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J. Biol. Chem., Vol. 278, Issue 26, 23270-23277, June 27, 2003

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Activation of Peroxisome Proliferator-activated Receptor- Inhibits the Runx2-mediated Transcription of Osteocalcin in Osteoblasts^*

Min Jae Jeon  , Jeong Ah Kim , Sung Hee Kwon ¶, Sang Wan Kim , Kyong Soo Park  ||, Sung-Woo Park ¶, Seong Yeon Kim  || and Chan Soo Shin  || **

From the Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea, the Hormone Research Center, Seoul National University Hospital Clinical Research Institute, Seoul 110-744, Korea, the ^||Institute of Endocrinology, Nutrition, and Metabolism, Seoul National University Medical Research Center, Seoul 110-744, Korea, and the ^¶Department of Internal Medicine, College of Medicine, Hallym University, Gyeonggi 431-070, Korea

Received for publication, November 14, 2002 , and in revised form, April 15, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mesenchymal cells are able to differentiate into several distinct cell types, including osteoblasts and adipocytes. The commitment to a particular lineage may be regulated by specific transcription factors. Peroxisome proliferator-activated receptor- (PPAR), acting in conjunction with CCAAT/enhancer-binding protein-, has been suggested as a key regulator of adipogenic differentiation. Previous studies have shown that the activation of PPAR in osteoblasts suppresses osteoblast differentiation and the expression of osteocalcin, an osteoblast-specific protein. However, the mechanism of this inhibition remains unclear. We investigated the effect of PPAR activation on the expression of osteocalcin and analyzed the molecular mechanism. Mouse osteoblastic MC3T3-E1 cells expressed PPAR, which was transcriptionally active, whereas rat osteosarcoma ROS 17/2.8 cells did not. Treatment of MC3T3-E1 osteoblasts and ROS 17/2.8 cells stably transfected with PPAR2 with the PPAR activator 15-deoxy-^12,14-prostaglandin J₂ inhibited the mRNA expression of osteocalcin and Runx2, the latter of which is a key transcription factor in osteoblast differentiation. This decreased expression of osteocalcin and Runx2 was partly explained by the decreased level of Runx2 resulting from the suppressed transcription from the Runx2 promoter. However, in addition to this indirect effect, the activation of PPAR by 15-deoxy-^12,14-prostaglandin J₂ directly suppressed the Runx2-mediated induction of the activities of the osteocalcin promoter and the artificial promoter p6OSE2, which contains six tandem copies of osteoblast-specific element-2, the Runx2-binding promoter sequence. This inhibition was mediated by a physical interaction between PPAR and Runx2 and the subsequent repression of the transcriptional activity at the osteoblast-specific element-2 sequence. Thus, this study demonstrates that the activation of PPAR inhibits osteocalcin expression both by suppressing the expression of Runx2 and by interfering with the transactivation ability of Runx2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mesenchymal cells are able to differentiate into several distinct cell types, including osteoblasts and adipocytes (1–3). The mechanisms directing the cells along a particular lineage and the suppression of alternative pathways are not well established, although signals derived from the extracellular environment and several key transcription factors have been identified (4–8).

Peroxisome proliferator-activated receptors (PPARs)¹ are a family of ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily (9, 10). PPAR is abundantly expressed in both white and brown adipose tissue and has been known to play a critical role in the regulation of adipocyte differentiation (10). The transfection of fibroblastic cells with PPAR2 and its subsequent activation with ligand have been shown to be sufficient to initiate adipogenesis (11). Moreover, determined myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into mature adipocytes by the ectopic expression of two adipogenic transcription factors, PPAR and CCAAT/enhancer-binding protein- (12). These results suggest that a developmental switch between these highly specialized cell types can be controlled by the expression of key adipogenic transcription factors. A few studies have suggested that PPAR also acts as a molecular switch between the osteogenic and adipogenic pathways. Lecka-Czernik et al. (13) showed that overexpression of PPAR2 in stromal cell lines results in the suppression of Osf2 (osteoblast-specific factor-2)/Runx2, a key transcription factor for osteoblast differentiation (6), and osteoblast-like biosynthetic activity while promoting terminal differentiation into adipocytes. Jackson and Demer (14) also reported that the treatment of MC3T3-E1 cells with high concentrations of PPAR ligands inhibits osteoblast maturation. These studies provide insights into the mechanisms underlying aging-related osteoporosis because a decrease in the number and differentiating potential of bone marrow precursors (15) and an alteration in the shunting of these cells between the osteoblast and adipocyte lineages (16) were demonstrated under this condition.

One of the osteoblast-specific proteins known to be suppressed by PPAR activation is osteocalcin (13, 14). Osteocalcin is an 6-kDa -carboxylated protein and composes up to 15% of the noncollagenous protein of mature bone (17). The expression of osteocalcin is largely restricted to the osteoblasts of bone and the odontoblasts and cementoblasts of teeth (18). The transcriptional control of osteocalcin gene expression has been extensively studied, and two different types of regulation have been identified, viz. hormonal regulation and tissue-specific regulation (19). Hormonal regulation is mediated by vitamin D and glucocorticoid through vitamin D-responsive element (20, 21) and glucocorticoid-responsive element (22), respectively, which are both located in the osteocalcin promoter. Recently, transcriptional and post-transcriptional stimulation by thyroid hormone has been also reported (23). Apart from hormone-responsive cis-acting elements, two osteoblast-specific elements, OSE1 and OSE2, have been identified in the mouse osteocalcin promoter, and these DNA sequences are known to be involved in the regulation of the tissue-specific expression of the osteocalcin gene (24). Similar sequences responsible for cell-specific regulation have also been identified in the rat osteocalcin promoter (25, 26). Of the two identified OSEs, OSE2 binds Runx2 (Cbfa1) (core-binding factor A1)/AML3/Pebp2A, a Runt-related transcription factor that is essential for osteoblast differentiation. Runx2 is the only osteoblast-specific transactivation factor identified to date (6, 27, 28), and an expression vector containing Runx2 has been shown to increase osteocalcin promoter activity through OSE2 (6). Thus, it is tempting to speculate that Runx2 might be a target of the PPAR-mediated suppression of osteocalcin expression observed in previous studies. In this study, we addressed the mechanism through which PPAR activation inhibits osteocalcin gene expression. The activation of PPAR by 15-deoxy-^12,14-prostaglandin J₂ (15-dPGJ₂) in osteoblasts inhibited osteocalcin expression by direct repression of osteocalcin promoter activity as well as an indirect effect through inhibition of Runx2 expression. We present evidence that PPAR interacts with Runx2 and that this leads to the decreased binding of Runx2 to OSE2 of the osteocalcin promoter. These results may help to explain why PPAR activation suppresses osteoblast differentiation and the expression of osteoblast-specific genes from mesenchymal precursors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—15-dPGJ₂, ciglitazone, and antisera to PPAR were purchased from BIOMOL Research Labs Inc. (Plymouth Meeting, PA); troglitazone was from Sankyo (Tokyo, Japan); and fenofibrate was from Sigma. TRI reagent was obtained from Molecular Research Center, Inc. (Cincinnati, OH), and Western blot detection reagents and [-³²P]dCTP were from Amersham Biosciences (Buckinghamshire, UK). Random priming kits and reagents for the luciferase assay were from Promega Corp. (Madison, WI), and nitrocellulose membranes were from Schleicher & Schüll (Dassel, Germany). LipofectAMINE Plus was obtained from Invitrogen, and anti-Cbfa1 (Runx2) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Oligonucleotides were synthesized by Bioneer Corp. (Chungwon, Korea); and unless otherwise indicated, all other chemicals, including tissue culture medium, were from Sigma.

Expression Vectors and Reporter Plasmids—An expression vector for PPAR2, pcDNA3-PPAR2, was constructed by isolating full-length PPAR2 cDNA from pSV-SPORT1-PPAR2 (a kind gift of Dr. Bruce Spiegelman, Harvard Medical School, Boston, MA) by digestion with KpnI and SnaBI and insertion into the KpnI/EcoRV sites of the pcDNA3 vector (Invitrogen). An expression vector for Runx2 (Cbfa1/Osf2), pCMV-Osf2, was obtained from Dr. Patricia Ducy (Baylor College of Medicine, Houston, TX). pCMX-mRXR, an expression vector for mouse retinoid X receptor- (mRXR), was obtained from Dr. David Mangelsdorf (University of Texas, Dallas, TX). The dominant-negative PPAR expression vector was kindly provided by Dr. V. Krishna Chatterjee (University of Cambridge, Cambridge, UK). The mouse osteocalcin II (OG2) promoter-luciferase reporter construct –1.3OG2-Luc, containing a 1.3-kb segment (positions –1316 to +13), has been described previously (29). The p6OSE2-Luc and p6OSE2m-Luc plasmids contain six copies of the wild-type and mutant OSE2 sequences of the osteocalcin promoter, respectively, followed by a minimal promoter, which directs the expression of luciferase (24). The pCbfa1-Luc plasmid contains a 135-bp fragment of the mouse Cbfa1/Runx2 promoter from positions –89 to +46, driving the expression of luciferase, and the OSE2 sites within this Cbfa1/Runx2 promoter segment are mutated in the pCbfa1m-Luc reporter (30). All these reporter plasmids were kindly provided by Dr. Patricia Ducy. PPREx3TK-Luc, containing three copies of the acyl-CoA oxidase PPAR-responsive element (PPRE) upstream of the herpesvirus thymidine kinase promoter, has been described previously (31). The GST-PPAR2 and GST-Runx2 constructs were kindly provided by Dr. Robert Roeder (The Rockefeller University, New York, NY) and Dr. Philip Hinds (Harvard Medical School), respectively.

Cell Culture—The osteogenic sarcoma cell line ROS 17/2.8 was provided by Dr. Roberto Civitelli (Washington University School of Medicine, St. Louis, MO). ROS 17/2.8 cells have been shown to express several osteoblastic features, including the production of osteocalcin and other matrix proteins (32). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Inc., Walkersville, MD). The mouse osteoblastic MC3T3-E1 cells were derived from spontaneously immortalized calvaria cells and represent immature osteogenic cells (33). MC3T3-E1 cells were maintained in DMEM/nutrient mixture F-12 containing 10% FBS. During osteoblast maturation studies, MC3T3-E1 cells were cultured in DMEM/nutrient mixture F-12 containing 10% FBS supplemented with 50 µg/ml ascorbic acid and 10 mM -glycerophosphate. Either 25 µM 15-dPGJ₂ or vehicle was added at confluence and with subsequent medium changes (every 3 days). Murine embryonic mesenchymal C3H10T1/2 cells (American Type Culture Collection, Manassas, VA) are pluripotent cells that retain an immature, fibroblast-like appearance under standard tissue culture conditions. C3H10T1/2 cells were grown in Eagle's basal medium containing 10% FBS. The 3T3-L1 preadipocytic cell line was a kind gift from Dr. Jae Bum Kim (Seoul National University, Seoul, Korea). 3T3-L1 cells were maintained in an immature state by culturing in DMEM supplemented with 20% FBS and 2.0 mM glutamine.

