Runx2 expression in bone marrow mesenchymal cell cultures

Bone 33 (2003) 652– 659

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Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPAR␥2 and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures Xudong Li,a,* Quanjun Cui,a Chinghai Kao,c,1 Gwo-Jaw Wang,a,d and Gary Baliana,b b

a Department of Orthopaedic Surgery, University of Virginia, School of Medicine, Charlottesville, VA 22908, USA Department of Biochemistry and Molecular Genetics, University of Virginia, School of Medicine, Charlottesville, VA 22908, USA c Department of Urology, University of Virginia, School of Medicine, Charlottesville, VA 22908, USA d Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan, ROC

Received 8 April 2003; revised 21 June 2003; accepted 24 June 2003

Abstract The mechanism whereby lovastatin can counteract steroid-induced osteonecrosis and osteoporosis is poorly understood. We assessed the effect of lovastatin on a multipotential cell line, D1, which is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPAR␥2, respectively, were determined. 422aP2 gene expression was analyzed. Osteocalcin promoter activity was measured by cotransfecting the cells with the phOC-luc and pSV ␤-Gal plasmids. Lovastatin enhanced osteoblast differentiation as assessed by a 1.8⫻ increase in expression of Cbfa1/Runx2 and by a 5⫻ increase in osteocalcin promoter activity. Expression of PPAR␥2 was decreased by 60%. By enhancing osteoblast gene expression and by inhibiting adipogenesis, lovastatin may shunt uncommitted osteoprogenitor cells in marrow from the adipocytic to the osteoblastic differentiation pathway. Future evaluation of lovastatin and other lipid-lowering drugs will help determine their potential as therapeutic agents for osteonecrosis and osteoporosis. © 2003 Elsevier Inc. All rights reserved. Keywords: Mesenchymal cells; Transcriptional Factors; Statins; Osteoblast; Adipocyte

Introduction Glucocorticoid-dependent bone loss resulting from changes in hormone levels causes clinically significant osteoporosis in approximately 50% of affected individuals [1]. Although changes in calcium metabolism, primary absorption, and effects on nonskeletal tissues contribute to the disease, the mechanism is unclear. In addition, despite recent successes with drugs that inhibit bone resorption [2], there is a need for nontoxic anabolic agents that will in-

* Corresponding author. Orthopaedic Research Laboratory, University of Virginia, School of Medicine, Box 800374, Charlottesville, VA 22908, USA. Fax: ⫹1-434-924-1691. E-mail address: [email protected] (X. Li). 1 Current address: Department of Urology, School of Medicine, Indianapolis, IN 46202. 8756-3282/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S8756-3282(03)00239-4

crease bone formation substantially in people who already suffer from significant bone loss [3]. Bone marrow stroma contains pluripotential cells that can differentiate into myocytes, chondrocytes, osteoblasts, and adipocytes [4 – 6]. Of these, the lineages for osteoblasts and adipocytes are most clearly elucidated and closely aligned. Fat represents 50% of the marrow space, and marrow adipocytes may supply energy for the differentiation and function of other marrow cell phenotypes [7]. Clinical studies have shown an inverse relationship between the amount of trabecular bone and adipose tissue in bone marrow [8,9]. Further, there is a reciprocal relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell culture [10]. Adipocytes isolated from cultures of rabbit bone marrow by limiting dilutions were shown to form bone in diffusion chamber implants [11]. Conversely, the addition of fatty acids to cultures of osteo-

