN promoter (–777/+79) in the absence or presence of increasing concentrations of pCMV-AML1/ETO (0.1, 0.2, 0.3 and 0.5 μg). (C) Repression of 1,25(OH)2D3 induction of OPN promoter activity by increasing concentrations of AML1/ETO in MC3T3-E1 cells (which also contain endogenous VDR and Runx2). Conditions for the transfection of MC3T3-E1 cells were the same as for ROS17/2.8 cells. Empty vectors were used to keep the total DNA concentration the same. Cells were treated with vehicle (open bar) or 10-8M 1,25(OH)2D3 (closed bar) for 24 h. OPN promoter activity is normalized to values for pRL-TK-Renilla luciferase activity as an internal control and is expressed as fold induction (mean ± SE; n = 3 or more experiments) by comparison to basal levels. For (A), (B) and (C) each concentration of AML-1/ETO resulted in a significant repression of 1,25(OH)2D3 induced OPN promoter activity (p<0.05). 0.5 μg AML-1/ETO [(B) and (C) open bar AML-1/ETO] resulted in a significant decrease in basal OPN transcription in ROS17/2.8 cells and in MC3T3-E1 cells [open bar AML-1/ETO vs. vec (vector transfected); p< 0.05]. Although basal levels were decreased by 36% and 56% by 0.5 μg AML-1 ETO in ROS 17/2.8 and MC3T3-E1 cells respectively, 1,25(OH)2D3 OPN transcription was decreased by 74.2 and 75.0% at 0.5 μg AML-1 ETO, indicating that not only basal but also 1,25(OH)2D3 induced OPN transcription is diminished by AML-1 ETO. In COS-7 cells or in the osteoblastic cells

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AML-1/ETO (0.5 μg) had no effect on the activity of a tk luciferase construct or on 1,25(OH)2D3 induced rat 25hydroxyvitaminD3 24(OH)ase transcription (not shown). Figure 3. Suppression of 1,25(OH)2D3 induction of OPN mRNA expression by AML1/ETO in osteoblastic cells. (A) ROS17/2.8 cells, plated in 100 mm tissue culture dishes, were transfected with pCMV or pCMV-AML1/ETO (1 μg, 4 μg or 5 μg) and treated with 10-8 M 1,25(OH)2D3 for 24 h. Northern blot analysis was performed as indicated in the Materials and Methods. Northern blots were hybridized with OPN cDNA followed by β−actin cDNA. Upper panel: representative autoradiogram. Lower panel: Graphic representation of Northern blot analyses. Data represent the means ± SE of 3 independent experiments. In the presence of 1, 4 or 5 μg pCMV-AML-1/ETO both basal (vehicle) and 1,25(OH)2D3 induced levels of OPN mRNA were significantly inhibited compared to similarly treated vector transfected cells (p< 0.05). (B) Northern blot analysis of OPN mRNA expression in MC3T3-E1 cells transfected with vector alone or 1 μg pCMV-AML1/ETO and treated with vehicle or 1,25(OH)2D3 as described for ROS17/2.8 cells. A representative autoradiogram is shown. In the presence of AML1/ETO 1,25(OH)2D3 induction of OPN mRNA in MC3T3-E1 cells is 54% of the OPN mRNA levels induced by 1,25(OH)2D3 in the absence of AML1/ETO and basal levels in the presence of AML-1/ETO are 75% of the basal levels of OPN mRNA in the absence of AML-1/ETO (data represent the averages from two experiments) (C) Western blot analysis was performed using 50 μg protein prepared from MC3T3-E1 cells transfected with vector alone or 1 μg pCMV-AML1/ETO and treated with vehicle (-D) or 1,25(OH)2D3 (10-8M) (+D) for 24 h. Detection was by immunoblotting using a polyclonal OPN antibody. Two additional experiments yielded similar results. Figure 4. Functional cooperation between VDR and Runx2 requires both the VDRE and the Runx2 site. (A) Illustration of mutations in the mouse OPN promoter. (B) COS-7 cells were plated in 24 well culture dishes and cells in each well were co-transfected with 0.3 μg OPN promoter construct with a mutation in the Runx2 site (OPN-Runx2-Mut) and 0.02 μg hVDR in the absence or presence of 0.1 μg Runx2 expression vector. (C) COS-7 cells were transfected with 0.3 μg OPN promoter construct with a mutation in the VDRE site (OPN-VDRE-Mut) and 0.02 μg hVDR in the absence or presence of 0.1 μg Runx2 expression vector. The total DNA content was kept constant by the addition of empty vector. COS-7 cells were treated with vehicle (open bar) or 1,25(OH)2D3 (10-8M) (closed bar). OPN promoter activity (normalized to values for pRL-TK-Renilla luciferase activity as an internal control) is expressed as fold induction (mean ± SE; n = 3 –10 observations per group) by comparison to basal levels. Figure 5. 1,25(OH)2D3 stimulates VDR and Runx2 binding to the osteopontin promoter in intact cells. (A) MC3T3-E1 cells were treated with vehicle or 1,25(OH)2D3 (10-8 M) for 30 min. And cells were cross-linked by 1% formaldehyde for 15 min. Cross-linked cell lysates were subjected to immunoprecipitation with IgG or VDR or Runx2 antibody (α-VDR or α-Runx2). DNA precipitates were isolated and then subjected to PCR using specific primers designed according to the VDRE site or the Runx2 site on the mouse OPN promoter (see Materials and Methods). Analysis of input DNA (0.2%) was

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taken prior to precipitation (input). (B) MC3T3-E1 cells were transfected with AML-1 ETO, treated with vehicle or 1,25(OH)2D3 and cross linked lysates were subjected to immunoprecipitation as described in (A). These experiments are representative of three separate experiments performed under the same conditions. Figure 6. Hes-1 enhancement of 1,25(OH)2D3 induced osteopontin transcription in osteoblastic cells. (A) ROS17/2.8 cells or (B) MC3T3-E1 cells were plated in 24 well culture dishes and cells in each well were transfected with 0.3 μg mouse OPN promoter firefly luciferase construct with increasing concentrations of pcDNA3-Hes1 expression vector (0.01, 0.05 and 0.1 μg). Empty vector was used to keep the total DNA concentration the same. pRL-TK-Renilla luciferase was co-transfected as an internal control. Transfection of Hes-1 had no effect on the activity of the tk luciferase construct (not shown). Cells were treated with vehicle or 1,25(OH)2D3 (10-8M) for 24 h. OPN transactivation was expressed as firefly/Renilla luciferase activity and is represented as fold induction (mean ± SE; n = at least 3 observations per group) by comparison to basal levels. In the presence of each concentration of Hes-1 both basal (vehicle treated) and 1,25(OH)2D3 induced OPN promoter activity were significantly enhanced compared to similarly treated vector transfected cells (p <0.05). (C) Northern blot analysis of MC3T3-E1 cells plated in 100 mm tissue culture dishes and transfected with vector alone (vec) or pcDNA3-Hes-1 expression vector (1 μg) and treated with vehicle (-D) or 1,25(OH)2D3 (+D) for 24 h. Two additional experiments yielded similar results. Figure 7. AML1/ETO inhibition of Hes-1 enhanced OPN transcription. ROS17/2.8 cells were transfected with 0.3 μg mouse OPN promoter and 0.1 μg pcDNA3-Hes1 expression plasmid with or without increasing concentrations of pCMV-AML1/ETO expression plasmid (0.02 – 0.2 μg) Empty vector was used to keep the total DNA concentration the same. pRL-TK-Renilla luciferase was cotransfected as an internal control. Cells were treated with vehicle (open bar) or 1,25(OH)2D3 (closed bar) for 24 h. OPN promoter activity was expressed as firefly/Renilla luciferase activity and is represented as fold induction (mean ± SE; n = at least 3 observations per group) by comparison to basal levels. In the presence of each concentration of AML-1/ETO (0.02-0.2 μg) Hes-1 enhancement of basal levels of OPN transcription was significantly inhibited (p< 0.05). In the presence of 0.05, 0.1 and 0.2 μg AML-1/ETO the Hes-1 enhancement of 1,25(OH)2D3 induced OPN transcription was significantly inhibited (p<0.05). Figure 8. 1,25(OH)2D3 enhancement of the interaction of Runx2 and Hes-1. (A) ROS 17/2.8 cell nuclear extracts were used for immunoprecipitation with Runx2 antibody, Hes-1 antibody or control rabbit immunoglobulin G (IgG). The pulled downed protein complex was boiled in SDS-containing buffer and loaded on 10% SDS-PAGE gel. Western blot was performed using Hes-1 antibody or Runx2 antibody. Treatment with 1,25(OH)2D3 (10-8M, 24h) increased the interaction between Hes-1 and Runx2. Three additional experiments yielded similar results. (B) ReChIP analysis of Hes-1 binding to the OPN promoter. MC3T3-E1 cells were treated with vehicle or 1,25(OH)2D3 and cross linked as described in Fig. 5 legend. Lysates were immunoprecipitated first with Runx2

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antibody and then with Hes-1 antibody. Eluted DNA was amplified by primers designed according to the Runx2 site. Figure 9. TSA rescues the AML1/ETO inhibition of the HES-1 enhanced OPN transcription. (A) ROS17/2.8 cells were transfected with 0.3 μg OPN promoter in the presence or absence of 0.1 μg HES-1 expression vector alone or Hes-1 expression vector and 0.2 μg pCMV-AML1/ETO. After 24 h, cells were treated with 1,25(OH)2D3 (10-8M, 24h; +D) in the absence or presence of 15 nM TSA. Empty vectors were used to keep the total DNA concentration the same. OPN promoter activity was expressed as firefly/Renilla luciferase activity and is represented as fold induction (mean + SE; n = at least 3 observations per group) by comparison to basal levels. (B) COS-7 cells were transfected with VDR and were cotransfected with vector or Runx2 and were treated with 1,25(OH)2D3.(10-8M, 24h). Nuclear extracts were prepared and 500 μg of nuclear protein was used for immunoprecipitation with HDAC1 antibody. Western blot was performed with Hes-1 antibody. The top panel shows the Western blot of cell extracts prior to immunoprecipitation probed with Hes-1 antibody.

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Vec-D

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Figure 2.

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B.

A. OPN

β-actin

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Figure 4.

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Figure 5.

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Figure 8. Hes-1 and Runx2 Interaction

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Mouse OPN(-777/+79)A.

Figure 9.

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Association of Hes1 and HDAC1

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Qi Shen and Sylvia Christakostranscriptional regulation of osteopontin

The vitamin D receptor, RUNX2 and the notch signaling pathway cooperate in the

published online September 29, 2005J. Biol. Chem. 

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