Generation of Stably Transfected Cell Lines—ROS 17/2.8 cells were seeded in p100 dishes (3 x 10⁶ cells/dish) in DMEM/nutrient mixture F-12 containing 10% (v/v) heat-inactivated FBS. After overnight recovery, the cells were transfected with either pcDNA3-PPAR2 or pcDNA3 without insert using LipofectAMINE Plus according to the manufacturer's protocol. Forty-eight hours later, the cells were diluted 10-fold and incubated with DMEM/nutrient mixture F-12 containing 10% (v/v) FBS and 400 µg/ml G418 (Sigma). Two weeks later, drug-resistant colonies were selected and expanded, and the expression of the exogenous gene was confirmed by Northern blot analysis as described below.

Northern Blotting—Total cellular RNA was isolated from cell mono-layers using TRI reagent according to the manufacturer's instructions. Samples (20 µg/lane) were separated on 1% formaldehyde-agarose gels by electrophoresis, blotted onto nylon membranes, and UV-cross-linked. The membranes were then hybridized using ³²P-labeled probes made by the random-primed oligonucleotide method (Label A Gene labeling kit, Promega Corp.) in ULTRAhyb solution (Ambion Inc., Austin, TX) at 42 °C overnight and washed twice with 2x SSC and 0.1% SDS at 42 °C, followed by one high stringency wash with 0.2x SSC and 0.1% SDS at 42 °C for 15 min. The following cDNA probes were used: 1.7-kb EcoRI fragment of mouse Runx2 (6), 470-bp EcoRI-PstI fragment of mouse osteocalcin, 600-bp XbaI-HindIII fragment of PPAR2, and 1.9-kb BamHI fragment of rat -actin. The level of mRNA was quantitated from digitized autoradiographic images using SigmaScan (SPSS Inc., Chicago, IL).

Reverse Transcription-PCR—First-strand cDNA was synthesized from 2 µg of total RNA using a reverse transcription system kit (Promega Corp.). PCR was performed using 2 µl of cDNA, 20 pmol of each primer (synthesized by Bioneer Corp.), 200 µM each dNTP, 1 mM MgCl₂, and 1 unit of Taq polymerase in a 50-µl reaction volume containing 1x Taq polymerase buffer using a PerkinElmer Life Sciences GeneAmp PCR System 2400. Primers 5'-CTCTGTCTCTCTGACCTCACAG-3' (sense) and 5'-GGAGCTGCTGTGACATCCATAC-3' (antisense), 5'-GAGGGCACAAGTTCTATCTGGA-3' (sense) and 5'-GGTGGTCCGCGATGATCTTC-3' (antisense), 5'-ATGGTTGACACAGAGATGCCA-3' (sense) and 5'-ATGCTTTATCCCCACAGAC-3' (antisense), 5'-GGGTGAAACTCTGGGAGATT-3' (sense) and 5'-ATGCTTTATCCCCACAGAC-3' (antisense), and 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TACAGCAACAGGGTGGTGGA-3' (antisense) were used to amplify osteocalcin, Runx2, PPAR1, PPAR2, and glyceraldehyde-3-phosphate dehydrogenase, producing bands of 359, 387, 348, 436, and 451 bp, respectively.

Western Blotting—Cell lysates were prepared by treating cells with lysis buffer (150 mM NaCl, 50 mM Tris-Cl (pH 7.4), 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors (Sigma)). Lysates were sonicated for 20 min on ice and centrifuged at 10,000 x g for 10 min to sediment particulate material. Protein concentrations of the supernatants were measured as described by Lowry et al. (34). SDS-PAGE was performed under reducing conditions on 10% polyacrylamide gels, and the resolved proteins were transferred onto nitrocellulose membranes. Membranes were blocked with 0.1% Tween 20 and Tris-buffered saline containing 2% bovine serum albumin and 3% dry milk at pH 7.4 for 1 h. Polyclonal antibody against PPAR was added, and the incubation was continued for an addition hour. After washing with 0.1% Tween 20 and Tris-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse antibodies for 1 h. After extensive washing, bands were visualized by chemiluminescence using an ECL kit (Amersham Biosciences) according to the manufacturer's instructions.

Transfections and Reporter Assays—Transient transfections were performed in triplicate, and the transfection efficiencies were monitored using pCMV--gal vectors (Promega Corp.) in parallel cultures. For these experiments, osteoblastic cells were plated at high density (3 x 10⁵ cells/well) onto 12-well plates. Appropriate plasmids were transfected into each well using LipofectAMINE Plus following the manufacturer's instructions. Cell lysates (0.25 ml/well) were prepared using the Promega luciferase assay system, and reporter activity was measured using a luminometer (Lumat LB 9507, Berthold, Wildbad, Germany). All luciferase values were normalized against the -galactosidase activities from the cotransfected pCMV--gal plasmid. All values and means ± S.D. are expressed as -fold induction relative to basal promoter activity.

Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared according to the method of Dignam et al. (35). Briefly, cells were washed with ice-cold phosphate-buffered saline and then resuspended in hypotonic lysis buffer containing 20 mM HEPES (pH 8.0), 25% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.3% Triton X-100, 0.6% ammonium sulfate, 1 mM dithiothreitol, and protease inhibitors. The protein concentrations of the nuclear extracts were determined by the Bradford assay (Bio-Rad) using bovine serum albumin as a standard. In vitro translated mouse PPAR2 and mRXR were obtained by transcribing and translating the pcDNA3-PPAR2 and pCMX-mRXR expression plasmids, respectively, using the TNT T7-coupled reticulocyte lysate system (Promega Corp.). Protein concentration was measured using parallel [³⁵S]methionine-labeled reactions.

Oligonucleotide probes corresponding to the OSE2 site in the mouse osteocalcin promoter (5'-GATCCGCTGCAATCACCAACCACAGCA-3') (24) and the optimal consensus PPRE sequence (5'-GATCAGCTACGTGACCTTTGACCTGGT-3') (36) were generated using an oligonucleotide synthesizer (Bioneer Corp.). The complementary oligonucleotides were annealed and labeled with [-³²P]dCTP. The binding reaction was performed by incubating 10 µg of nuclear protein from cultured cells or in vitro translated proteins in 20 mM HEPES (pH 8.0), 25% glycerol, 1.5 mM MgCl₂, 300 mg of bovine serum albumin, and 1 mg of poly(dI-dC) in a final volume of 10 µl for 10 min at 25 °C. The labeled oligonucleotide was added to the reaction mixture and allowed to incubate for an additional 20 min in ice. To prove specific binding of Runx2 to the oligonucleotide, the nuclear lysates were preincubated for 1 h at 4 °C with anti-Cbfa1 (Runx2) antibody prior to the addition of poly(dI-dC) and radiolabeled probe DNA. The samples were electrophoresed on a 4% nondenaturing polyacrylamide gel. The gel was then dried and autoradiographed.

GST Pull-down Analyses—GST fusion proteins were induced in Escherichia coli BL21 for 3 h at 25 °C by the addition of isopropyl-1-thio--D-galactopyranoside (100 µM final concentration) to a 100-ml bacterial culture (A₆₀₀ 0.5). After induction, bacteria were pelleted for 20 min at 3000 x g and resuspended in 20 ml of ice-cold binding buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA). Bacteria were lysed by freeze-thawing for 5 min in liquid nitrogen, followed by thawing for 10 min at 37 °C. The lysis procedure was repeated three times. The freeze-thawed bacteria were then subjected at 4 °C to three 10-s rounds of sonication, and the bacterial debris was pelleted by centrifugation at 15,000 x g for 30 min at 4 °C. Supernatants were stored frozen at –20 °C in 100-µl aliquots until needed. Free GST lysates were prepared in a similar manner from E. coli BL21 transformed with a pGEX-3 vector. Free GST and GST fusion proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's recommendations and dialyzed against binding buffer.

For GST pull-down assays, equal amounts of purified recombinant GST or GST fusion proteins were immobilized on glutathione-Sepharose beads (Amersham Biosciences) and washed four times with 1 ml of wash buffer (20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 1 mM NaF, 2 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride) at 4 °C. ³⁵S-Labeled PPAR2 and Runx2 were synthesized in rabbit reticulocyte lysate by coupled in vitro transcription and translation (TNT T7-coupled reticulocyte lysate system), added to immobilized GST or GST fusion proteins, and incubated for 2 h. After binding, proteins bound to the beads were eluted with elution buffer (10 mM reduced glutathione, 20 mM Tris-HCl (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, and 2 µg/ml aprotinin), and the samples were separated by SDS-PAGE and analyzed by autoradiography.

Immunoprecipitations—MC3T3-E1 cells were lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate containing a mixture of protease inhibitors. Lysates were then precleared for 3 h at 4 °C with protein G-Sepharose (Roche Applied Science, Mannheim, Germany). For immunoprecipitation of endogenous Runx2 from MC3T3-E1 cells, following incubation with goat anti-Cbfa1 (Runx2) antibody or an isotype-matched control (anti-thyroglobulin antibody), rabbit anti-goat Ig secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used prior to precipitation. PPAR was immunoprecipitated with anti-PPAR anti-body or an isotype-matched control (anti-hemagglutinin antibody, Santa Cruz Biotechnology). The lysates were incubated for 3 h at 4 °C prior to incubation with protein G-Sepharose. After extensive washing, the immunoprecipitates were subjected to SDS-PAGE, and the expression levels of the proteins of interest were verified by Western analyses of the cell lysates using specific antibodies.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
PPAR Is Expressed in MC3T3-E1 Cells, but Not in ROS 17/2.8 Cells—We determined the expression of PPAR in rodent osteoblastic and mesenchymal cell lines. MC3T3-E1 cells represent immature osteoblasts derived from mouse calvarial cells, which undergo osteoblast differentiation in culture (33). ROS 17/2.8 cells are a rat osteosarcoma cell line often used for the study of osteoblast function (32). Western blot analysis revealed that PPAR was expressed in MC3T3-E1 cells, but not in ROS 17/2.8 cells (Fig. 1A). Embryonic mesenchymal C3H10T1/2 cells also expressed PPAR under basal conditions, and the level was increased after treatment with the PPAR activator 15-dPGJ₂. Because PPAR exists as two isoforms (1 and 2) as a result of alternative splicing, we investigated which isoform was expressed in the MC3T3-E1 cells by reverse transcription-PCR. We were able to demonstrate the expression of PPAR1 mRNA in this cell line; however, PPAR2 mRNA was not detectable even up to 35 cycles (data not shown). To determine whether the PPAR expressed in the MC3T3-E1 cells was transcriptionally active, a PPRE cloned upstream of luciferase (PPREx3TK-Luc) was transiently transfected into MC3T3-E1 cells, and the cells were then treated with 15-dPGJ₂. Expression of luciferase activity was significantly induced after 15-dPGJ₂ treatment (Fig. 1B), suggesting that MC3T3-E1 cells express functionally active PPAR.

View larger version (26K): [in this window] [in a new window]   FIG. 1. PPAR expression in osteoblastic cell lines. A, cell lysate proteins (20 µg/lane) from 3T3-L1 preadipocytes and MC3T3-E1, ROS 17/2.8, and C3H10T1/2 cells were loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred to nitrocellulose. C3H10T1/2 cells were treated with either vehicle or 25 µM 15-dPGJ2 for 48 h. The immunoblot was probed with antibody for PPAR. B, MC3T3-E1 cells express functional PPAR. MC3T3-E1 cells were transiently transfected with a PPRE-luciferase (Luc) construct and the pCMV--gal plasmid and treated with the PPAR activator 15-dPGJ2 (25 µM) for 48 h. Cells were then lysed, and luciferase activity was measured. Values normalized for -galactosidase activity are shown as -fold induction relative to basal promoter activity as described under ``Experimental Procedures.'' Values are presented as means ± S.D., and results are representative of three experiments, each performed in triplicate.

PPAR Activators Inhibit Osteocalcin Gene Expression in Rodent Osteoblasts—The possibility that PPAR activators inhibit osteocalcin expression was investigated. ROS 17/2.8 cells, which constitutively express osteocalcin (24), were stably transfected with a PPAR2 expression construct or an empty vector. Exposure of the cells with an empty vector to 15-dPGJ₂ did not alter the level of osteocalcin expression. However, ROS 17/2.8 cells stably transfected with the PPAR2 construct showed a reduced level of osteocalcin mRNA in the presence of 15-dPGJ₂ (Fig. 2A). Mouse osteoblastic MC3T3-E1 cells expressed only low levels of osteocalcin under basal conditions, but the induction of osteoblast maturation by -glycerophosphate and ascorbic acid increased the expression of osteocalcin mRNA by severalfold (Fig. 2B). This increment was, however, obliterated in the presence of 15-dPGJ₂ (Fig. 2B). These data support the notion that PPAR activated by 15-dPGJ₂ inhibits the expression of osteocalcin. Because Runx2 is a key transcription factor in the regulation of osteocalcin expression, we investigated the effect of activated PPAR on the expression of Runx2 in ROS 17/2.8 and MC3T3-E1 cells. As shown in Fig. 2 (A and B), Runx2 mRNA levels were reduced when PPAR was activated, in parallel with the changes observed in osteocalcin mRNA levels. To further confirm the role of PPAR activation in the expression of Runx2, we analyzed the luciferase activity in cells transfected with pCbfa1-Luc, which encompassed the –89/+46 segment of the Runx2 promoter harboring three conserved consensus OSE2 elements (30). Transient transfection of pCbfa1-Luc into ROS 17/2.8 cells resulted in constitutive reporter activity, and this activity was reduced in cells cotransfected with the PPAR2 construct in the presence of 15-dPGJ₂ (Fig. 3A). However, cotransfection of the PPAR2 construct and treatment with 15-dPGJ₂ failed to suppress the luciferase activity in cells transfected with pCbfa1m-Luc, which carries mutations in all three OSE elements (Fig. 3A). Likewise, constitutive Runx2 promoter activity in MC3T3-E1 cells was decreased by 15-dPGJ₂ treatment, whereas introducing mutations into all three OSE2 sites resulted in lower reporter activity, which was not repressed by 15-dPGJ₂ (Fig. 3B). Taken together, these results indicate that the activation of PPAR in osteoblasts results in the suppression of osteocalcin expression, which could be attributed to the decreased transcription and expression of Runx2.

View larger version (29K): [in this window] [in a new window]   FIG. 2. PPAR- inhibits the expression of osteocalcin and Runx2 and suppresses transcription from the Runx2 promoter. A, ROS 17/2.8 cells transfected with either PPAR2 or an empty vector were cultured in the presence of 25 µM 15-dPGJ2 or vehicle for 48 h. Total cellular RNA was isolated, and 20 µg of RNA was added to each lane. The Northern blot was probed for PPAR, Runx2, osteocalcin, and -actin. B, shown are the results from reverse transcription-PCR analysis of osteocalcin and Runx2 expression in MC3T3-E1 cells. Cells were cultured in DMEM/nutrient mixture F-12 with 10% FBS and harvested at confluence (first lane) or after induction of osteoblast differentiation with 50 µg/ml ascorbic acid (Asc) and 10 mM -glycerophosphate (-gp) with either vehicle (second lane) or 25 µM 15-dPGJ2 (third lane) for 6 days. The amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for cDNA quality and PCR efficiency in each lane.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Activation of PPAR inhibits the transcription of the Cbfa1/Runx2 promoter. ROS 17/2.8 (A) or MC3T3-E1 (B) cells were transfected with the pCbfa1-Luc or pCbfa1m-Luc reporter plasmid as indicated. Cells were treated with 15-dPGJ2 (25 µM) or vehicle 24 h after transfection. Forty-eight hours after transfection, the cells were harvested, and luciferase activity was measured. Luciferase activity values were normalized for transfection efficiency against -galactosidase activity from the cotransfected pCMV--gal plasmid. All values are expressed as -fold induction relative to basal promoter activity. Values are presented as means ± S.D., and results are representative of three experiments, each performed in triplicate.

Runx2 Is Essential in the PPAR-mediated Repression of Osteocalcin—Because Runx2 governs the transcription of osteocalcin, we assessed the effect of PPAR activators on transcription from the osteocalcin promoter in the Runx2-expressing osteoblastic cell lines ROS 17/2.8 and MC3T3-E1 and the mesenchymal cell line C3H10T1/2, which does not express Runx2 (6). Transient transfection of –1.3OG2-Luc, which carries one binding site for Runx2 (24), resulted in constitutive reporter activity in ROS 17/2.8 cells, and this transcription was decreased by cotransfection of the PPAR2 construct in the presence of 15-dPGJ₂ (Fig. 4A). The same pattern of suppression of OG2 promoter activity was observed when we treated MC3T3-E1 cells with 15-dPGJ₂ (Fig. 4B). In contrast, 15-dPGJ₂ failed to suppress OG2 promoter activity in C3H10T1/2 cells. In these cells, the transfection of –1.3OG2-Luc showed only a basal level of transactivation, and no effect of 15-dPGJ₂ was observed (Fig. 4C, first two bars). However, cotransfection of Runx2 enhanced transcription from the OG2 promoter, and this induction was inhibited by treatment with 15-dPGJ₂ (Fig. 4C, ?last two bars). These results suggest that the expression of Runx2 is necessary for the inhibitory effect of PPAR activation. To determine whether other PPAR activators also suppress OG2 promoter activity in C3H10T1/2 cells, we cotransfected the cells with the Runx2 and OG2 promoter and added a number of PPAR activators (troglitazone, ciglitazone, and 15-dPGJ₂) and also fenofibrate, a PPAR activator. As shown in Fig. 4D, the OG2 promoter activity was suppressed by treatment of other PPAR activators as well, whereas the PPAR activator fenofibrate showed minimal suppression. This result confirms the notion that the suppression of OG2 promoter activity by 15-dPGJ₂ resulted from the activation of PPAR.

View larger version (29K): [in this window] [in a new window]   FIG. 4. Activation of PPAR inhibits the transcription of the osteocalcin promoter. A and B, ROS 17/2.8 cells (overexpressing PPAR2) and MC3T3-E1 cells, respectively, were transfected with the –1.3OG2-Luc plasmid and treated with either vehicle or 25 µM 15-dPGJ2. C, C3H10T1/2 cells were cotransfected with the Runx2 expression plasmid (pCMV-Osf2/Cbfa1) or an empty control plasmid (pCMV5) and treated with either vehicle or 25 µM 15-dPGJ2. D, C3H10T1/2 cells were transfected with the –1.3OG2-Luc plasmid and treated with vehicle (control (C)), 25 µM troglitazone (Tz), 25 µM ciglitazone (Cg), 25 µM 15-dPGJ2 (Pg), or 25 µM fenofibrate (Ff; a PPAR activator). Luciferase activity was assayed as described in the legend to Fig. 3. Values are presented as means ± S.D., and results are representative of three experiments, each performed in triplicate.

PPAR Decreases the Transcriptional Activity of Runx2— Murine OSE2 is a cis-acting element in the OG2 promoter that specifically binds Runx2, which is particularly critical for the transcriptional activity of osteocalcin (37). To confirm that the inhibition of Runx2 activity is essential in the suppression of osteocalcin by PPAR, we examined the role of this sequence in PPAR-mediated repression. We used an artificial promoter reporter plasmid, p6OSE2-Luc, in which luciferase expression is controlled by six tandem copies of OSE2 (24). In ROS 17/2.8 cells, this promoter showed a high level of transcription, which was inhibited by cotransfection of the PPAR2 construct in the presence of 15-dPGJ₂ (Fig. 5A). Likewise, exposure of MC3T3-E1 cells to 15-dPGJ₂ suppressed the promoter activity (Fig. 5B). However, the suppressive effects of 15-dPGJ₂ were partially blocked by overexpressing a dominant-negative form of PPAR, confirming the role of PPAR in mediating the inhibitory effects of 15-dPGJ₂ (Fig. 5B). We also tested the transcription from p6OSE2m-Luc, in which the Runx2-binding site was mutated to prevent Runx2 binding. As shown in Fig. 5, the mutated promoter markedly decreased reporter activity in both ROS 17/2.8 cells (Fig. 5A) and MC3T3-E1 cells (Fig. 5B) and abolished 15-dPGJ₂-mediated repression. Together, these results strongly suggest that PPAR activation suppresses the transactivation of the osteocalcin promoter by directly affecting the Runx2 binding to OSE2 in the OG2 promoter.

View larger version (28K): [in this window] [in a new window]   FIG. 5. Inhibition of transcription from the OSE2 sequence by the activation of PPAR A and B, ROS 17/2.8 cells (overexpressing PPAR2) and MC3T3-E1 cells, respectively, were transfected with the p6OSE2-Luc or p6OSE2m-Luc plasmid and treated with either vehicle or 25 µM 15-dPGJ2. Some cells received 1 µg of expression vector for dominant-negative PPAR (DN PPAR). C, C3H10T1/2 cells were transfected with increasing amounts of pCMV-Osf2 and assayed for luciferase activity. D, C3H10T1/2 cells were cotransfected with p6OSE2-Luc and 500 ng of pCMV-Osf2 and treated with vehicle or 25 µM 15-dPGJ2. Luciferase activity was assayed as described in the legend to Fig. 3. Values are presented as means ± S.D., and results are representative of three experiments, each performed in triplicate.

As has been noted for –1.3OG2-Luc plasmids in C3H10T1/2 cells, transfection of p6OSE2-Luc resulted in only a low level of luciferase activity in C3H10T1/2 cells in the absence of Runx2 (Fig. 5C). However, cotransfection of the Runx2 expression vector (pCMV-Osf2) enhanced transcription in a dose-dependent manner (Fig. 5C). Treatment of the cells with 15-dPGJ₂ in this setting inhibited Runx2-dependent transcription (Fig. 5D). Mutation of the OSE2 sites in the p6OSE2m-Luc plasmid abolished the induction of transcription by Runx2 as well as PPAR-mediated repression (Fig. 5D). These findings further confirm that the PPAR-mediated repression of osteocalcin promoter activity depends on the expression of Runx2 as well as the presence of an intact OSE2 site, the Runx2-binding sequence in the osteocalcin promoter.

PPAR Physically Associates with Runx2 and Prevents the Binding of Runx2 to OSE2—To examine whether activation of PPAR prevents Runx2 from binding to the OSE2 binding sequence, we evaluated the binding of endogenous Runx2 to the OSE2 DNA sequence in MC3T3-E1 cells using gel shift assays. As shown in Fig. 6, we detected a Runx2·DNA complex in MC3T3-E1 cell lysates (first and fifth lanes), which could be supershifted using anti-Runx2 antibody (sixth lane). However, when we treated these cells with 15-dPGJ₂, Runx2 complex formation decreased in a dose-dependent manner (second through fourth lanes). This complex was not present in C3H10T1/2 cells, which lack Runx2 expression (seventh lane).

View larger version (71K): [in this window] [in a new window]   FIG. 6. The PPAR activator inhibits the binding of Runx2 to the OSE2 site of the osteocalcin promoter. Five micrograms of nuclear protein from MC3T3-E1 cells was incubated with a 32P-labeled oligonucleotide containing the OSE2 site of the osteocalcin promoter. MC3T3-E1 cells were treated with vehicle (first and fifth lanes) or 15-dPGJ2 (5, 10, and 25 µM; second through fourth lanes, respectively) for 4 h. The identity of the Runx2-containing complex was confirmed by its partial disappearance and supershift (SS) after incubation with anti-Runx2 antibody (sixth lane) and its absence in nuclear extracts of C3H10T1/2 cells, which lack Runx2 expression (seventh lane).

To determine the mechanism of the suppression of Runx2 binding to OSE2 by activated PPAR, we examined the possibility of a direct interaction between PPAR and OSE2 in the osteocalcin promoter. A gel shift assay was performed by incubating in vitro translated PPAR·mRXR heterodimer with radiolabeled OSE2 probes. As shown in Fig. 7, we were unable to detect any binding of the PPAR·mRXR heterodimer to OSE2 (fourth lane). This result indicates that the suppression of Runx2-stimulated OG2 activity by PPAR is not derived from the competition between Runx2 and PPAR for binding to the OSE2 site.

View larger version (56K): [in this window] [in a new window]   FIG. 7. Lack of binding of PPAR to the OSE2 site of the osteocalcin promoter. An electrophoretic mobility shift assay was performed on end-labeled PPRE or OSE2 site oligonucleotide in the presence of in vitro translated mRXR (first lane), mouse PPAR2 (mPPAR2; second lane), or both mouse PPAR2 and mRXR (third and fourth lanes). The binding activity of the nuclear extracts from MC3T3-E1 cells with radiolabeled OSE2 oligonucleotides is demonstrated in the fifth lane as a control.

We therefore examined the possibility of a physical interaction between PPAR and Runx2, thereby sequestering Runx2 and preventing it from binding to OSE2. First, we assayed directly the protein interactions in vitro using GST-PPAR2 and in vitro translated Runx2 labeled with [³⁵S]methionine. ³⁵S-Labeled Runx2 associated with GST-PPAR2, but not with GST alone (Fig. 8A, left panel). Conversely, ³⁵S-labeled PPAR2 showed robust interaction with Runx2 (right panel).

View larger version (45K): [in this window] [in a new window]   FIG. 8. PPAR and Runx2 physically interact in vitro and in vivo. A, pull-down experiments were performed to examine the in vitro binding of PPAR and Runx2 using GST-PPAR2 and in vitro translated Runx2 labeled with [35S]methionine (left panel) and GST-Runx2 and in vitro translated PPAR2 labeled with [35S]methionine (right panel). Interacting protein was visualized following gel electrophoresis and autoradiography. B, shown is the in vivo association of endogenous PPAR and endogenous Runx2 in MC3T3-E1 cells as assessed by coimmunoprecipitation. Nuclear extracts from MC3T3-E1 cells were incubated with anti-Runx2, anti-PPAR, or control (Cntr) antibody. Protein A-Sepharose beads were added to all reactions. Proteins bound to the beads were resolved by SDS-PAGE and Western-blotted (WB) with anti-Runx2 (left panel) or anti-PPAR (right panel) antibody. IP, immunoprecipitation.

To further evaluate the ability of endogenous PPAR to interact with endogenous Runx2 in vivo, we performed a coimmunoprecipitation experiment, followed by Western blot analyses. As shown in Fig. 8B (left panel), immunoprecipitation of MC3T3-E1 lysates either with anti-Runx2 or anti-PPAR antibody could pull down Runx2. We also found that immunoprecipitation of Runx2 or PPAR coprecipitated PPAR (right panel). These results suggest that a physical interaction between PPAR and Runx2 perturbs the binding of Runx2 to OSE2, leading to a decrease in the transactivation of the osteocalcin promoter.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
This study demonstrates that the mouse osteoblastic cell line MC3T3-E1 expresses functionally active PPAR and that the activation of PPAR down-regulates Runx2-mediated osteocalcin expression. We found that Runx2, a transcription factor essential for osteoblast differentiation, is a target of inhibition by PPAR. PPAR activation not only inhibited the transcription and abundance of Runx2, but also suppressed the transcriptional activity of Runx2, which was mediated by a physical interaction between Runx2 and PPAR. These results provide a mechanistic basis for the inhibition of osteoblast differentiation and osteoblast gene expression by key molecules in the adipogenic differentiation pathways.

We found that mouse calvarial osteoblastic MC3T3-E1 cells express PPAR, whereas the rat osteosarcoma cell line ROS 17/2.8 does not. Moreover, PPAR expressed in MC3T3-E1 cells could transactivate the PPRE, suggesting a functional role of this nuclear receptor in osteoblasts. Of the two PPAR isoforms, only PPAR1 was detectable by reverse transcription-PCR, confirming the result of Jackson and Demer (14). The absence of PPAR expression in ROS 17/2.8 cells has also been reported previously, whereas significant levels of aP2, a late adipocytic marker, were observed in this cell line (38). In light of the fact that MC3T3-E1 cells represent immature osteoblasts or preosteoblasts, whereas ROS 17/2.8 cells are from an osteosarcoma cell line, these results suggest that PPAR has a role in the early stage of differentiation of osteoblasts as a developmental switch from their mesenchymal progenitors.

As a key transcription factor of adipogenic differentiation, the effect of PPAR activation on the reciprocal inhibition of osteogenic differentiation and on the suppression of osteoblast-specific gene expression has been demonstrated in previous studies (13, 14). However, the mechanism of this inhibition has yet to be characterized. Because Runx2 plays an essential role in osteoblast differentiation and in the expression of osteoblast-specific genes, it would be prudent to consider this molecule as a target of PPAR-mediated inhibition. Runx2, also called Pebp2A or AML3, is a member of the Runt family of transcription factors and has a Runt DNA-binding domain (39). During osteoblast differentiation, mesenchymal cells activate Runx2 expression, whereas ectopic Runx2 expression in mesenchymal cells induces osteoblast differentiation (6). The critical role of Runx2 in osteoblast differentiation and activation of osteoblast gene expression has been demonstrated in a Runx2 knockout mouse model, in which osteoblast differentiation and bone formation were completely lost (40, 41). In addition, Runx2 has been shown to activate osteocalcin gene expression by binding to OSE2, located in the osteocalcin promoter (6). Predicted OSE2 sites are also present in the promoters of other osteoblast differentiation-associated genes such as alkaline phosphatase (42), bone sialoprotein, and 1(I) and 2(I) collagens (43). Moreover, Runx2 regulates positively the activity of its own promoter, which has three conserved consensus OSE2 elements (30). In view of these observations, it is likely that the PPAR-mediated suppression of osteocalcin and Runx2 expression, as demonstrated by our study, results from the inhibition of Runx2 transcription, thus decreasing Runx2 mRNA and protein levels. Furthermore, we have provided evidence that, in addition to the indirect mechanisms, PPAR activation down-regulates osteocalcin expression via its direct effect on Runx2 function. First, 15-dPGJ₂ was found to inhibit osteocalcin promoter activity, and this was mediated by OSE2 present in this promoter. Second, the presence of both Runx2 and an intact OSE2 site was found to be necessary for the PPAR-mediated repression of the osteocalcin promoter. Third, the overexpression of a dominant-negative form of PPAR was found to relieve the suppressive effect of PPAR activators on osteocalcin promoter activity. Finally, we demonstrated an interaction between PPAR and Runx2 in MC3T3-E1 cells in which PPAR-mediated repression was observed, leading to decreased binding of Runx2 to its OSE2 binding sequence. Therefore, we conclude that the PPAR-mediated repression of transcription by Runx2 results from decreased Runx2 binding to the OSE2 sequence. Which of the PPAR isoforms mediates this repression is still unclear. Because both MC3T3-E1 cells (expressing only PPAR1) and ROS 17/2.8 cells (overexpressing PPAR2) showed similar repression of osteocalcin expression upon treatment with 15-dPGJ₂, we believe it is likely that both isoforms are responsible for the repression of Runx2 function.

Although we found evidence of a physical interaction between PPAR and Runx2, the precise mechanism of the repression of osteocalcin transcription is still unclear. Activation of PPAR has been shown to interfere negatively with the NF-B, STAT1 (signal transducer and activator of transcription-1), and AP-1 pathways (44). However, the regulation of PPAR activation is extremely complex, involving heterodimerization with retinoid X receptors, the presence of different coactivators/repressors, and binding to different PPREs (45). Recently, it was also reported that TLE1 (transducin-like enhancer of split-1), a mammalian homolog of Drosophila Groucho, interacts with Runx2 and represses the Runx2-dependent activation of osteocalcin gene transcription (46), suggesting that proteins related to Groucho may play a role in the PPAR-mediated repression of RUNX2.

The evidence presented in this report raises clinical issues regarding aging-related osteoporosis. Previous studies have shown that increases in the number of adipocytes at the expense of a decrease in the number and differentiation potential of osteoblast progenitors lead to suppression of bone formation rate in aging-related osteoporosis (47–49). Moreover, it has been recently shown that the adipose tissue PPAR mRNA level is higher in middle-aged men than in younger individuals (50). If a similar phenomenon is observed in the bone marrow compartment of older men, PPAR would be one of the target molecules of intervention in aging-related osteoporosis. Additional studies will be required to analyze the roles of the PPAR pathway components with respect to osteoblast differentiation and function in vitro and in vivo.

	FOOTNOTES

 
^* This work was supported by Grant 01-PJ1-PG1-01CH08-0001 from the Ministry of Health and Welfare of Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Internal Medicine, Seoul National University College of Medicine, 28 Yungun-Dong, Chongno-Gu, Seoul 110-744, Korea. Tel.: 82-2-760-3734; Fax: 82-2-762-9662; E-mail: csshin{at}plaza.snu.ac.kr.

¹ The abbreviations used are: PPARs, peroxisome proliferator-activated receptors; OSE, osteoblast-specific element; 15-dPGJ₂, 15-deoxy-^12,14 prostaglandin J₂; mRXR, mouse retinoid X receptor-; PPRE, PPAR-responsive element; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.

	ACKNOWLEDGMENTS

 
We sincerely thank Drs. Roberto Civitelli, Su-Li Cheng (Washington University School of Medicine), and Jae Bum Kim for providing the ROS 17/2.8, MC3T3-E1, and 3T3-L1 cells, respectively. We are also grateful to Dr. Patricia Ducy for providing all the reporter plasmids and the Runx2 expression vector, Dr. Bruce Spiegelman for the expression vector for PPAR2, Dr. David Mangelsdorf for the expression vector for mRXR, Dr. V. Krishna Chatterjee for the dominant-negative PPAR construct, Dr. Robert Roeder for GST-PPAR2, and Dr. Philip Hinds for GST-Runx2. We are also indebted to Drs. Roberto Civitelli, Su-Li Cheng, and Fernando Lecanda (School of Medicine, University of Navarra, Spain) for helpful criticism during the preparation of this manuscript.

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