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blastic cells leads to their differentiation into adipocyte-like cells [12]. Our laboratory has demonstrated that single-cell clones of stromal cells can differentiate into either osteoblasts or adipocytes depending on culture conditions [13– 15]. These findings strongly support the hypothesis that osteoblast and adipocyte phenotypes are regulated in a reciprocal process [16,17]. It is likely that interconversion of stromal cells among phenotypes, as well as commitment to a particular lineage with suppression of alternative phenotypes, is governed by specific transcription factors [18]. Indeed, core binding factor a1 (Cbfa1/Runx2) is a transcription factor that is required for commitment of mesenchymal progenitors to the osteoblast lineage [18 –21]. Mice that are deficient in this factor lack osteoblasts and mineralized bone matrix [22]. Expression of Cbfa1/Runx2 in fibroblastic cells induces transcription of osteoblast specific genes [23]. By contrast, peroxisome proliferator activated receptor ␥2 (PPAR␥2) gene expression destines cells to adipocyte differentiation [24,25]. Transfection of fibroblastic cells with PPAR␥2, and subsequent activation with an appropriate ligand, causes the development of adipocytes, supporting the idea that PPAR␥2 plays a crucial role in the differentiation of mesenchymal cells to adipocytes [26]. Most of the interest in lovastatin has centered on its action in the treatment of hypercholestrolemia [27]. Data from our laboratory show that lovastatin improves bone formation and prevents dexamethasone-mediated adipogenesis by mesenchymal cells in vitro [28,29]. However, the molecular basis for these observations has not been elucidated. Therefore, we hypothesized that lovastatin affects the differentiation of osteoblast precursor cells through a mechanism that involves cell lineage specific transcription factors. We tested this hypothesis on a multipotential cell line, D1, and defined the effects of lovastatin on sequential molecular and cellular events during osteoblastic and adipocytic commitment and differentiation of the cell in culture. This commitment to either differentiation pathway was assessed by measuring the expression of Cbfa1/ Runx2 and PPAR␥2 gene expression.

Materials and methods Steroid-induced adipogenesis in culture and treatment with lovastatin Pluripotent mesenchymal cells, D1, which were cloned from Balb/c mouse bone marrow cells, were maintained in Dulbecco’s modified Eagle medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, VT), 50 ␮g/mL sodium ascorbate, and 100 U penicillin G and 100 ␮g of streptomycin per milliliter of culture media, in a humidified atmosphere of 5% carbon dioxide at 37°C. For all experiments, cells were seeded at a density of 5 ⫻ 103 cells/cm2 and the experiments were started when the cells reached 80% confluence.

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D1 cells were treated with 10⫺7 mol/L dexamethasone (Sigma Chemical Company, St. Louis, MO). To examine the effect of a lipid-lowering agent, 1– 6 ␮mol/L lovastatin (Merck & Company, Inc., West Point, PA) was added to the culture medium at the same time as dexamethasone. Cell morphology and the appearance of cytoplasmic lipid droplets were monitored with a phase-contrast microscope. To determine the number of adipocytes, cells in culture were stained with Sudan IV, a stain for fat, and counterstained with hematoxylin, and scanned using a Nikon 35-mm slide scanner (Nikon Inc., Melville, NY). Semiquantitative RT-PCR Semiquantitative RT-PCR was carried out according to the Ambion (Ambion, Austin, TX) protocol based on the Competimer Technique. This method has been validated to be semiquantitative with data that has correlation coefficients of ⫺0.97 [30]. By mixing primers for 18S rRNA with Ambion’s exclusive competimers (primers of the same sequence but that cannot be extended), the 18S rRNA signal can be reduced even to the level of rare messages. It provides semiquantitative data on relative changes of a given mRNA when amplified with the optimal ratio of 18S primers: competimers for a fixed number of PCR cycles within the liner range [31]. RNA (0.5 ␮g) was reverse transcribed in 20 ␮l buffer containing AMV reverse transcriptase 5⫻, 2.5 ␮M poly dT, 1 mM each of dATP, dCTP, dGTP, and dTTP, 20 U of RNAse inhibitor, and 20 U of AMV RT. The reverse transcription reaction was incubated at room temperature for 10 min and then in a Perkin–Elmer Cetus DNA thermal cycler at 42°C for 15 min, 99°C for 5 min, and then at 5°C for 5 min. Aliquots of cDNA were amplified in a 100-␮l PCR reaction mixture which contained 0.3 ␮M 5⬘ and 3⬘ oligoprimers, in high-fidelity PCR buffer containing 15 mM MgCl2, 0.1 nM each of dATP, dCTP, dGTP, and dTTP, 0.35 U of high-fidelity Taq DNA polymerase. For each cDNA sample, PCR amplification was performed in duplicate. The identity of PCR products was confirmed by sequence analysis in an automated DNA sequencer (Perkin– Elmer, Norwalk, CT). The primer sequences for PCR were as follows: for Cbfa1/Runx2: mCbfa1/Runx2 ⫹ 515.F-5⬘ ACG ACA ACC GCA CCA TGG T-3⬘ mCbfa1/Runx2 ⫹ 1382.R-5⬘ CGG CTC TCA GTG AGG GAT G-3⬘ for PPAR2: mPPAR␥2 ⫹ 1111.F-5⬘ CTG GCC TCC CTG ATG AAT AA-3⬘ mPPAR␥2 ⫹ 1315.R-5⬘ GGC GGT CTC CAC TGA GAA TA-3⬘ For mCbfa1/Runx2, amplification by PCR was performed at thermal cycling parameters of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 34 cycles followed by a final extension at 72°C for 7 min. For mPPAR␥2, amplification

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was carried out with 30 cycles of 94°C for 15 s, 50°C for 15 s, and 65°C for 30 s followed by a final extension at 72°C for 2 min. For each PCR product mixture, 5 ␮l was analyzed by electrophoresis in 3% (w/v) agarose gels. The amplified DNA fragments were visualized by staining with ethidium bromide. Photographs were scanned using a ScanJet II cx (Hewlett Packard, CA). Relative quantitative data were obtained using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Quantitative differences were normalized with the QuantumRNA 18S PCR products (Ambion, Austin, TX). RNA isolation and Northern blot analysis Total RNA was prepared from cells in culture using RNeasy (Qiagen, Chatsworth, CA) kit, and RNA concentrations were determined by measuring absorbance at 260 nm. For Northern blot hybridization, 20-␮g RNA samples, denatured in 2.2 M formaldehyde/50% formamide in 1 ⫻ 3-(N-morpholino) propanesulfonic acid buffer at 65°C for 10 min, were separated by electrophoresis on 1.2% agarose gels containing 2.2% formaldehyde/50% formamide in 1 ⫻ 3-(N-morpholino) propanesulfonic acid running buffer (125 V for 2 h), transferred overnight by capillary action onto a Zeta-Probe nylon membrane (Bio-Rad Laboratories, Hercules, CA) in 20⫻ saline sodium phosphate and EDTA buffer (3.6 M NaCl, 0.2 M Na2HPO4 · 7H2O, and 20 mM EDTA), and crosslinked to the membrane by irradiation with ultraviolet light for 1 min. All cDNA probes were labeled with [32P]dCTP using a random primed DNA labeling kit (Boehringer Mannheim, Germany) to at least 1 ⫻ 108 counts per minute. Hybridization was performed at 65°C overnight in a hybridization buffer consisting of 1% bovine serum albumin, 0.25 M Na2HPO47H2O, 7% sodium dodecyl sulfate, 1 mM EDTA, and 100 mg/mL of denatured salmon sperm deoxyribonucleic acid (DNA). Membranes were washed twice for 10 min each in 2 ⫻ 3.6 M saline, 0.2 M sodium phosphate, and 20 mM EDTA– 0.1% sodium dodecyl sulfate at room temperature, then three washes in 0.1 ⫻ 3.6 M saline, 0.2 M sodium phosphate, and 20 mM EDTA– 0.1% sodium dodecyl sulfate with the first two washes of these three washes at room temperature for 10 min each and the final wash for 1 h at 65°C. Membranes were exposed to intensifying screens, which were scanned on a phosphorimager and quantitated using ImageQuant software (Molecular Dynamics). Probes were removed by washing the membrane twice for 1 h each at 70°C with a solution containing 0.25 M Tris, 0.25 M ethylenediameintetraacetic acid, and 20% sodium dodecyl sulfate. The RNA on the membrane was then hybridized with other DNA probes. Transient reporter gene assays D1 cells at a density of 3 ⫻ 105 per well of six-well culture plate were maintained in culture media until they

reached 60 – 80% confluence. Cells were cotransfected with two DNA constructs: (1) human osteocalcin promoter luciferase-fusion plasmid (phOC-luc; 3 ␮g), and (2) pSV ␤-Gal plasmid (0.5 ␮g), using a DOSPER Liposomal Transfection protocol (Roche, Boehringer Mannheim). The cells were transfected with 60 ␮l DOSPER/DNA complexes for 6 h in 1 ml fresh culture medium without serum and maintained in culture for an additional 24 – 48 h in complete growth media. Luciferase activity was measured using a luciferase assay kit (Promega, Madison, WI) and luminescence was detected with an Optocom I luminometer (MGM Instruments, Cambridge, MA). ␤-Galactosidase activity was measured with a commercial kit using a colorimetric assay (Promega, Madison, WI). Luciferase activity was normalized against ␤-galactosidase activity. Statistical methods All experiments were performed in triplicate. Data presented as means ⫾ SD at a significance level of P ⬍ 0.05. Statistical differences between groups were calculated using a Student’s t-test.

Results Counteraction of steroid-induced adipogenesis in D1 cells by lovastatin Upon treatment with 10⫺7 M dexamethasone, the D1 cells accumulated lipid vesicles that were smaller initially and increased in size with time. At Day 7, the cells containing lipid vesicles were distinguishable clearly from the surrounding cells by examination with a phase-contrast microscope (Fig. 1A and B) and after staining with Sudan IV (Fig. 1C and D), a stain that is characteristic of neutral lipids in fat cells. Adipogenic changes were not found in D1 cells in the absence of dexamethasone. Treatment of D1 cells with lovastatin, which was added to culture dishes at the same time as dexamethasone, decreased the appearance of fat cells (Fig. 1) with the maximal effect seen at a concentration of 6 ␮M. Lovastatin-mediated PPAR␥2 down-regulation is concentration and time dependent To determine the effect of different concentrations of lovastatin on PPAR␥2 mRNA, D1 cells were incubated for 48 h with lovastatin concentrations from 0.5 to 10 ␮M. RT-PCR analysis was quantified and is presented graphically in Fig. 2A. At 0.5 ␮M lovastatin, the lowest concentration employed, PPAR␥2 mRNA levels were almost the same as those of untreated control cells. Lovastatin concentrations above 8 ␮M showed cytotoxic effects and 10 ␮M caused most of the cells to detach from the dishes (data not shown). The range between 1 and 6 ␮M lovastatin (concen-

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Fig. 1. Phase-contrast micrographs of D1 cells in culture showing adipogenesis when the cells are treated with dexamethasone. Adipogenesis is diminished while lovastatin is combined with dexamethasone. (A) and (C) After treatment with 10⫺7 M dexamethasone showing accumulation of lipid vesicles; (C) cells stained with Sudan IV. (B) and (D) After combined treatment with 10⫺7 M dexamethasone and 6 ␮M lovastatin showing diminished adipogenesis; (D) cells stained with Sudan IV. (Original magnification, 200.)

trations of 1, 2, 4, and 6 ␮M) decreased PPAR␥2 mRNA levels to 92, 71, 60, and 48% of controls, respectively. To examine the time course effect, RNA was prepared from D1 cells after culture in the presence of 6 ␮M lovastatin for 12 h and 1, 2, and 4 days, and from untreated control cells at the same time point. The RT-PCR analysis shown in Fig. 2B indicates that a decrease in PPAR␥2 mRNA is first noted at 12 h, and it decreases through the final time point of 4 days. At this time PPAR␥2 mRNA decreased to approximately 35% of control levels. Up-regulation of lovastatin-mediated Cbfa1/Runx2 gene expression is concentration and time dependent To determine the dose response of Cbfa1/Runx2 mRNA to lovastatin, D1 cells were incubated for 48 h with lovastatin concentrations from 1 to 6 ␮M. RT-PCR analysis was quantified and is presented graphically in Fig. 3A. At 1 ␮M lovastatin, the Cbfa1/Runx2 mRNA level was essentially

the same as those of untreated control cells. At 6 ␮M lovastatin, the expression of Cbfa1/Runx2 mRNA was approximately 1.6-fold higher than the untreated control cells. To examine the time course of up-regulation of Cbfa1/ Runx2 mRNA, RNA was prepared from D1 cells after culture in the presence of 6 ␮M lovastatin for 12 h and 1, 2, and 4 days, and from untreated control cells at the same time point. The RT-PCR analysis showed that very little increase in Cbfa1/Runx2 mRNA at 12 h, and it increased through the final time point of 4 days to 180% of control levels (Fig. 3B). Lovastatin reverses dexamethasone-mediated upregulation of PPAR␥2, and down-regulation Cbfa1/Runx2 RT-PCR analysis of PPAR␥2 and Cbfa1/Runx2 mRNA levels after incubation of cells for 4 days either with 10⫺7 M dexamethasone (Dex) or 6 ␮M lovastatin (Lov) or with a combination of Dex and Lov is shown in Fig. 4. Cells

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Fig. 2. PPAR␥2 down-regulation by lovastatin in a concentration- and time-dependent manner. Quantitation of the relative PPAR␥2 gene expression of the D1 cells treated with different concentrations of lovastatin (A) for 48 h and 6 ␮M of lovastatin for different time points (B). Graph depicting quantitative data from densitometric scanning of the gel stained with ethidium bromide. Values are based on band density relative to internal control 18S ribosomal RNA band density and plotted as a percentage of the control (zero lovastatin). Error bars represent the standard deviation of triplicate experiments.

treated with dexamethasone for 48 h increased PPAR␥2 mRNA and decreased Cbfa1/Runx2 compared with control (P ⬍ 0.05). PPAR␥2 mRNA levels in lovastatin-treated cells decreased to approximately 36% of that of untreated controls, while Cbfa1/Runx2 expression increased to approximately 180% of that in untreated controls (P ⬍ 0.05). There was no significant difference of PPAR␥2 and Cbfa1/ Runx2 gene expression between cells treated with the combinations of Dex/Lov and the cells that were not treated with either Dex or Lov.

Fig. 3. Concentration and time response of Cbfa1/Runx2 mRNA upregulation by lovastatin. Quantitation of the relative Cbfa1/Runx2 gene expression of the D1 cells treated with different concentrations of lovastatin (A) for 48 h and 6 ␮M of lovastatin for different time points (B). Graph depicting quantitative data from densitometric scanning of the gel stained with ethidium bromide. Values are based on band density relative to internal control 18S ribosomal RNA band density and plotted as a percentage of the control (zero lovastatin). Error bars represent the standard deviation of triplicate experiments.

Lovastatin stimulates OC-promoter activity, but dexamethasone represses it To characterize the osteoblastic transactivation potential of lovastatin, we studied the effect of lovastatin on osteo-

Dexamethasone increases 422(aP2) mRNA, while lovastatin decreases 422(aP2) mRNA To investigate the effect of dexamethasone and lovastatin on the adipose cell specific gene 422aP2, Northern blots were performed on RNA extracted from D1 cells incubated for 4 days with 10⫺7 M Dex, 6 ␮M Lov, and with both Dex and Lov. Dex increased aP2 expression eightfold while lovastatin decrease it to 20% of control (Fig. 5). Cells treated both Dex and Lov showed an approximately twofold increase in 422aP2 mRNA compared with control cells.

Fig. 4. Lovastatin reverses dexamethasone-mediated down-modulation of Cbfa1/Runx2 and up-modulation of PPAR␥2. Quantitation of the relative gene expression of the D1 cells treated with 10⫺7 M dexamethasone, 6 ␮M lovastatin, or both dexamethasone and lovastatin for 4 days. Band densitometry was performed with the use of ImageQuant software. Values are based on band density relative to internal control 18S ribosomal RNA band density. Error bars represent the standard deviation of triplicate experiments. Significance is denoted by an asterisk (*) compared to the control.

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Fig. 5. Lovastatin-dependent decrease of steroid-induced 422(aP2) mRNA. Northern blot analysis of 422(aP2) mRNA. RNA prepared from cells treated with 10⫺7 M dexamethasone (Dex), 6 ␮M lovastatin (Lov), or both Dex and Lov and control cells was transferred to nylon membranes and hybridized using 32P-labeled 422aP2 cDNA. (A) Autoradiogram from hybridization with 422(aP2) cDNA probe. Dex, 10⫺7 M dexamethasone; Lov, 6 ␮M lovastatin; Dex⫹Lov, 10⫺7 M dexamethasone and 6 ␮M lovastatin. (B) Densitometric analysis. Densitometry was performed with the use of ImageQuant software and normalized to 18S ribosomal RNA band density.

calcin promoter activity. Treatment of cells with 6 ␮M lovastatin resulted in a significant increase in osteocalcin promoter activity (P ⬍ 0.01). By contrast, after D1 cells were treated with Dex for 2 days, a marked decrease of osteocalcin promoter activity occurred compared with untreated cells (P ⬍ 0.05) (Fig. 6).

Discussion The D1 cell was cloned from mouse bone marrow and is multipotential with the capacity for osteogenic and adipocytic differentiation in vivo and in vitro [32]. These multipotential characteristics are similar to stem-like cells (mesenchymal stem cells) that give rise to adipocytes and the osteoprogenitors in marrow stroma. Such a multipotential cell has been recognized as a common precursor to adipocytes and osteogenitors, as well as to myoblasts and chondroprogenitors [11]. The pathogenesis of steroid-induced osteoporosis has been intensively investigated but is not completely understood. Consistent with other reports [26,33–35], our results demonstrate that dexamethasone down-regulates the expression of Cbfa1/Runx2 and osteocalcin promoter activity while it increases the expression of PPAR␥2 and the adipocytic specific gene 422aP2. The previously unexplained effects of glucocorticoids on bone loss may result from down-regulation of osteoblast transcription factor expres-

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sion with concomitant up-regulation of the adipocyte transcription factor leading to the differentiation of bone marrow stromal cells along the adipocytic lineage. Osteoporosis occurs because the amount of bone removed from the skeleton by bone-resorbing osteoclasts exceeds that laid down by osteoblasts. Restoring the balance between bone resorption and formation is therefore a key goal of pharmacological intervention in osteoporosis. Inhibitors of osteoclastic bone resorption, such as bisphosphonates, estrogen, or selective estrogen receptor modulators, are already widely used in the treatment and prevention of osteoporosis. These agents reduce the incidence of fractures but lack the ability to replace the amounts of bone, which may be substantial, that have been lost already by the time osteoporosis is detected clinically. Drugs that stimulate new bone formation or inhibit steroid-induced adipogenesis would therefore be a significant alternative to prevent osteoporosis. Statins are commonly prescribed drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme reductase to decrease cholesterol biosynthesis by the liver, thereby reducing serum cholesterol concentrations and lowering the risk of heart attacks [27]. Lovastatin counteracts the effect of dexamethasone on the differentiation of bone marrow precursor cells into adipocytes [28,29]. Lovastatin has been associated with increased bone morphogenetic protein-2 (BMP-2) gene expression [36,37], alkaline phosphatase activity [38,39], matrix mineralization, and enhanced osteogenesis by bone cells in vitro [37,38]. A retrospective study of older women taking lipid-lowering agents demonstrated that statins increase hip bone mineral density and lower the risk of hip fracture, although the effect of statins on osteoporosis was not conclusive at that time [40]. We have demonstrated that upon treatment of multipotential cells in culture, the expression of Cbfa1/Runx2 mRNA increased by 180%, and the activity of the osteocalcin promoter was fivefold higher while lovastatin decreased PPAR␥2 expression by 60% and 422aP2 by 80%. This may

Fig. 6. OC promoter activity. Cells were cotransfected with the osteocalcin promoter luciferase cDNA construct and the ␤-Gal expression plasmid. Incubated for 48 h, before luciferase, ␤-Gal activity was measured. Relative luciferase activity was determined by dividing the luciferase activity by the ␤-Gal activity. Error bars represent the standard deviation of triplicate experiments. Significance is denoted by an asterisk (*) compared to the control.

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partially explain the up-regulation of BMP-2 gene expression and ALP activity upon treatment with lovastatin. The overall effect of lovastatin on stromal precursor cells is to enhance their differentiation into osteoblasts and to inhibit their differentiation to adipocytes. Thus lovastatin may stimulate shunting of uncommitted precursor cells from the adipocyte differentiation pathway into the osteoblast differentiation pathway. Nevertheless, the precise mechanism of action of lovastatin on mesenchymal cells is not known. While we selected lovastatin for our studies because it was available at the time our study began, it may not be the ideal drug to use as a systemic bone-activation agent. Lovastatin was selected for its capacity to lower serum cholesterol, which requires targeting HMG Co-A reductase in liver cells. Thus the concentration of lovastatin in other tissues may be lower than in the liver. The statins that are most effective at preventing marrow mesenchymal cell differentiation to adipocytes would be those that preferentially target bone or bone marrow. