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The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin *

The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 49, pp. 40589 –40598, December 9, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin Received for publication, April 18, 2005, and in revised form, September 6, 2005 Published, JBC Papers in Press, September 29, 2005, DOI 10.1074/jbc.M504166200 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 Osteopontin (OPN), a glycosylated phosphoprotein that binds aspartate (RGD) integrin binding motifs and promotes attachment of calcium, is present in bone extracellular matrix and has been bone cells to the bone surface through binding to OPN receptors such as reported to modulate both mineralization and bone resorption. the   integrin and CD44 (1–3). OPN has been suggested to be v 3 Targeted disruption in mice of the vitamin D receptor (VDR) or involved in the attachment of osteoclasts during bone resorption, to Runx2 results in marked inhibition of OPN expression in osteo- play a role in osteogenesis by attachment of osteoblasts when they form blasts. In this study, we addressed possible cross-talk between VDR bone matrix, and to act to regulate crystal size during bone mineraliza- and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvita- tion (2). In addition, OPN has been suggested to be a mediator of bone min D (1,25(OH) D ) or Runx2 stimulated OPN transcription remodeling in response to mechanical strain (4). OPN null mice are 3 2 3 (mouse OPN promoter 777/79) 2–3-fold. However, coexpres- resistant to mineral loss and bone resorption upon estrogen deprivation sion of Runx2 and VDR in COS-7 cells and treatment with and have impaired activation of osteoclasts (3, 5–7). Also, vasculariza- 1,25(OH) D resulted in an 8-fold induction of OPN transcription, tion and resorption of bone discs have been reported to be significantly 2 3 indicating for the first time functional cooperation between Runx2 impaired in the absence of OPN (8). Although recent studies using OPN and VDR in the regulation of OPN transcription. In ROS 17/2.8 and null mice have provided new insight into the role of OPN in vivo in bone MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO, metabolism, the factors that affect the regulation of OPN are not yet which acts as a repressor of Runx2, significantly inhibited clearly defined. 1,25-Dihydroxyvitamin D (1,25(OH) D ), the active 3 2 3 1,25(OH) D induction of OPN transcription, OPN mRNA, and 2 3 form of vitamin D, is a major calcitropic hormone involved in calcium protein expression. Both a Runx2 site (136/130) and the vitamin homeostasis (9). One of its functions in bone is to regulate the synthesis D response element (757/743) in the OPN promoter are needed of the bone calcium-binding proteins osteocalcin (OC) and OPN (9). for cooperative activation. Chromatin immunoprecipitation analy- 1,25(OH) D modulates the expression of these genes through tran- 2 3 ses showed that 1,25(OH) D can enhance VDR and Runx2 recruit- 2 3 scriptional regulation. The actions of 1,25(OH) D are mediated 2 3 ment on the OPN promoter, further indicating cooperation through the vitamin D receptor (VDR). Liganded VDR heterodimerizes between these two factors in the regulation of OPN. In osteoblastic with the retinoid X receptor and interacts with a vitamin D response cells, Hes-1, a downstream factor of the Notch signaling pathway, element (VDRE). The VDRE in the mouse OPN promoter (at 757/ was found to enhance basal and 1,25(OH) D -induced OPN tran- 2 3 743) is a perfect direct repeat of the motif GGTTCA spaced by three scription. This enhancement was inhibited by AML-1/ETO, an nucleotides (10). Transcription proceeds through the interaction of inhibitor of Runx2. Immunoprecipitation assays indicated that VDR with coactivators and coregulators, including SRC-1/NcoA1, Hes-1 and Runx2 interact and that 1,25(OH) D can enhance this 2 3 SRC-2/GRIP-1 (GR-interacting protein)/NcoA2, SRC-3/ACTR, and interaction. Taken together, these findings define novel mecha- the multisubunit DRIP (vitamin D receptor-interacting protein) com- nisms involving the intersection of three pathways, Runx2, plex (11). Although a VDRE has been identified in the mouse OPN 1,25(OH) D , and Notch signaling, that play a major role in the 2 3 promoter (10) and VDR null mice show marked inhibition of OPN regulation of OPN in osteoblastic cells and therefore in the process expression in osteoblasts (12), the exact mechanisms, including protein- of bone remodeling. protein and protein-DNA interactions, involved in 1,25(OH) D -regu- 2 3 lated OPN transcription are not well understood. Runx2/Cbfa1 is a member of the runt/Cbfa family of transcription Osteopontin (OPN) is a sialic acid-rich glycosylated phosphopro- factors that was first identified as an osteoblast-specific transcription tein, comprising about 2% of the noncollagenous protein in bone (1, 2). factor and a regulator of osteoblast differentiation (13, 14). Runx2 / OPN is produced by osteoblasts when they form bone matrix (1, 2). mice die shortly after birth and show a complete lack of mineralized OPN is an extracellular matrix protein that contains arginine-glycine- bone tissue (13, 14). Marked decreases in the expression of osteopontin and osteocalcin are observed in Runx2 / mice, indicating the regu- * The costs of publication of this article were defrayed in part by the payment of page lation of these genes by Runx2 (13). Three Runx2 binding motifs have charges. This article must therefore be hereby marked “advertisement” in accordance been identified in the rat OC promoter (15). In addition, Runx2 has been with 18 U.S.C. Section 1734 solely to indicate this fact. shown to play a key role in the 1,25(OH) D regulation of rat OC (15, To whom correspondence should be addressed: Dept. of Biochemistry and Molecular 2 3 Biology, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 16). However, it is not yet known whether a similar cooperation occurs 07103. Tel.: 973-972-4033; Fax: 973-972-5594; E-mail: [email protected]. 2 between VDR and Runx2 in the regulation of OPN. The abbreviations used are: OPN, osteopontin; 1,25(OH) D , 1,25-dihydroxyvitamin D ; 2 3 3 OC, osteocalcin; VDR, vitamin D receptor; FBS, fetal bovine serum; PBS, phosphate- Hes-1 (Hairy and enhancer of split homologue-1), a downstream tar- buffered saline; ChIP, chromatin immunoprecipitation; VDRE, vitamin D response ele- get of the Notch signaling pathway, is a helix-loop-helix transcription ment; hVDR, human VDR; WT, wild type; BSP, bone sialoprotein; Pipes, 1,4-pipera- zinediethanesulfonic acid. factor that has been reported to play a role in developmental processes, DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40589 This is an Open Access article under the CC BY license. Transcriptional Regulation of Osteopontin including 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 con- tribute 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 find- ings define, for the first time, novel mechanisms involving the intersec- tion of Runx2, 1,25(OH) D and Notch signaling that are involved in the 2 3 regulation of the OPN gene. EXPERIMENTAL PROCEDURES Materials—[- P]ATP (3,000 Ci (111 TBq)/mmol), nylon mem- 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 brane, and the enhanced chemiluminescent detection system (ECL) were cotransfected with 0.3 g of mouse OPN promoter firefly luciferase construct were purchased from PerkinElmer Life Sciences. Dulbecco’s modified (777/79) and 0.02 g of hVDR expression plasmid in the absence or presence of 0.1 Eagle’s medium plus Ham’s F-12 nutrient mixture, Dulbecco’s modified g of pCMV-Runx2. Empty vectors were used to keep the total DNA concentration the same. Cells were treated with vehicle (D)or10 M 1,25(OH) D (D) for 24 h and 2 3 Eagle’s medium, fetal bovine serum (FBS), and PSN antibiotic mixture harvested, and luciferase activity was determined. The data were normalized to values were purchased from Invitrogen. -Minimal essential medium was pur- for pRL-TK-Renilla luciferase as an internal control. OPN promoter activity (firefly/Renilla luciferase activity) is represented as -fold induction (mean S.E.; n 3–10 observations) chased from Sigma. VDR antiserum (C-20), mouse OPN antiserum and quantitated by comparison with basal levels. 1,25(OH) D treatment or expression 2 3 (P-18), and histone deacetylase-1 antiserum (H-51) were purchased of Runx2 led to a significant increase in OPN promoter activity compared with basal from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Runx2 anti- levels (p  0.05). 1,25(OH) D treatment in the presence of Runx2 led to a significant 2 3 enhancement of OPN promoter activity compared with treatment with 1,25(OH) D 2 3 serum was purchased from Oncogene Research Products (San Diego, (vector (Vec) D) or expression of Runx2 (Runx2 D)(p  0.05). CA). The antiserum reacting to Hes-1 (a gift from T. Sudo, Kamakura, Japan) was produced by immunizing rabbits with a fusion protein con- performed according to the protocol of the manufacturer and normal- sisting of the C-terminal 19 amino acids (SPSSGSSLTSDSMWRP- ized to values for pRL-TK-Renilla luciferase. For all transcription stud- WRN) of mouse Hes-1 coupled to keyhole limpet hemocyanin. 1,25- ies, OPN promoter activity (firefly/Renilla luciferase) is represented as Dihydroxyvitamin D3 was a generous gift from Dr. Milan Uskokovic -fold induction by comparison with basal levels (basal levels refer to (Hoffmann-LaRoche, Nutley, NJ). levels of OPN promoter activity in cells transfected with vector alone Cell Culture—COS-7 African green monkey kidney cells were and treated with vehicle). obtained from the American Type Culture Collection (Manassas, VA) Site-directed Mutagenesis—Mutant mouse OPN promoter (777/ and were cultured in Dulbecco’s modified Eagle’s medium supple- 79) luciferase reporter constructs were generated by site directed mented with 10% FBS. ROS17/2.8 cells (a gift of S. Rodan and G. Rodan mutagenesis using the QuikChange site-directed mutagenesis kit (Merck)) were maintained in Dulbecco’s modified Eagle’s medium/F-12 (Stratagene, La Jolla, CA). The oligonucleotides used to generate the medium supplemented with 5% FBS, 1% PSN. MC3T3-E1 cells (Riken Runx2 mutated site (shown in lowercase) were as follows: 5-CCT TTT Cell Bank, Tsukuba, Japan) were cultured in -minimal essential TTT TTT TTT AAg aAC AAA ACC AGA GGA GG-3 (top strand) medium supplemented with 10% FBS, 1% PSN. All cells were cultured in and 5-CCT CCT CTG GTT TTG Ttc TTA AAA AAA AAA AAA a humidified atmosphere of 95% air, 5% CO at 37 °C. Cells were seeded GG-3 (lower strand). The oligonucleotides used to generate the VDRE at 70–80% confluence 24 h before experiments. Treatments with mutated site (shown in lowercase) were as follows: 5-CAG AGC AAC 1,25(OH) D were performed in medium supplemented with 2% char- AAG Gcc CAC GAG GTT CAC GTC-3 (top strand) and 5-GAC 2 3 coal-stripped serum. GTG AAC CTC GTG ggC CTT GTT GCT CTG-3 (bottom strand). Transient Transfection and Dual Luciferase Assay—The mouse Northern Blot Analysis—ROS17/2.8 cells or MC3T3-E1 cells, plated osteopontin promoter (777/79) firefly luciferase reporter construct at 70% confluence in 100-mm tissue culture dishes, were transfected was kindly provided by D. Denhardt (Rutgers University, Piscataway, using Lipofectamine 2000 reagent, with AML-1/ETO or Hes-1 expres- NJ). pCMV-Runx2 was a gift of G. Karsenty (Baylor College of Medi- sion vector or vector alone. 24 h after transfection, cells were treated for cine, Houston, TX), and pCMV-AML-1/ETO expression vector was 24 h with 1,25(OH) D (10 M) or vehicle control. The treated cells 2 3 from S. W. Hiebert (Vanderbilt University School of Medicine, Nash- were then harvested by trypsinization, pelleted, and washed with PBS. ville, TN). pcDNA3-Hes1 expression vector was a gift from Dr. S. Stifani Total RNA was isolated by RNA-bee RNA extraction solution (Tel- (McGill University, Montreal, Canada). Cells were seeded in a 24-well Test, Friendswood, TX) and precipitated by chloroform and isopropyl culture dish 24 h prior to transfection at 70% confluence. Cells in each alcohol. 20 g of total RNA from each sample was used for Northern well were transfected using Lipofectamine 2000 reagent (Invitrogen) blot analysis as previously described (24). P-Labeled cDNA was pre- according to the manufacturer’s instructions. Empty vectors were used pared using the Random Primers DNA labeling system (Invitrogen) to keep the total DNA concentration the same. Efficiency of transfec- according to the random primer method (25). The mouse osteopontin tion, as assessed by green fluorescent protein cotransfection and subse- cDNA was generated by HindIII digestion and was a gift from D. Den- quent visualization, was estimated at 60–70%. 1,25(OH) D (10 M)or hardt (Rutgers University, Piscataway, NJ). The -actin cDNA was pur- 2 3 TSA (15 nM) was added to cells 24 h post-transfection for another 24 h. chased from Clontech. The blots were hybridized with the P-labeled Cells were washed twice with phosphate-buffered saline (PBS) and har- mouse OPN cDNA probes for 16 h at 42 °C, washed, air-dried and vested by incubating with 1 passive lysis buffer, supplied by the Dual- exposed to Eastman Kodak Co. BIOMAX MR film at 80 °C for 1 day. Luciferase reporter assay kit (Promega). The luciferase activity assay was The same blots were stripped and probed with P-labeled -actin 40590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin FIGURE 2. AML1/ETO inhibits cooperative effects between VDR and Runx2 in COS-7 cells and 1,25(OH) D -induced OPN transcription in 2 3 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 g of mouse OPN pro- moter luciferase construct (777/79) and 0.02 g of hVDR expression vector in the absence or presence of pCMV-Runx2 (0.1 g) 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) D -induced levels of 2 3 OPN transcription even at high concentrations (0.5 g) of pCMV-AML-1/ETO (open bar, vector-trans- fected (Vec) versus vector  AML-1 ETO (p  0.5) and 1,25(OH) D and 1,25(OH) D  AML-1 2 3 2 3 ETO; p  0.5). B, ROS17/2.8 cells, containing endogenous VDR and Runx2, were transfected with 0.3 g of mouse OPN promoter (777/79) in the absence or presence of increasing concen- trations of pCMV-AML1/ETO (0.1, 0.2, 0.3, and 0.5 g). C, repression of 1,25(OH) D induction of OPN 2 3 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)or10 M 1,25(OH) D (closed bar) for 24 h. OPN promoter 2 3 activity is normalized to values for pRL-TK-Renilla luciferase activity as an internal control and is expressed as -fold induction (mean  S.E.; n  3 experiments) by comparison with basal levels. For A–C, each concentration of AML-1/ETO resulted in a significant repression of 1,25(OH) D -induced 2 3 OPN promoter activity (p  0.05). 0.5 g of 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 versus vector (vector-transfected); p 0.05). Although basal levels were decreased by 36 and 56% by 0.5g of AML-1 ETO in ROS 17/2.8 and MC3T3-E1 cells, respectively, 1,25(OH) D induced 2 3 OPN transcription was decreased by 74.2 and 75.0% at 0.5 g of AML-1 ETO, indicating that not only basal but also 1,25(OH) D -induced OPN 2 3 transcription is diminished by AML-1 ETO. In COS-7 cells or in the osteoblastic cells, AML-1/ETO (0.5 g) had no effect on the activity of a thymidine kinase luciferase construct or on 1,25(OH) D -induced rat 2 3 25-hydroxyvitamin D 24(OH)ase transcription (not shown). cDNA. Autoradiograms were analyzed by densitometric scanning using Western blotting detection system (PerkinElmer Life Sciences) was the Dual-Wavelength Flying Spot Scanner. The relative optical density used to detect the antigen-antibody complex. obtained using the OPN probe was divided by the relative optical den- Chromatin Immunoprecipitation (ChIP) Assay—MC3T3-E1 cells sity obtained after probing with -actin to normalize for sample were cultured in -minimal essential medium supplemented with 10% variation. FBS to 95% confluence prior to the experiment and then treated in OPN Western Blot Analysis—MC3T3-E1 cells, plated at 70% conflu- -minimal essential medium supplemented with 2% charcoal-stripped ence in 100-mm tissue culture dishes, were transfected with vector serum under the conditions and for the times indicated. Treated cells alone or pCMV-AML1/ETO and treated with vehicle or 1,25(OH) D were used for the ChIP assay (26, 27). Briefly, cells were first washed with 2 3 (10 M) for 24 h and harvested by trypsinization. For Western blot PBS and subjected to a cross-link reaction with 1% formaldehyde for 15 analysis, 50 g of protein from total cell lysates was loaded onto a 10% min. The cross-link reaction was stopped by adding glycine to a final SDS-polyacrylamide gel and separated by electrophoresis. Protein was concentration of 0.125 M. Cells were washed with ice-cold PBS twice. transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The cells were collected by scraping and lysed sequentially in 5 mM Membranes were incubated overnight at 4 °C with mouse OPN poly- Pipes, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40 and then in 1% SDS, 10 mM clonal antibody (P-18; Santa Cruz Biotechnology) at a 1:1000 dilution in EDTA, 50 mM Tris-HCl, pH 8.1, for 20 min individually. The chromatin PBS containing 5% nonfat milk. The membrane was washed with PBS pellets were sonicated to an average DNA size of 500 bp DNA (assessed and incubated for 1 h with the corresponding secondary antibody con- by 1% agarose gel electrophoresis) using a Fisher model 100 sonic dis- jugated with horseradish peroxidase. The enhanced chemiluminescent membranator at a power setting of 1. The sonicated extract was centri- DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40591 Transcriptional Regulation of Osteopontin FIGURE 3. Suppression of 1,25(OH) D induc- 2 3 tion 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, 4, or 5 g) and treated with 10 M 1,25(OH) D for 24 h. Northern 2 3 blot analysis was performed as indicated under “Experimental Procedures.” Northern blots were hybridized with OPN cDNA followed by -actin cDNA. Upper panel, representative autoradiogram. Lower panel, graphic representation of Northern blot analyses. Data represent the means  S.E. of three independent experiments. In the presence of 1, 4, or 5 g of pCMV-AML-1/ETO both basal (vehicle) and 1,25(OH) D induced levels of OPN 2 3 mRNA were significantly inhibited compared with similarly treated vector-transfected cells (p 0.05). B, Northern blot analysis of OPN mRNA expression in MC3T3-E1 cells transfected with vec- tor alone or 1 g pCMV-AML1/ETO and treated with vehicle or 1,25(OH) D as described for 2 3 ROS17/2.8 cells. A representative autoradiogram is shown. In the presence of AML1/ETO 1,25(OH) D 2 3 induction of OPN mRNA in MC3T3-E1 cells is 54% of the OPN mRNA levels induced by 1,25(OH) D in 2 3 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 g of protein prepared from MC3T3-E1 cells trans- fected with vector alone or 1 g of pCMV-AML1/ ETO and treated with vehicle (D) or 1,25(OH) D 2 3 (10 M)(D) for 24 h. Detection was by immuno- blotting using a polyclonal OPN antibody. Two additional experiments yielded similar results. fuged for 10 min at maximum speed and then diluted into ChIP dilution (pH 7.4), 1.5 mM MgCl ,10mM KCl, 0.5 mM dithiothreitol, phosphatase buffer (16.7 mM Tris-HCl, pH 8.1, 150 mM NaCl, 0.01% SDS, 1.1% Tri- inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, ton X-100, 1.2 mM EDTA). Immunoprecipitations were performed at 2 mg/ml leupeptin, 2 mg/ml aprotinin), and 1% Triton X-100. Nuclei 4 °C overnight with the indicated antibody overnight. After a 1-h incu- were pelleted at 4,000 rpm for 4 min, and cytoplasmic supernatants bation with salmon sperm DNA and bovine serum albumin-pretreated were separated. Nuclei were resuspended in hypertonic buffer contain- Zysorbin (Zymed Laboratories Inc., San Francisco, CA), the precipitates ing 0.42 mM NaCl, 0.2 mM EDTA, 25% glycerol, and the phosphatase were collected by centrifugation. Precipitates were washed sequentially and protease inhibitors indicated above. After a 2-h incubation at 4 °C, in buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, nuclear soluble proteins were collected by centrifuging at 13,000 rpm pH 8.1, 150 mM NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mM for 10 min. Protein concentration of the supernatant was measured by EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), buffer III (0.25 M LiCl, the method of Bradford (28), and aliquots were stored at 80 °C. 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH Immunoprecipitation—To examine the association of Runx2 and 8.1), and TE buffer (10 mM Tris, 1 mM EDTA) twice. The protein-DNA Hes-1 in the presence or absence of 1,25(OH) D coimmunoprecipita- 2 3 was then eluted by using 1% SDS and 0.1 M NaHCO for 15 min twice. tion experiments were done. Nuclear extracts were prepared as indi- Cross-links were reversed by incubating at 65 °C overnight in elution cated above from ROS17/2.8 cells or MC3T3-E1 cells, and protein con- buffer with 0.2 M NaCl. DNA fragments were purified using Qiagen centration was detected by the Bradford method (28). 500 g of each QIAquick PCR purification kits (Valencia, CA) and subjected to PCR preparation was used for immunoprecipitation with the addition of 4 g using the primers designed to amplify fragments of murine osteopontin of Hes-1 antiserum or 4 g of Runx2 antiserum in the presence or promoter VDRE motif (upper, 5-ACC ACC TCT TCT GCT CTA TAT absence of 1,25(OH) D (10 M) for 24 h at 4 °C. Then 30 l of protein 2 3 GGC-3; lower, 5-TGA CAC TTG AAC TAT GCA GCC GC-3) and A-Sepharose 4 Fast Flow Beads (Amersham Biosciences) were added to the primers designed to amplify the Runx2 motif (upper, 5-TTC CGG each sample, and, after further incubation by rotating at 4 °C for 1 h, the GAT TCT AAA TGC AGT CTA-3; lower, 5-CTC CCA GAA TTT immunoprecipitated complex was collected by centrifuging at 3,000 AAA TGC TGG TCC-3). PCR analysis was carried out in the linear rpm for 5 min. The complex was separated by 12% SDS-PAGE and range of DNA amplification. PCR products were resolved in 5% TBE probed with Runx2 antibody or Hes-1 antibody. Immunoprecipitation acrylamide gel and visualized using ethidium bromide staining. DNA experiments were also done as described above using COS-7 cells trans- acquired prior to precipitation was collected and used as the input. 10% fected with VDR and treated with 1,25(OH) D (10 M for 24 h) and 2 3 of input was used for PCR evaluation. cotransfected with vector alone (pCMV) or 2 g of pCMV-Runx2 to In re-ChIP experiments, complexes were eluted by incubation for 30 examine the association of Hes-1 with histone deacetylase-1 in the pres- minat37 °Cin60 l of elution buffer containing 10 mM dithiothreitol. ence or absence of Runx2. For these studies, 500 g of nuclear extract The eluted samples were diluted 50 times with ChIP dilution buffer and was used for immunoprecipitation with the addition of 4 g of histone subjected again to the ChIP procedure with specific antibodies. deacetylase-1 antiserum followed by the addition of protein A-Sepha- Nuclear Extracts—Cells were washed with cold PBS twice, harvested rose 4 Fast Flow Beads incubated and collected by centrifugation as by scraping, pelleted by centrifuging at 4,000 rpm for 4 min. The pellets described above. The complex was separated by 12% SDS-PAGE and were washed and lysed in hypotonic buffer containing 10 mM HEPES probed with Hes-1 antibody. 40592 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin fected with the mouse OPN promoter (777/79; VDRE 757/743) and hVDR and/or Runx2 expression vectors. In 1,25(OH) D -treated 2 3 (10 M 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) D (Fig. 1). Coexpression of Runx2 and VDR and treatment 2 3 with 1,25(OH) D (10 M 24 h) resulted in an 8.3  0.8-fold induction 2 3 of OPN transcription (Fig. 1), suggesting functional cooperation between Runx2 and VDR in the regulation of OPN. The chimeric protein AML-1/ETO can efficiently block Runx2-me- diated transcriptional activation (29). In COS-7 cells, the enhancement of the inductive action by 1,25(OH) D and Runx2 was inhibited by 2 3 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 sig- nificantly diminished the 1,25(OH) D induction of OPN transcription 2 3 (Fig. 2, B and 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) D -induced OPN mRNA 2 3 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) D -induced OPN mRNA (Fig. 3A). Note (Fig. 3A, last two 2 3 bars) that although there is a 50% decrease in basal OPN mRNA, there is a 75% decrease in 1,25(OH) D -induced OPN mRNA. Similar results 2 3 were observed using MC3T3-E1 cells (Fig. 3B). In addition, inhibition of 1,25(OH) D OPN protein expression was also observed in the presence 2 3 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 Acti- vation 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 FIGURE 4. Functional cooperation between VDR and Runx2 requires both the VDRE assays indicated, similar to previous studies (23), that 136/130 in 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 the mouse OPN promoter is a binding site for Runx2. To investigate 0.3 g of OPN promoter construct with a mutation in the Runx2 site (OPN-Runx2-Mut) the specific contribution of the VDRE and the Runx2 site to the and 0.02 g of hVDR in the absence or presence of 0.1 g of Runx2 expression vector. C, cooperative activation of OPN transcription, mutant OPN promoter COS-7 cells were transfected with a 0.3-g OPN promoter construct with a mutation in the VDRE site (OPN-VDRE-Mut) and 0.02g of hVDR in the absence or presence of 0.1g constructs were generated with either the Runx2 site (136/130) of Runx2 expression vector. The total DNA content was kept constant by the addition of mutated or the VDRE (757/743) mutated (Fig. 4A). Mutation of empty vector. COS-7 cells were treated with vehicle (open bar) or 1,25(OH) D (10 M) 2 3 (closed bar). OPN promoter activity (normalized to values for pRL-TK-Renilla luciferase the Runx2 site did not affect the induction by 1,25(OH) D of OPN 2 3 activity as an internal control) is expressed as -fold induction (mean  S.E.; n  3–10 transcription in VDR-transfected COS-7 cells (Fig. 4B, vector-trans- observations/group) by comparison with basal levels. fected, vehicle- and 1,25(OH) D -treated) and resulted in a 2 3 decreased (but not abolished) 1,25(OH) D response in ROS 17/2.8 Statistical Analysis—Results are expressed as the mean  S.E., and 2 3 cells (not shown). However, in COS-7 cells Runx2 could no longer significance was determined by analysis with Student’s t test for two- activate OPN transcription (Fig. 4B, Runx2-transfected, vehicle- group comparison or analysis of variance for multiple group treated; p  0.4 compared with vector-transfected vehicle-treated comparison. (lane 3 versus lane 1)), indicating that Runx2 acts through this site in RESULTS the mouse OPN promoter 777/79 and not through additional sites (unlike the regulation of OC by Runx2) (15). Also, mutation of Runx2 Cooperates with VDR in Regulating OPN—Targeted disrup- tion in mice of VDR or Runx2 results in a marked inhibition of OPN the Runx2 site resulted in a loss of the cooperative response (Fig. 4B, expression in osteoblasts (12, 13). In order to address possible cross-talk lane 4; compare with Fig. 1, lane 4). The decrease in the response to between VDR and Runx2 in regulating OPN transcription, studies were 1,25(OH) D in the presence of Runx2 using the OPN promoter with 2 3 done using COS-7 cells (that lack endogenous VDR and Runx2) trans- the mutated Runx2 site may be due to the reported binding of Runx2 DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40593 Transcriptional Regulation of Osteopontin AML-1/ETO resulted in decreased recruitment of Runx2 to the OPN promoter (Fig. 5B). The 1,25(OH) D enhancement of Runx2 as well as 2 3 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 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) D - and Runx2-mediated regulation of 2 3 OPN transcription. In ROS17/2.8 cells and MC3T3-E1 cells, that con- tain endogenous Runx2, transfection of Hes-1 (0.1–1 g) resulted in an enhancement of both basal and 1,25(OH) D -induced OPN transcrip- 2 3 tion (Fig. 6, A and B). Expression of Hes-1 also resulted in an enhance- ment of basal and 1,25(OH) D -induced OPN mRNA expression (Fig. 2 3 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 transfected Runx2, expression of Hes-1 resulted in a repression of 1,25(OH) D -dependent induction 2 3 of OPN transcription, and co-transfection of Runx2 in COS-7 cells reversed the inhibition by Hes-1 (not shown), further suggesting func- tional 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) D can increase this inter- 2 3 action (Fig. 8A), suggesting that 1,25(OH) D may enhance functional 2 3 cooperation between Hes-1 and Runx2 by enhancing Hes-1/Runx2 FIGURE 5. 1,25(OH) D stimulates VDR and Runx2 recruitment to the osteopontin 2 3 promoter in intact cells. A, MC3T3-E1 cells were treated with vehicle or 1,25(OH) D interaction. Similar results were observed using MC3T3-E1 cells (not 2 3 (10 M) for 30 min, and cells were cross-linked by 1% formaldehyde for 15 min. Cross- shown). Further, re-ChIP analysis shows that Runx2 and Hes-1 bind linked cell lysates were subjected to immunoprecipitation with IgG or VDR or Runx2 simultaneously to the OPN promoter (Fig. 8B). antibody (-VDR or -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 Since both Runx2 and Hes-1 can interact with TLE (transducin-like mouse OPN promoter (see “Experimental Procedures”). Analysis of input DNA (0.2%) was enhancer of split) proteins, which can recruit histone deacetylases (20, taken prior to precipitation (INPUT). B, MC3T3-E1 cells were transfected with AML-1 ETO, treated with vehicle or 1,25(OH) D and cross-linked lysates were subjected to immuno- 30), we asked whether inhibition of histone deacetylation may be 2 3 precipitation as described in A. These experiments are representative of three separate involved in the activation by Hes-1. In ROS17/2.8 cells, TSA, a histone experiments performed under the same conditions. CON, control. deacetylase inhibitor, was able to rescue the inhibition by AML-1/ETO of Hes-1-enhanced 1,25(OH) D -induced OPN transcription (Fig. 9A). 2 3 to VDR (16). Runx2, in the presence of a mutated Runx2 site in the In addition, in COS-7 cells, in the absence of transfected Runx2, inhibi- OPN promoter, may bind to VDR, and thus less VDR would be tion of 1,25(OH) D -induced OPN transcription by Hes-1 was reversed 2 3 available for 1,25(OH) D induced transcription. Using the OPN 2 3 in the presence of TSA (not shown). These findings suggest that Hes-1/ promoter construct bearing a mutation in the VDRE, 1,25(OH) D 2 3 Runx2 binding may interfere with Runx2-TLE and Hes-1-TLE interac- was unable to activate the OPN promoter in VDR-transfected COS-7 tions, thus preventing repression, which may be mediated, at least in cells (Fig. 4C, vector-transfected (Vec), 1,25(OH) D -treated) or in 2 3 part, by histone deacetylation. Coimmunoprecipitation studies showed ROS 17/2.8 cells (not shown). However, transfection of COS-7 cells the association of Hes-1 and histone deacetylase-1 and a decrease in this with Runx2 could still result in enhanced OPN transcription, and, association in the presence of Runx2 (Fig. 9B). Taken together, these similar to the mutation of the Runx2 site, the cooperative response findings show that Hes-1 can potentiate VDR-mediated OPN transcrip- was not observed (Fig. 4C). These findings suggest that the Runx2 tion in the presence of Runx2 and define new mechanisms and func- site at 136/130 and the VDRE are essential for cooperative effects tional interactions that are involved in the regulation of OPN and may of Runx2 and VDR in activating mouse OPN transcription. therefore affect the process of bone remodeling. Runx2 and VDR Interact with the OPN Promoter in Intact Osteoblas- tic Cells—In order to further understand mechanisms involved in acti- DISCUSSION vation of OPN transcription, we examined VDR and Runx2 complex formation on the OPN promoter in MC3T3-E1 cells using the ChIP This study describes for the first time cooperative effects between assay and specific antibodies against Runx2 and VDR. The antibodies Runx2, VDR, and Hes-1 in the transcriptional regulation of OPN. Func- tional cooperation was demonstrated between Runx2 and VDR in the were used to precipitate sonicated chromatin cross-links from whole cell lysates after formaldehyde cross-linking of DNA to transcription regulation of OPN transcription, OPN mRNA, and protein expression. factors. DNA was amplified using specific primers directed against the 1,25(OH) D was found to enhance both VDR and Runx2 recruitment 2 3 VDRE or the Runx2 binding region of the OPN promoter. In the PCR on the OPN promoter in vivo, further indicating cooperation between these two factors in the regulation of OPN. Hes-1, a downstream target procedure, the number of cycles was chosen so that the amplification was conducted in the linear range of amplification efficiency. No signal of the Notch signaling pathway, was found to act as an enhancer of basal was detected in the presence of IgG (Fig. 5A). The ChIP analysis showed and 1,25(OH) D -induced OPN transcription and OPN mRNA in the 2 3 that 1,25(OH) D can enhance both VDR and Runx2 recruitment to the presence of Runx2. Coimmunoprecipitation analysis indicated that 2 3 OPN promoter (Fig. 5A). Note that transfection of MC3T3-E1 cells with Hes-1 and Runx2 interact, and 1,25(OH) D enhances this interaction. 2 3 40594 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin FIGURE 6. Hes-1 enhancement of 1,25(OH) D - 2 3 induced osteopontin transcription in osteo- blastic cells. ROS17/2.8 cells (A) or MC3T3-E1 cells (B) were plated in 24-well culture dishes, and cells in each well were transfected with 0.3g of mouse OPN promoter firefly luciferase construct with increasing concentrations of pcDNA3-Hes1 expression vector (0.01, 0.05, and 0.1 g). Empty vector was used to keep the total DNA concentra- tion 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 thymi- dine kinase luciferase construct (not shown). Cells were treated with vehicle or 1,25(OH) D (10 M) 2 3 for 24 h. OPN transactivation was expressed as fire- fly/Renilla luciferase activity and is represented as -fold induction (mean  S.E.; n  3 observations/ group) by comparison with basal levels. In the presence of each concentration of Hes-1, both basal (vehicle-treated) and 1,25(OH) D -induced 2 3 OPN promoter activity were significantly enhanced compared with similarly treated vector transfected cells (p  0.05). C, Northern blot anal- ysis of MC3T3-E1 cells plated in 100-mm tissue cul- ture dishes and transfected with vector alone (Vec) or pcDNA3-Hes-1 expression vector (1 g) and treated with vehicle (D) or 1,25(OH) D (D) for 2 3 24 h. Two additional experiments yielded similar results. We propose that these three major pathways, Runx2, 1,25(OH) D , and block of the activation of OPN transcription by Runx2 (Fig. 4A), indi- 2 3 Notch signaling, intersect and play a major role in the regulation of OPN cating that Runx2 can act through this single site in the OPN promoter. in osteoblastic cells and therefore in the process of bone remodeling. This is unlike the regulation of rat OC. The rat OC promoter contains Runx2 was found not only to up-regulate OPN basal promoter activ- two distal Runx2 sites (A and B) and a proximal Runx2 site (C). All three ity but also to enhance 1,25(OH) D -induced OPN transcription. sites are required for maximal OC promoter activity. Mutation of the 2 3 Runx2 has been reported to be essential for osteogenic differentiation proximal site C has the least effect on basal OC promoter activity (15, (31, 32). 1,25(OH) D promotes osteoblastic differentiation and directly 16). Three Runx sites have also been noted in the BSP promoter (38). 2 3 stimulates the production of OC and OPN (33, 34). OPN has been The Runx sites in the BSP promoter mediate repression of BSP (38). reported to be present in preosteoblasts and is present in high concen- 1,25(OH) D also represses BSP expression (39). It has been suggested 2 3 trations in the osteoblast (35, 36). Bone sialoprotein (BSP), another cal- that the context of the multiple Runx2 motifs within a promoter may cium-binding protein present in bone matrix that shares structural fea- contribute to the formation of Runx2 regulatory complexes and second- tures with OPN, is expressed after OPN but earlier than OC in the ary interactions that mediate either repression or activation (38). How- development of the osteoblast phenotype (35, 36). OC is the latest of the ever, previous studies have also indicated, similar to our study, that differentiation markers to be expressed. OC is abundantly expressed in multiple Runx2 sites are not always required for regulation by Runx2. mature osteoblasts (35, 36). These calcium-binding proteins may func- For example, Drissi et al. (40) reported that, although more than one tion in regulating the ordered deposition of mineral (2, 37). Although Runx2 site is present in the Runx2 promoter, a single site within the much work has been done concerning the regulation of OC, we are only proximal promoter is sufficient to confer negative autoregulation. It is of beginning to understand mechanisms involved in the regulation of OPN interest that although the function of all three calcium-binding proteins, and BSP. Two Runx2 sites had previously been suggested in the OPN BSP, OC, and OPN, is associated with ordered deposition of mineral, promoter (at136/130) (23) and on the reverse strand at695/690 OC and OPN are induced by 1,25(OH) D and are positively regulated 2 3 (14). Mutation of the Runx2 site at 136/130 resulted in a complete by Runx2 and BSP is inhibited by 1,25(OH) D , and Runx mediates its 2 3 DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40595 Transcriptional Regulation of Osteopontin FIGURE 7. AML1/ETO inhibition of Hes-1 enhanced OPN transcription. ROS17/2.8 cells were transfected with 0.3 g of mouse OPN promoter and 0.1 g of pcDNA3-Hes1 expression plasmid with or without increasing concentrations of pCMV-AML1/ETO expression plasmid (0.02– 0.2g). Empty vector was used to keep the total DNA concen- tration the same. pRL-TK-Renilla luciferase was cotransfected as an internal control. Cells were treated with vehicle (open bar) or 1,25(OH) D (closed bar) for 24 h. OPN promoter 2 3 activity was expressed as firefly/Renilla luciferase activity and is represented as -fold induction (mean  S.E.; n  3 observations/group) by comparison with basal levels. In the presence of each concentration of AML-1/ETO (0.02– 0.2 g), 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 g of AML-1/ETO, the Hes-1 enhancement of 1,25(OH) D -induced 2 3 OPN transcription was significantly inhibited (p  0.05). FIGURE 9. TSA rescues the AML1/ETO inhibition of the HES-1-enhanced OPN tran- scription. A, ROS17/2.8 cells were transfected with 0.3 g of OPN promoter in the pres- ence or absence of 0.1 g of HES-1 expression vector alone or Hes-1 expression vector and 0.2 g of pCMV-AML1/ETO. After 24 h, cells were treated with 1,25(OH) D (10 M, 2 3 24 h;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/Re- nilla luciferase activity and is represented as -fold induction (mean  S.E.; n  3 obser- vations/group) by comparison with basal levels. B, COS-7 cells were transfected with VDR and were cotransfected with vector or Runx2 and were treated with 1,25(OH) D (10 M, 2 3 24 h). Nuclear extracts were prepared, and 500 g of nuclear protein was used for immu- noprecipitation (IP) with histone deacetylase-1 antibody. Western blot was performed with Hes-1 antibody. The top panel shows the Western blot of cell extracts prior to immu- noprecipitation probed with Hes-1 antibody. FIGURE 8. 1,25(OH) D enhancement of the interaction of Runx2 and Hes-1. A, ROS 2 3 17/2.8 cell nuclear extracts were used for immunoprecipitation (IP) with Runx2 antibody, Hes-1 antibody, or control rabbit IgG. The pulled down protein complex was boiled in cells, indicating the involvement of tissue-specific factors and a cooper- SDS-containing buffer and loaded on a 10% SDS-polyacrylamide gel. Western blot was ative effect of VDR and Runx2 in bone cells. However, in osteoblastic performed using Hes-1 antibody or Runx2 antibody. Treatment with 1,25(OH) D (10 2 3 cells, mutation of the Runx2 sites in the rat OC promoter blocks M, 24 h) increased the interaction between Hes-1 and Runx2. Three additional experi- ments yielded similar results. B, re-ChIP analysis of Hes-1 binding to the OPN promoter. 1,25(OH) D OC transcription (15, 16). The 1,25(OH) D regulation of 2 3 2 3 MC3T3-E1 cells were treated with vehicle or 1,25(OH) D and cross-linked as described in 2 3 rat OC requires a functional Runx2 site B, which is adjacent to the OC the legend to Fig. 5. Lysates were immunoprecipitated first with Runx2 antibody and then with Hes-1 antibody. Eluted DNA was amplified by primers designed according to VDRE (15, 16). In addition, both Runx2 and AP1 binding sites are the Runx2 site. required for parathyroid hormone stimulation of collagenase 3 tran- scription (41, 42). In the collagenase 3 promoter, there is an overlapping repression. Thus, differential regulation by Runx2 and 1,25(OH) D AP1 and Runx2 site, and Runx2 has been reported to interact with c-Fos 2 3 may be needed to regulate the timing of the expression of these proteins and c-Jun (43, 44). It has been suggested that parathyroid hormone-de- and to accommodate their functional role at various stages of osteoblast pendent collagenase 3 expression involves cooperation between Runx2 differentiation. and AP1 transcription factors and the composite Runx2/TRE element Although Runx2 enhanced VDR-mediated OPN transcription, the as well as a distal Runx2 site (43). For the regulation of OPN, the Runx2 Runx2 site was not required for 1,25(OH) D induction of OPN tran- site is not adjacent or overlapping the VDRE (VDRE 757/743; 2 3 scription. Mutation of the Runx2 site in the OPN promoter did not Runx2 site 136/130). It is possible that the Runx2 site in the OPN affect the 1,25(OH) D response in COS-7 cells (Fig. 4B) and resulted in promoter is not critical for 1,25(OH) D regulation of OPN, since the 2 3 2 3 a decreased (but not abolished) 1,25(OH) D response in ROS 17/2.8 Runx2 site is not adjacent or overlapping the VDRE, and that it is the 2 3 40596 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin K. A., Higashio, K., Enomoto, S., Nifuji, A., Rittling, S. R., and Noda, M. (2001) J. Biol. organization of the Runx2 motifs that is important in the regulation of Chem. 276, 13065–13071 gene expression and the responsiveness to physiological regulation. 7. Shapses, S. A., Cifuentes, M., Spevak, L., Chowdhury, H., Brittingham, J., Boskey, In our study, we also examined the effect on OPN transcription of the A. L., and Denhardt, D. T. (2003) Calcif. Tissue Int. 73, 86–92 transcription factor Hes-1, a downstream target of the Notch signaling 8. Asou, Y., Rittling, S. R., Yoshitake, H., Tsuji, K., Shinomiya, K., Nifuji, A., Denhardt, pathway, which is known to bind and modulate the transactivating D. T., and Noda, M. (2001) Endocrinology 142, 1325–1332 9. Christakos, S. (2002) in Principles of Bone Biology (Bilezikian, J. P., Raisz, L. G., and function of Runx2 (20). We found that Hes-1 is able to enhance basal Rodan, G. A., eds) pp. 573–586, Academic Press, San Diego, CA and VDR-mediated OPN transcription and OPN protein expression in 10. Noda, M., Vogel, R. L., Craig, A. M., Prahl, J., DeLuca, H. F., and Denhardt, D. T. the presence of Runx2. Although Hes-1 null mice die during late gesta- (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9995–9999 tion, we found that OPN mRNA (determined by reverse transcription- 11. Rachez, C., and Freedman, L. P. (2000) Gene (Amst.) 246, 9–21 12. Yoshizawa, T., Handa, Y., Uematsu, Y., Takeda, S., Sekine, K., Yoshihara, Y., PCR analysis) is not significantly different in Hes-1 and WT 15-day- / 3 Kawakami, T., Arioka, K., Sato, H., Uchiyama, Y., Masushige, S., Fukamizu, A., Mat- old embryo littermates (n  3 WT and 3 Hes-1 embryos, p  0.5 ; sumoto, T., and Kato, S. (1997) Nat. Genet. 16, 391–396 heterozygote mating pairs were obtained from Dr. Q. Al-Awqati at the 13. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., College of Physicians and Surgeons of Columbia University, and the Bronson, R. T., Gao, Y. H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S., Hes-1 mice were originally generated in the laboratory of R. and Kishimoto, T. (1997) Cell 89, 755–764 14. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, Kageyama (Kyoto University) (45)). It is possible that cell type-specific 747–754 differences in OPN mRNA expression may be observed that were unde- 15. Javed, A., Gutierrez, S., Montecino, M., van Wijnen, A. J., Stein, J. L., Stein, G. S., and tectable using whole embryos or that there may be compensation by Lian, J. B. (1999) Mol. Cell. Biol. 19, 7491–7500 Hes-5 in the regulation of OPN. Hes-5 has been reported to compensate 16. Paredes, R., Arriagada, G., Cruzat, F., Villagra, A., Olate, J., Zaidi, K., van Wijnen, A., Lian, J. B., Stein, G. S., Stein, J. L., and Montecino, M. (2004) Mol. Cell. Biol. 24, for the lack of Hes-1 in studies examining neuronal differentiation (46). 8847–8861 Hes-1 is generally thought to act as a negative regulator (47–50). In 17. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., and Nakanishi, S. (1992) Genes Drosophila, Hes proteins are known to interact with the transcriptional Dev. 6, 2620–2634 corepressor Groucho (51, 52), and mutations that inhibit the Groucho/ 18. 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Chem. 2221–2231 40598 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin *

Shen, Qi; Christakos, Sylvia
Journal of Biological Chemistry , Volume 280 (49) – Dec 9, 2005
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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m504166200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 49, pp. 40589 –40598, December 9, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin Received for publication, April 18, 2005, and in revised form, September 6, 2005 Published, JBC Papers in Press, September 29, 2005, DOI 10.1074/jbc.M504166200 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 Osteopontin (OPN), a glycosylated phosphoprotein that binds aspartate (RGD) integrin binding motifs and promotes attachment of calcium, is present in bone extracellular matrix and has been bone cells to the bone surface through binding to OPN receptors such as reported to modulate both mineralization and bone resorption. the   integrin and CD44 (1–3). OPN has been suggested to be v 3 Targeted disruption in mice of the vitamin D receptor (VDR) or involved in the attachment of osteoclasts during bone resorption, to Runx2 results in marked inhibition of OPN expression in osteo- play a role in osteogenesis by attachment of osteoblasts when they form blasts. In this study, we addressed possible cross-talk between VDR bone matrix, and to act to regulate crystal size during bone mineraliza- and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvita- tion (2). In addition, OPN has been suggested to be a mediator of bone min D (1,25(OH) D ) or Runx2 stimulated OPN transcription remodeling in response to mechanical strain (4). OPN null mice are 3 2 3 (mouse OPN promoter 777/79) 2–3-fold. However, coexpres- resistant to mineral loss and bone resorption upon estrogen deprivation sion of Runx2 and VDR in COS-7 cells and treatment with and have impaired activation of osteoclasts (3, 5–7). Also, vasculariza- 1,25(OH) D resulted in an 8-fold induction of OPN transcription, tion and resorption of bone discs have been reported to be significantly 2 3 indicating for the first time functional cooperation between Runx2 impaired in the absence of OPN (8). Although recent studies using OPN and VDR in the regulation of OPN transcription. In ROS 17/2.8 and null mice have provided new insight into the role of OPN in vivo in bone MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO, metabolism, the factors that affect the regulation of OPN are not yet which acts as a repressor of Runx2, significantly inhibited clearly defined. 1,25-Dihydroxyvitamin D (1,25(OH) D ), the active 3 2 3 1,25(OH) D induction of OPN transcription, OPN mRNA, and 2 3 form of vitamin D, is a major calcitropic hormone involved in calcium protein expression. Both a Runx2 site (136/130) and the vitamin homeostasis (9). One of its functions in bone is to regulate the synthesis D response element (757/743) in the OPN promoter are needed of the bone calcium-binding proteins osteocalcin (OC) and OPN (9). for cooperative activation. Chromatin immunoprecipitation analy- 1,25(OH) D modulates the expression of these genes through tran- 2 3 ses showed that 1,25(OH) D can enhance VDR and Runx2 recruit- 2 3 scriptional regulation. The actions of 1,25(OH) D are mediated 2 3 ment on the OPN promoter, further indicating cooperation through the vitamin D receptor (VDR). Liganded VDR heterodimerizes between these two factors in the regulation of OPN. In osteoblastic with the retinoid X receptor and interacts with a vitamin D response cells, Hes-1, a downstream factor of the Notch signaling pathway, element (VDRE). The VDRE in the mouse OPN promoter (at 757/ was found to enhance basal and 1,25(OH) D -induced OPN tran- 2 3 743) is a perfect direct repeat of the motif GGTTCA spaced by three scription. This enhancement was inhibited by AML-1/ETO, an nucleotides (10). Transcription proceeds through the interaction of inhibitor of Runx2. Immunoprecipitation assays indicated that VDR with coactivators and coregulators, including SRC-1/NcoA1, Hes-1 and Runx2 interact and that 1,25(OH) D can enhance this 2 3 SRC-2/GRIP-1 (GR-interacting protein)/NcoA2, SRC-3/ACTR, and interaction. Taken together, these findings define novel mecha- the multisubunit DRIP (vitamin D receptor-interacting protein) com- nisms involving the intersection of three pathways, Runx2, plex (11). Although a VDRE has been identified in the mouse OPN 1,25(OH) D , and Notch signaling, that play a major role in the 2 3 promoter (10) and VDR null mice show marked inhibition of OPN regulation of OPN in osteoblastic cells and therefore in the process expression in osteoblasts (12), the exact mechanisms, including protein- of bone remodeling. protein and protein-DNA interactions, involved in 1,25(OH) D -regu- 2 3 lated OPN transcription are not well understood. Runx2/Cbfa1 is a member of the runt/Cbfa family of transcription Osteopontin (OPN) is a sialic acid-rich glycosylated phosphopro- factors that was first identified as an osteoblast-specific transcription tein, comprising about 2% of the noncollagenous protein in bone (1, 2). factor and a regulator of osteoblast differentiation (13, 14). Runx2 / OPN is produced by osteoblasts when they form bone matrix (1, 2). mice die shortly after birth and show a complete lack of mineralized OPN is an extracellular matrix protein that contains arginine-glycine- bone tissue (13, 14). Marked decreases in the expression of osteopontin and osteocalcin are observed in Runx2 / mice, indicating the regu- * The costs of publication of this article were defrayed in part by the payment of page lation of these genes by Runx2 (13). Three Runx2 binding motifs have charges. This article must therefore be hereby marked “advertisement” in accordance been identified in the rat OC promoter (15). In addition, Runx2 has been with 18 U.S.C. Section 1734 solely to indicate this fact. shown to play a key role in the 1,25(OH) D regulation of rat OC (15, To whom correspondence should be addressed: Dept. of Biochemistry and Molecular 2 3 Biology, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 16). However, it is not yet known whether a similar cooperation occurs 07103. Tel.: 973-972-4033; Fax: 973-972-5594; E-mail: [email protected]. 2 between VDR and Runx2 in the regulation of OPN. The abbreviations used are: OPN, osteopontin; 1,25(OH) D , 1,25-dihydroxyvitamin D ; 2 3 3 OC, osteocalcin; VDR, vitamin D receptor; FBS, fetal bovine serum; PBS, phosphate- Hes-1 (Hairy and enhancer of split homologue-1), a downstream tar- buffered saline; ChIP, chromatin immunoprecipitation; VDRE, vitamin D response ele- get of the Notch signaling pathway, is a helix-loop-helix transcription ment; hVDR, human VDR; WT, wild type; BSP, bone sialoprotein; Pipes, 1,4-pipera- zinediethanesulfonic acid. factor that has been reported to play a role in developmental processes, DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40589 This is an Open Access article under the CC BY license. Transcriptional Regulation of Osteopontin including 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 con- tribute 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 find- ings define, for the first time, novel mechanisms involving the intersec- tion of Runx2, 1,25(OH) D and Notch signaling that are involved in the 2 3 regulation of the OPN gene. EXPERIMENTAL PROCEDURES Materials—[- P]ATP (3,000 Ci (111 TBq)/mmol), nylon mem- 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 brane, and the enhanced chemiluminescent detection system (ECL) were cotransfected with 0.3 g of mouse OPN promoter firefly luciferase construct were purchased from PerkinElmer Life Sciences. Dulbecco’s modified (777/79) and 0.02 g of hVDR expression plasmid in the absence or presence of 0.1 Eagle’s medium plus Ham’s F-12 nutrient mixture, Dulbecco’s modified g of pCMV-Runx2. Empty vectors were used to keep the total DNA concentration the same. Cells were treated with vehicle (D)or10 M 1,25(OH) D (D) for 24 h and 2 3 Eagle’s medium, fetal bovine serum (FBS), and PSN antibiotic mixture harvested, and luciferase activity was determined. The data were normalized to values were purchased from Invitrogen. -Minimal essential medium was pur- for pRL-TK-Renilla luciferase as an internal control. OPN promoter activity (firefly/Renilla luciferase activity) is represented as -fold induction (mean S.E.; n 3–10 observations) chased from Sigma. VDR antiserum (C-20), mouse OPN antiserum and quantitated by comparison with basal levels. 1,25(OH) D treatment or expression 2 3 (P-18), and histone deacetylase-1 antiserum (H-51) were purchased of Runx2 led to a significant increase in OPN promoter activity compared with basal from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Runx2 anti- levels (p  0.05). 1,25(OH) D treatment in the presence of Runx2 led to a significant 2 3 enhancement of OPN promoter activity compared with treatment with 1,25(OH) D 2 3 serum was purchased from Oncogene Research Products (San Diego, (vector (Vec) D) or expression of Runx2 (Runx2 D)(p  0.05). CA). The antiserum reacting to Hes-1 (a gift from T. Sudo, Kamakura, Japan) was produced by immunizing rabbits with a fusion protein con- performed according to the protocol of the manufacturer and normal- sisting of the C-terminal 19 amino acids (SPSSGSSLTSDSMWRP- ized to values for pRL-TK-Renilla luciferase. For all transcription stud- WRN) of mouse Hes-1 coupled to keyhole limpet hemocyanin. 1,25- ies, OPN promoter activity (firefly/Renilla luciferase) is represented as Dihydroxyvitamin D3 was a generous gift from Dr. Milan Uskokovic -fold induction by comparison with basal levels (basal levels refer to (Hoffmann-LaRoche, Nutley, NJ). levels of OPN promoter activity in cells transfected with vector alone Cell Culture—COS-7 African green monkey kidney cells were and treated with vehicle). obtained from the American Type Culture Collection (Manassas, VA) Site-directed Mutagenesis—Mutant mouse OPN promoter (777/ and were cultured in Dulbecco’s modified Eagle’s medium supple- 79) luciferase reporter constructs were generated by site directed mented with 10% FBS. ROS17/2.8 cells (a gift of S. Rodan and G. Rodan mutagenesis using the QuikChange site-directed mutagenesis kit (Merck)) were maintained in Dulbecco’s modified Eagle’s medium/F-12 (Stratagene, La Jolla, CA). The oligonucleotides used to generate the medium supplemented with 5% FBS, 1% PSN. MC3T3-E1 cells (Riken Runx2 mutated site (shown in lowercase) were as follows: 5-CCT TTT Cell Bank, Tsukuba, Japan) were cultured in -minimal essential TTT TTT TTT AAg aAC AAA ACC AGA GGA GG-3 (top strand) medium supplemented with 10% FBS, 1% PSN. All cells were cultured in and 5-CCT CCT CTG GTT TTG Ttc TTA AAA AAA AAA AAA a humidified atmosphere of 95% air, 5% CO at 37 °C. Cells were seeded GG-3 (lower strand). The oligonucleotides used to generate the VDRE at 70–80% confluence 24 h before experiments. Treatments with mutated site (shown in lowercase) were as follows: 5-CAG AGC AAC 1,25(OH) D were performed in medium supplemented with 2% char- AAG Gcc CAC GAG GTT CAC GTC-3 (top strand) and 5-GAC 2 3 coal-stripped serum. GTG AAC CTC GTG ggC CTT GTT GCT CTG-3 (bottom strand). Transient Transfection and Dual Luciferase Assay—The mouse Northern Blot Analysis—ROS17/2.8 cells or MC3T3-E1 cells, plated osteopontin promoter (777/79) firefly luciferase reporter construct at 70% confluence in 100-mm tissue culture dishes, were transfected was kindly provided by D. Denhardt (Rutgers University, Piscataway, using Lipofectamine 2000 reagent, with AML-1/ETO or Hes-1 expres- NJ). pCMV-Runx2 was a gift of G. Karsenty (Baylor College of Medi- sion vector or vector alone. 24 h after transfection, cells were treated for cine, Houston, TX), and pCMV-AML-1/ETO expression vector was 24 h with 1,25(OH) D (10 M) or vehicle control. The treated cells 2 3 from S. W. Hiebert (Vanderbilt University School of Medicine, Nash- were then harvested by trypsinization, pelleted, and washed with PBS. ville, TN). pcDNA3-Hes1 expression vector was a gift from Dr. S. Stifani Total RNA was isolated by RNA-bee RNA extraction solution (Tel- (McGill University, Montreal, Canada). Cells were seeded in a 24-well Test, Friendswood, TX) and precipitated by chloroform and isopropyl culture dish 24 h prior to transfection at 70% confluence. Cells in each alcohol. 20 g of total RNA from each sample was used for Northern well were transfected using Lipofectamine 2000 reagent (Invitrogen) blot analysis as previously described (24). P-Labeled cDNA was pre- according to the manufacturer’s instructions. Empty vectors were used pared using the Random Primers DNA labeling system (Invitrogen) to keep the total DNA concentration the same. Efficiency of transfec- according to the random primer method (25). The mouse osteopontin tion, as assessed by green fluorescent protein cotransfection and subse- cDNA was generated by HindIII digestion and was a gift from D. Den- quent visualization, was estimated at 60–70%. 1,25(OH) D (10 M)or hardt (Rutgers University, Piscataway, NJ). The -actin cDNA was pur- 2 3 TSA (15 nM) was added to cells 24 h post-transfection for another 24 h. chased from Clontech. The blots were hybridized with the P-labeled Cells were washed twice with phosphate-buffered saline (PBS) and har- mouse OPN cDNA probes for 16 h at 42 °C, washed, air-dried and vested by incubating with 1 passive lysis buffer, supplied by the Dual- exposed to Eastman Kodak Co. BIOMAX MR film at 80 °C for 1 day. Luciferase reporter assay kit (Promega). The luciferase activity assay was The same blots were stripped and probed with P-labeled -actin 40590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin FIGURE 2. AML1/ETO inhibits cooperative effects between VDR and Runx2 in COS-7 cells and 1,25(OH) D -induced OPN transcription in 2 3 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 g of mouse OPN pro- moter luciferase construct (777/79) and 0.02 g of hVDR expression vector in the absence or presence of pCMV-Runx2 (0.1 g) 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) D -induced levels of 2 3 OPN transcription even at high concentrations (0.5 g) of pCMV-AML-1/ETO (open bar, vector-trans- fected (Vec) versus vector  AML-1 ETO (p  0.5) and 1,25(OH) D and 1,25(OH) D  AML-1 2 3 2 3 ETO; p  0.5). B, ROS17/2.8 cells, containing endogenous VDR and Runx2, were transfected with 0.3 g of mouse OPN promoter (777/79) in the absence or presence of increasing concen- trations of pCMV-AML1/ETO (0.1, 0.2, 0.3, and 0.5 g). C, repression of 1,25(OH) D induction of OPN 2 3 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)or10 M 1,25(OH) D (closed bar) for 24 h. OPN promoter 2 3 activity is normalized to values for pRL-TK-Renilla luciferase activity as an internal control and is expressed as -fold induction (mean  S.E.; n  3 experiments) by comparison with basal levels. For A–C, each concentration of AML-1/ETO resulted in a significant repression of 1,25(OH) D -induced 2 3 OPN promoter activity (p  0.05). 0.5 g of 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 versus vector (vector-transfected); p 0.05). Although basal levels were decreased by 36 and 56% by 0.5g of AML-1 ETO in ROS 17/2.8 and MC3T3-E1 cells, respectively, 1,25(OH) D induced 2 3 OPN transcription was decreased by 74.2 and 75.0% at 0.5 g of AML-1 ETO, indicating that not only basal but also 1,25(OH) D -induced OPN 2 3 transcription is diminished by AML-1 ETO. In COS-7 cells or in the osteoblastic cells, AML-1/ETO (0.5 g) had no effect on the activity of a thymidine kinase luciferase construct or on 1,25(OH) D -induced rat 2 3 25-hydroxyvitamin D 24(OH)ase transcription (not shown). cDNA. Autoradiograms were analyzed by densitometric scanning using Western blotting detection system (PerkinElmer Life Sciences) was the Dual-Wavelength Flying Spot Scanner. The relative optical density used to detect the antigen-antibody complex. obtained using the OPN probe was divided by the relative optical den- Chromatin Immunoprecipitation (ChIP) Assay—MC3T3-E1 cells sity obtained after probing with -actin to normalize for sample were cultured in -minimal essential medium supplemented with 10% variation. FBS to 95% confluence prior to the experiment and then treated in OPN Western Blot Analysis—MC3T3-E1 cells, plated at 70% conflu- -minimal essential medium supplemented with 2% charcoal-stripped ence in 100-mm tissue culture dishes, were transfected with vector serum under the conditions and for the times indicated. Treated cells alone or pCMV-AML1/ETO and treated with vehicle or 1,25(OH) D were used for the ChIP assay (26, 27). Briefly, cells were first washed with 2 3 (10 M) for 24 h and harvested by trypsinization. For Western blot PBS and subjected to a cross-link reaction with 1% formaldehyde for 15 analysis, 50 g of protein from total cell lysates was loaded onto a 10% min. The cross-link reaction was stopped by adding glycine to a final SDS-polyacrylamide gel and separated by electrophoresis. Protein was concentration of 0.125 M. Cells were washed with ice-cold PBS twice. transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The cells were collected by scraping and lysed sequentially in 5 mM Membranes were incubated overnight at 4 °C with mouse OPN poly- Pipes, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40 and then in 1% SDS, 10 mM clonal antibody (P-18; Santa Cruz Biotechnology) at a 1:1000 dilution in EDTA, 50 mM Tris-HCl, pH 8.1, for 20 min individually. The chromatin PBS containing 5% nonfat milk. The membrane was washed with PBS pellets were sonicated to an average DNA size of 500 bp DNA (assessed and incubated for 1 h with the corresponding secondary antibody con- by 1% agarose gel electrophoresis) using a Fisher model 100 sonic dis- jugated with horseradish peroxidase. The enhanced chemiluminescent membranator at a power setting of 1. The sonicated extract was centri- DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40591 Transcriptional Regulation of Osteopontin FIGURE 3. Suppression of 1,25(OH) D induc- 2 3 tion 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, 4, or 5 g) and treated with 10 M 1,25(OH) D for 24 h. Northern 2 3 blot analysis was performed as indicated under “Experimental Procedures.” Northern blots were hybridized with OPN cDNA followed by -actin cDNA. Upper panel, representative autoradiogram. Lower panel, graphic representation of Northern blot analyses. Data represent the means  S.E. of three independent experiments. In the presence of 1, 4, or 5 g of pCMV-AML-1/ETO both basal (vehicle) and 1,25(OH) D induced levels of OPN 2 3 mRNA were significantly inhibited compared with similarly treated vector-transfected cells (p 0.05). B, Northern blot analysis of OPN mRNA expression in MC3T3-E1 cells transfected with vec- tor alone or 1 g pCMV-AML1/ETO and treated with vehicle or 1,25(OH) D as described for 2 3 ROS17/2.8 cells. A representative autoradiogram is shown. In the presence of AML1/ETO 1,25(OH) D 2 3 induction of OPN mRNA in MC3T3-E1 cells is 54% of the OPN mRNA levels induced by 1,25(OH) D in 2 3 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 g of protein prepared from MC3T3-E1 cells trans- fected with vector alone or 1 g of pCMV-AML1/ ETO and treated with vehicle (D) or 1,25(OH) D 2 3 (10 M)(D) for 24 h. Detection was by immuno- blotting using a polyclonal OPN antibody. Two additional experiments yielded similar results. fuged for 10 min at maximum speed and then diluted into ChIP dilution (pH 7.4), 1.5 mM MgCl ,10mM KCl, 0.5 mM dithiothreitol, phosphatase buffer (16.7 mM Tris-HCl, pH 8.1, 150 mM NaCl, 0.01% SDS, 1.1% Tri- inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, ton X-100, 1.2 mM EDTA). Immunoprecipitations were performed at 2 mg/ml leupeptin, 2 mg/ml aprotinin), and 1% Triton X-100. Nuclei 4 °C overnight with the indicated antibody overnight. After a 1-h incu- were pelleted at 4,000 rpm for 4 min, and cytoplasmic supernatants bation with salmon sperm DNA and bovine serum albumin-pretreated were separated. Nuclei were resuspended in hypertonic buffer contain- Zysorbin (Zymed Laboratories Inc., San Francisco, CA), the precipitates ing 0.42 mM NaCl, 0.2 mM EDTA, 25% glycerol, and the phosphatase were collected by centrifugation. Precipitates were washed sequentially and protease inhibitors indicated above. After a 2-h incubation at 4 °C, in buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, nuclear soluble proteins were collected by centrifuging at 13,000 rpm pH 8.1, 150 mM NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mM for 10 min. Protein concentration of the supernatant was measured by EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), buffer III (0.25 M LiCl, the method of Bradford (28), and aliquots were stored at 80 °C. 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH Immunoprecipitation—To examine the association of Runx2 and 8.1), and TE buffer (10 mM Tris, 1 mM EDTA) twice. The protein-DNA Hes-1 in the presence or absence of 1,25(OH) D coimmunoprecipita- 2 3 was then eluted by using 1% SDS and 0.1 M NaHCO for 15 min twice. tion experiments were done. Nuclear extracts were prepared as indi- Cross-links were reversed by incubating at 65 °C overnight in elution cated above from ROS17/2.8 cells or MC3T3-E1 cells, and protein con- buffer with 0.2 M NaCl. DNA fragments were purified using Qiagen centration was detected by the Bradford method (28). 500 g of each QIAquick PCR purification kits (Valencia, CA) and subjected to PCR preparation was used for immunoprecipitation with the addition of 4 g using the primers designed to amplify fragments of murine osteopontin of Hes-1 antiserum or 4 g of Runx2 antiserum in the presence or promoter VDRE motif (upper, 5-ACC ACC TCT TCT GCT CTA TAT absence of 1,25(OH) D (10 M) for 24 h at 4 °C. Then 30 l of protein 2 3 GGC-3; lower, 5-TGA CAC TTG AAC TAT GCA GCC GC-3) and A-Sepharose 4 Fast Flow Beads (Amersham Biosciences) were added to the primers designed to amplify the Runx2 motif (upper, 5-TTC CGG each sample, and, after further incubation by rotating at 4 °C for 1 h, the GAT TCT AAA TGC AGT CTA-3; lower, 5-CTC CCA GAA TTT immunoprecipitated complex was collected by centrifuging at 3,000 AAA TGC TGG TCC-3). PCR analysis was carried out in the linear rpm for 5 min. The complex was separated by 12% SDS-PAGE and range of DNA amplification. PCR products were resolved in 5% TBE probed with Runx2 antibody or Hes-1 antibody. Immunoprecipitation acrylamide gel and visualized using ethidium bromide staining. DNA experiments were also done as described above using COS-7 cells trans- acquired prior to precipitation was collected and used as the input. 10% fected with VDR and treated with 1,25(OH) D (10 M for 24 h) and 2 3 of input was used for PCR evaluation. cotransfected with vector alone (pCMV) or 2 g of pCMV-Runx2 to In re-ChIP experiments, complexes were eluted by incubation for 30 examine the association of Hes-1 with histone deacetylase-1 in the pres- minat37 °Cin60 l of elution buffer containing 10 mM dithiothreitol. ence or absence of Runx2. For these studies, 500 g of nuclear extract The eluted samples were diluted 50 times with ChIP dilution buffer and was used for immunoprecipitation with the addition of 4 g of histone subjected again to the ChIP procedure with specific antibodies. deacetylase-1 antiserum followed by the addition of protein A-Sepha- Nuclear Extracts—Cells were washed with cold PBS twice, harvested rose 4 Fast Flow Beads incubated and collected by centrifugation as by scraping, pelleted by centrifuging at 4,000 rpm for 4 min. The pellets described above. The complex was separated by 12% SDS-PAGE and were washed and lysed in hypotonic buffer containing 10 mM HEPES probed with Hes-1 antibody. 40592 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin fected with the mouse OPN promoter (777/79; VDRE 757/743) and hVDR and/or Runx2 expression vectors. In 1,25(OH) D -treated 2 3 (10 M 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) D (Fig. 1). Coexpression of Runx2 and VDR and treatment 2 3 with 1,25(OH) D (10 M 24 h) resulted in an 8.3  0.8-fold induction 2 3 of OPN transcription (Fig. 1), suggesting functional cooperation between Runx2 and VDR in the regulation of OPN. The chimeric protein AML-1/ETO can efficiently block Runx2-me- diated transcriptional activation (29). In COS-7 cells, the enhancement of the inductive action by 1,25(OH) D and Runx2 was inhibited by 2 3 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 sig- nificantly diminished the 1,25(OH) D induction of OPN transcription 2 3 (Fig. 2, B and 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) D -induced OPN mRNA 2 3 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) D -induced OPN mRNA (Fig. 3A). Note (Fig. 3A, last two 2 3 bars) that although there is a 50% decrease in basal OPN mRNA, there is a 75% decrease in 1,25(OH) D -induced OPN mRNA. Similar results 2 3 were observed using MC3T3-E1 cells (Fig. 3B). In addition, inhibition of 1,25(OH) D OPN protein expression was also observed in the presence 2 3 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 Acti- vation 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 FIGURE 4. Functional cooperation between VDR and Runx2 requires both the VDRE assays indicated, similar to previous studies (23), that 136/130 in 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 the mouse OPN promoter is a binding site for Runx2. To investigate 0.3 g of OPN promoter construct with a mutation in the Runx2 site (OPN-Runx2-Mut) the specific contribution of the VDRE and the Runx2 site to the and 0.02 g of hVDR in the absence or presence of 0.1 g of Runx2 expression vector. C, cooperative activation of OPN transcription, mutant OPN promoter COS-7 cells were transfected with a 0.3-g OPN promoter construct with a mutation in the VDRE site (OPN-VDRE-Mut) and 0.02g of hVDR in the absence or presence of 0.1g constructs were generated with either the Runx2 site (136/130) of Runx2 expression vector. The total DNA content was kept constant by the addition of mutated or the VDRE (757/743) mutated (Fig. 4A). Mutation of empty vector. COS-7 cells were treated with vehicle (open bar) or 1,25(OH) D (10 M) 2 3 (closed bar). OPN promoter activity (normalized to values for pRL-TK-Renilla luciferase the Runx2 site did not affect the induction by 1,25(OH) D of OPN 2 3 activity as an internal control) is expressed as -fold induction (mean  S.E.; n  3–10 transcription in VDR-transfected COS-7 cells (Fig. 4B, vector-trans- observations/group) by comparison with basal levels. fected, vehicle- and 1,25(OH) D -treated) and resulted in a 2 3 decreased (but not abolished) 1,25(OH) D response in ROS 17/2.8 Statistical Analysis—Results are expressed as the mean  S.E., and 2 3 cells (not shown). However, in COS-7 cells Runx2 could no longer significance was determined by analysis with Student’s t test for two- activate OPN transcription (Fig. 4B, Runx2-transfected, vehicle- group comparison or analysis of variance for multiple group treated; p  0.4 compared with vector-transfected vehicle-treated comparison. (lane 3 versus lane 1)), indicating that Runx2 acts through this site in RESULTS the mouse OPN promoter 777/79 and not through additional sites (unlike the regulation of OC by Runx2) (15). Also, mutation of Runx2 Cooperates with VDR in Regulating OPN—Targeted disrup- tion in mice of VDR or Runx2 results in a marked inhibition of OPN the Runx2 site resulted in a loss of the cooperative response (Fig. 4B, expression in osteoblasts (12, 13). In order to address possible cross-talk lane 4; compare with Fig. 1, lane 4). The decrease in the response to between VDR and Runx2 in regulating OPN transcription, studies were 1,25(OH) D in the presence of Runx2 using the OPN promoter with 2 3 done using COS-7 cells (that lack endogenous VDR and Runx2) trans- the mutated Runx2 site may be due to the reported binding of Runx2 DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40593 Transcriptional Regulation of Osteopontin AML-1/ETO resulted in decreased recruitment of Runx2 to the OPN promoter (Fig. 5B). The 1,25(OH) D enhancement of Runx2 as well as 2 3 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 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) D - and Runx2-mediated regulation of 2 3 OPN transcription. In ROS17/2.8 cells and MC3T3-E1 cells, that con- tain endogenous Runx2, transfection of Hes-1 (0.1–1 g) resulted in an enhancement of both basal and 1,25(OH) D -induced OPN transcrip- 2 3 tion (Fig. 6, A and B). Expression of Hes-1 also resulted in an enhance- ment of basal and 1,25(OH) D -induced OPN mRNA expression (Fig. 2 3 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 transfected Runx2, expression of Hes-1 resulted in a repression of 1,25(OH) D -dependent induction 2 3 of OPN transcription, and co-transfection of Runx2 in COS-7 cells reversed the inhibition by Hes-1 (not shown), further suggesting func- tional 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) D can increase this inter- 2 3 action (Fig. 8A), suggesting that 1,25(OH) D may enhance functional 2 3 cooperation between Hes-1 and Runx2 by enhancing Hes-1/Runx2 FIGURE 5. 1,25(OH) D stimulates VDR and Runx2 recruitment to the osteopontin 2 3 promoter in intact cells. A, MC3T3-E1 cells were treated with vehicle or 1,25(OH) D interaction. Similar results were observed using MC3T3-E1 cells (not 2 3 (10 M) for 30 min, and cells were cross-linked by 1% formaldehyde for 15 min. Cross- shown). Further, re-ChIP analysis shows that Runx2 and Hes-1 bind linked cell lysates were subjected to immunoprecipitation with IgG or VDR or Runx2 simultaneously to the OPN promoter (Fig. 8B). antibody (-VDR or -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 Since both Runx2 and Hes-1 can interact with TLE (transducin-like mouse OPN promoter (see “Experimental Procedures”). Analysis of input DNA (0.2%) was enhancer of split) proteins, which can recruit histone deacetylases (20, taken prior to precipitation (INPUT). B, MC3T3-E1 cells were transfected with AML-1 ETO, treated with vehicle or 1,25(OH) D and cross-linked lysates were subjected to immuno- 30), we asked whether inhibition of histone deacetylation may be 2 3 precipitation as described in A. These experiments are representative of three separate involved in the activation by Hes-1. In ROS17/2.8 cells, TSA, a histone experiments performed under the same conditions. CON, control. deacetylase inhibitor, was able to rescue the inhibition by AML-1/ETO of Hes-1-enhanced 1,25(OH) D -induced OPN transcription (Fig. 9A). 2 3 to VDR (16). Runx2, in the presence of a mutated Runx2 site in the In addition, in COS-7 cells, in the absence of transfected Runx2, inhibi- OPN promoter, may bind to VDR, and thus less VDR would be tion of 1,25(OH) D -induced OPN transcription by Hes-1 was reversed 2 3 available for 1,25(OH) D induced transcription. Using the OPN 2 3 in the presence of TSA (not shown). These findings suggest that Hes-1/ promoter construct bearing a mutation in the VDRE, 1,25(OH) D 2 3 Runx2 binding may interfere with Runx2-TLE and Hes-1-TLE interac- was unable to activate the OPN promoter in VDR-transfected COS-7 tions, thus preventing repression, which may be mediated, at least in cells (Fig. 4C, vector-transfected (Vec), 1,25(OH) D -treated) or in 2 3 part, by histone deacetylation. Coimmunoprecipitation studies showed ROS 17/2.8 cells (not shown). However, transfection of COS-7 cells the association of Hes-1 and histone deacetylase-1 and a decrease in this with Runx2 could still result in enhanced OPN transcription, and, association in the presence of Runx2 (Fig. 9B). Taken together, these similar to the mutation of the Runx2 site, the cooperative response findings show that Hes-1 can potentiate VDR-mediated OPN transcrip- was not observed (Fig. 4C). These findings suggest that the Runx2 tion in the presence of Runx2 and define new mechanisms and func- site at 136/130 and the VDRE are essential for cooperative effects tional interactions that are involved in the regulation of OPN and may of Runx2 and VDR in activating mouse OPN transcription. therefore affect the process of bone remodeling. Runx2 and VDR Interact with the OPN Promoter in Intact Osteoblas- tic Cells—In order to further understand mechanisms involved in acti- DISCUSSION vation of OPN transcription, we examined VDR and Runx2 complex formation on the OPN promoter in MC3T3-E1 cells using the ChIP This study describes for the first time cooperative effects between assay and specific antibodies against Runx2 and VDR. The antibodies Runx2, VDR, and Hes-1 in the transcriptional regulation of OPN. Func- tional cooperation was demonstrated between Runx2 and VDR in the were used to precipitate sonicated chromatin cross-links from whole cell lysates after formaldehyde cross-linking of DNA to transcription regulation of OPN transcription, OPN mRNA, and protein expression. factors. DNA was amplified using specific primers directed against the 1,25(OH) D was found to enhance both VDR and Runx2 recruitment 2 3 VDRE or the Runx2 binding region of the OPN promoter. In the PCR on the OPN promoter in vivo, further indicating cooperation between these two factors in the regulation of OPN. Hes-1, a downstream target procedure, the number of cycles was chosen so that the amplification was conducted in the linear range of amplification efficiency. No signal of the Notch signaling pathway, was found to act as an enhancer of basal was detected in the presence of IgG (Fig. 5A). The ChIP analysis showed and 1,25(OH) D -induced OPN transcription and OPN mRNA in the 2 3 that 1,25(OH) D can enhance both VDR and Runx2 recruitment to the presence of Runx2. Coimmunoprecipitation analysis indicated that 2 3 OPN promoter (Fig. 5A). Note that transfection of MC3T3-E1 cells with Hes-1 and Runx2 interact, and 1,25(OH) D enhances this interaction. 2 3 40594 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin FIGURE 6. Hes-1 enhancement of 1,25(OH) D - 2 3 induced osteopontin transcription in osteo- blastic cells. ROS17/2.8 cells (A) or MC3T3-E1 cells (B) were plated in 24-well culture dishes, and cells in each well were transfected with 0.3g of mouse OPN promoter firefly luciferase construct with increasing concentrations of pcDNA3-Hes1 expression vector (0.01, 0.05, and 0.1 g). Empty vector was used to keep the total DNA concentra- tion 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 thymi- dine kinase luciferase construct (not shown). Cells were treated with vehicle or 1,25(OH) D (10 M) 2 3 for 24 h. OPN transactivation was expressed as fire- fly/Renilla luciferase activity and is represented as -fold induction (mean  S.E.; n  3 observations/ group) by comparison with basal levels. In the presence of each concentration of Hes-1, both basal (vehicle-treated) and 1,25(OH) D -induced 2 3 OPN promoter activity were significantly enhanced compared with similarly treated vector transfected cells (p  0.05). C, Northern blot anal- ysis of MC3T3-E1 cells plated in 100-mm tissue cul- ture dishes and transfected with vector alone (Vec) or pcDNA3-Hes-1 expression vector (1 g) and treated with vehicle (D) or 1,25(OH) D (D) for 2 3 24 h. Two additional experiments yielded similar results. We propose that these three major pathways, Runx2, 1,25(OH) D , and block of the activation of OPN transcription by Runx2 (Fig. 4A), indi- 2 3 Notch signaling, intersect and play a major role in the regulation of OPN cating that Runx2 can act through this single site in the OPN promoter. in osteoblastic cells and therefore in the process of bone remodeling. This is unlike the regulation of rat OC. The rat OC promoter contains Runx2 was found not only to up-regulate OPN basal promoter activ- two distal Runx2 sites (A and B) and a proximal Runx2 site (C). All three ity but also to enhance 1,25(OH) D -induced OPN transcription. sites are required for maximal OC promoter activity. Mutation of the 2 3 Runx2 has been reported to be essential for osteogenic differentiation proximal site C has the least effect on basal OC promoter activity (15, (31, 32). 1,25(OH) D promotes osteoblastic differentiation and directly 16). Three Runx sites have also been noted in the BSP promoter (38). 2 3 stimulates the production of OC and OPN (33, 34). OPN has been The Runx sites in the BSP promoter mediate repression of BSP (38). reported to be present in preosteoblasts and is present in high concen- 1,25(OH) D also represses BSP expression (39). It has been suggested 2 3 trations in the osteoblast (35, 36). Bone sialoprotein (BSP), another cal- that the context of the multiple Runx2 motifs within a promoter may cium-binding protein present in bone matrix that shares structural fea- contribute to the formation of Runx2 regulatory complexes and second- tures with OPN, is expressed after OPN but earlier than OC in the ary interactions that mediate either repression or activation (38). How- development of the osteoblast phenotype (35, 36). OC is the latest of the ever, previous studies have also indicated, similar to our study, that differentiation markers to be expressed. OC is abundantly expressed in multiple Runx2 sites are not always required for regulation by Runx2. mature osteoblasts (35, 36). These calcium-binding proteins may func- For example, Drissi et al. (40) reported that, although more than one tion in regulating the ordered deposition of mineral (2, 37). Although Runx2 site is present in the Runx2 promoter, a single site within the much work has been done concerning the regulation of OC, we are only proximal promoter is sufficient to confer negative autoregulation. It is of beginning to understand mechanisms involved in the regulation of OPN interest that although the function of all three calcium-binding proteins, and BSP. Two Runx2 sites had previously been suggested in the OPN BSP, OC, and OPN, is associated with ordered deposition of mineral, promoter (at136/130) (23) and on the reverse strand at695/690 OC and OPN are induced by 1,25(OH) D and are positively regulated 2 3 (14). Mutation of the Runx2 site at 136/130 resulted in a complete by Runx2 and BSP is inhibited by 1,25(OH) D , and Runx mediates its 2 3 DECEMBER 9, 2005• VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40595 Transcriptional Regulation of Osteopontin FIGURE 7. AML1/ETO inhibition of Hes-1 enhanced OPN transcription. ROS17/2.8 cells were transfected with 0.3 g of mouse OPN promoter and 0.1 g of pcDNA3-Hes1 expression plasmid with or without increasing concentrations of pCMV-AML1/ETO expression plasmid (0.02– 0.2g). Empty vector was used to keep the total DNA concen- tration the same. pRL-TK-Renilla luciferase was cotransfected as an internal control. Cells were treated with vehicle (open bar) or 1,25(OH) D (closed bar) for 24 h. OPN promoter 2 3 activity was expressed as firefly/Renilla luciferase activity and is represented as -fold induction (mean  S.E.; n  3 observations/group) by comparison with basal levels. In the presence of each concentration of AML-1/ETO (0.02– 0.2 g), 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 g of AML-1/ETO, the Hes-1 enhancement of 1,25(OH) D -induced 2 3 OPN transcription was significantly inhibited (p  0.05). FIGURE 9. TSA rescues the AML1/ETO inhibition of the HES-1-enhanced OPN tran- scription. A, ROS17/2.8 cells were transfected with 0.3 g of OPN promoter in the pres- ence or absence of 0.1 g of HES-1 expression vector alone or Hes-1 expression vector and 0.2 g of pCMV-AML1/ETO. After 24 h, cells were treated with 1,25(OH) D (10 M, 2 3 24 h;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/Re- nilla luciferase activity and is represented as -fold induction (mean  S.E.; n  3 obser- vations/group) by comparison with basal levels. B, COS-7 cells were transfected with VDR and were cotransfected with vector or Runx2 and were treated with 1,25(OH) D (10 M, 2 3 24 h). Nuclear extracts were prepared, and 500 g of nuclear protein was used for immu- noprecipitation (IP) with histone deacetylase-1 antibody. Western blot was performed with Hes-1 antibody. The top panel shows the Western blot of cell extracts prior to immu- noprecipitation probed with Hes-1 antibody. FIGURE 8. 1,25(OH) D enhancement of the interaction of Runx2 and Hes-1. A, ROS 2 3 17/2.8 cell nuclear extracts were used for immunoprecipitation (IP) with Runx2 antibody, Hes-1 antibody, or control rabbit IgG. The pulled down protein complex was boiled in cells, indicating the involvement of tissue-specific factors and a cooper- SDS-containing buffer and loaded on a 10% SDS-polyacrylamide gel. Western blot was ative effect of VDR and Runx2 in bone cells. However, in osteoblastic performed using Hes-1 antibody or Runx2 antibody. Treatment with 1,25(OH) D (10 2 3 cells, mutation of the Runx2 sites in the rat OC promoter blocks M, 24 h) increased the interaction between Hes-1 and Runx2. Three additional experi- ments yielded similar results. B, re-ChIP analysis of Hes-1 binding to the OPN promoter. 1,25(OH) D OC transcription (15, 16). The 1,25(OH) D regulation of 2 3 2 3 MC3T3-E1 cells were treated with vehicle or 1,25(OH) D and cross-linked as described in 2 3 rat OC requires a functional Runx2 site B, which is adjacent to the OC the legend to Fig. 5. Lysates were immunoprecipitated first with Runx2 antibody and then with Hes-1 antibody. Eluted DNA was amplified by primers designed according to VDRE (15, 16). In addition, both Runx2 and AP1 binding sites are the Runx2 site. required for parathyroid hormone stimulation of collagenase 3 tran- scription (41, 42). In the collagenase 3 promoter, there is an overlapping repression. Thus, differential regulation by Runx2 and 1,25(OH) D AP1 and Runx2 site, and Runx2 has been reported to interact with c-Fos 2 3 may be needed to regulate the timing of the expression of these proteins and c-Jun (43, 44). It has been suggested that parathyroid hormone-de- and to accommodate their functional role at various stages of osteoblast pendent collagenase 3 expression involves cooperation between Runx2 differentiation. and AP1 transcription factors and the composite Runx2/TRE element Although Runx2 enhanced VDR-mediated OPN transcription, the as well as a distal Runx2 site (43). For the regulation of OPN, the Runx2 Runx2 site was not required for 1,25(OH) D induction of OPN tran- site is not adjacent or overlapping the VDRE (VDRE 757/743; 2 3 scription. Mutation of the Runx2 site in the OPN promoter did not Runx2 site 136/130). It is possible that the Runx2 site in the OPN affect the 1,25(OH) D response in COS-7 cells (Fig. 4B) and resulted in promoter is not critical for 1,25(OH) D regulation of OPN, since the 2 3 2 3 a decreased (but not abolished) 1,25(OH) D response in ROS 17/2.8 Runx2 site is not adjacent or overlapping the VDRE, and that it is the 2 3 40596 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 49 •DECEMBER 9, 2005 Transcriptional Regulation of Osteopontin K. A., Higashio, K., Enomoto, S., Nifuji, A., Rittling, S. R., and Noda, M. (2001) J. Biol. organization of the Runx2 motifs that is important in the regulation of Chem. 276, 13065–13071 gene expression and the responsiveness to physiological regulation. 7. Shapses, S. A., Cifuentes, M., Spevak, L., Chowdhury, H., Brittingham, J., Boskey, In our study, we also examined the effect on OPN transcription of the A. L., and Denhardt, D. T. (2003) Calcif. Tissue Int. 73, 86–92 transcription factor Hes-1, a downstream target of the Notch signaling 8. Asou, Y., Rittling, S. R., Yoshitake, H., Tsuji, K., Shinomiya, K., Nifuji, A., Denhardt, pathway, which is known to bind and modulate the transactivating D. T., and Noda, M. (2001) Endocrinology 142, 1325–1332 9. Christakos, S. (2002) in Principles of Bone Biology (Bilezikian, J. P., Raisz, L. G., and function of Runx2 (20). We found that Hes-1 is able to enhance basal Rodan, G. A., eds) pp. 573–586, Academic Press, San Diego, CA and VDR-mediated OPN transcription and OPN protein expression in 10. Noda, M., Vogel, R. L., Craig, A. M., Prahl, J., DeLuca, H. F., and Denhardt, D. T. the presence of Runx2. Although Hes-1 null mice die during late gesta- (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9995–9999 tion, we found that OPN mRNA (determined by reverse transcription- 11. Rachez, C., and Freedman, L. P. (2000) Gene (Amst.) 246, 9–21 12. Yoshizawa, T., Handa, Y., Uematsu, Y., Takeda, S., Sekine, K., Yoshihara, Y., PCR analysis) is not significantly different in Hes-1 and WT 15-day- / 3 Kawakami, T., Arioka, K., Sato, H., Uchiyama, Y., Masushige, S., Fukamizu, A., Mat- old embryo littermates (n  3 WT and 3 Hes-1 embryos, p  0.5 ; sumoto, T., and Kato, S. (1997) Nat. Genet. 16, 391–396 heterozygote mating pairs were obtained from Dr. Q. Al-Awqati at the 13. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., College of Physicians and Surgeons of Columbia University, and the Bronson, R. T., Gao, Y. H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S., Hes-1 mice were originally generated in the laboratory of R. and Kishimoto, T. (1997) Cell 89, 755–764 14. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, Kageyama (Kyoto University) (45)). It is possible that cell type-specific 747–754 differences in OPN mRNA expression may be observed that were unde- 15. Javed, A., Gutierrez, S., Montecino, M., van Wijnen, A. J., Stein, J. L., Stein, G. S., and tectable using whole embryos or that there may be compensation by Lian, J. B. (1999) Mol. Cell. Biol. 19, 7491–7500 Hes-5 in the regulation of OPN. Hes-5 has been reported to compensate 16. Paredes, R., Arriagada, G., Cruzat, F., Villagra, A., Olate, J., Zaidi, K., van Wijnen, A., Lian, J. B., Stein, G. S., Stein, J. L., and Montecino, M. (2004) Mol. Cell. Biol. 24, for the lack of Hes-1 in studies examining neuronal differentiation (46). 8847–8861 Hes-1 is generally thought to act as a negative regulator (47–50). In 17. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., and Nakanishi, S. (1992) Genes Drosophila, Hes proteins are known to interact with the transcriptional Dev. 6, 2620–2634 corepressor Groucho (51, 52), and mutations that inhibit the Groucho/ 18. Matsue, M., Kageyama, R., Denhardt, D. T., and Noda, M. (1997) Bone 20, 329–334 Hes interaction interfere with the ability of the Hes proteins to repress 19. McLarren, K. W., Theriault, F. M., and Stifani, S. (2001) J. Biol. Chem. 276, 1578–1584 transcription (50). The mammalian homolog of Groucho, TLE, associ- 20. McLarren, K. W., Lo, R., Grbavec, D., Thirunavukkarasu, K., Karsenty, G., and Stifani, ates with Hes-1. It has been suggested that TLE can mediate transcrip- S. (2000) J. Biol. Chem. 275, 530–538 tional repression by Hes-1 by recruiting histone deacetylases (20, 30). 21. Jimenez, G., Pinchin, S. M., and Ish-Horowicz, D. (1996) EMBO J. 15, 7088–7098 However, Runx2 also interacts with Hes-1, and Hes-1 and Runx2 were 22. Tsai, C., and Gergen, P. (1995) Development 121, 453–462 23. Sato, M., Morii, E., Komori, T., Kawahata, H., Sugimoto, M., Terai, K., Shimizu, H., reported to colocalize to the nuclear matrix in osteoblastic cells (19). Yasui, T., Ogihara, H., Yasui, N., Ochi, T., Kitamura, Y., Ito, Y., and Nomura, S. (1998) TLEs also associate with the nuclear matrix (53). Our transcription Oncogene 17, 1517–1525 assay results as well as the coimmunoprecipitation studies in the pres- 24. Barletta, F., Freedman, L. P., and Christakos, S. (2002) Mol. Endocrinol. 16, 301–314 ence or absence of Runx2 suggest that Runx2/Hes-1 binding may inhibit 25. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and the Hes-1/TLE interaction and therefore the association with histone Struhl, K. (2005) Current Protocol in Molecular Biology, pp. 6.3.1–6.3.4, John Wiley & Sons, Inc., New York deacetylases, thereby acting as a negative regulator of the inhibitory 26. Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000) Cell 103, 843–852 activity of Hes1. Runx2 also interacts with TLE (20), and Hes-1 binding 27. Yamamoto, H., Shevde, N. K., Warrier, A., Plum, L. A., DeLuca, H. F., and Pike, J. W. to Runx2 may also interfere with this interaction. It has been reported (2003) J. Biol. Chem. 278, 31756–31765 that the binding of Hes-1 to Runx2 potentiates Runx2-mediated trans- 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254 29. Meyers, S., Lenny, N., and Hiebert, S. W. (1995) Mol. Cell. Biol. 15, 1974–1982 activation of OC by interfering with TLE-mediated transcriptional 30. Choi, C. Y., Kim, Y. H., Kwon, H. J. and Kim, Y. (1999) J. Biol. Chem. 274, repression (20). In our study, we found that 1,25(OH) D enhances 2 3 33194–33197 Runx2/Hes-1 binding. Thus the enhancement of the interaction of 31. Franceschi, R. T., and Xiao, G. (2003) J. Cell. Biochem. 88, 446–454 Runx2 with Hes-1 may be an additional mechanism involved in the 32. Karsenty, G. (2000) Semin. Cell Dev. Biol. 11, 343–346 activation of OPN transcription by 1,25(OH) D in osteoblastic cells. 33. van Driel, M., Pols, H. A., and van Leeuwen, J. P. (2004) Curr. Pharm. Des. 10, 2 3 2535–2555 These findings define novel mechanisms involving the intersection of 34. Christakos, S., Dhawan, P., Liu, Y., Peng, X., and Porta, A. (2003) J. Cell. Biochem. 88, Runx2, 1,25(OH) D , and Notch signaling that are involved in the reg- 2 3 695–705 ulation of OPN. Our findings suggest that VDR-mediated transcrip- 35. Aubin, J. E., Liu, F., Malaval, L., and Gupta, A. K. (1995) Bone 17, Suppl. 2, 77–83 tional regulation of OPN is modulated by both Runx2 and Hes-1 and 36. Malaval, L., Modrowski, D., Gupta, A. K., and Aubin, J. E. (1994) J. Cell. Physiol. 158, 555–572 that 1,25(OH) D may have a role in osteoblast differentiation by alter- 2 3 37. Pockwinse, S. M., Wilming, L. G., Conlon, D. M., Stein, G. S., and Lian, J. B. (1992) ing the balance of transcription factors affecting their interaction as well J. Cell. Biochem. 49, 310–323 as their recruitment to the OPN promoter. 38. Javed, A., Barnes, G. L., Jasanya, B. O., Stein, J. L., Gerstenfeld, L., Lian, J. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Dec 9, 2005

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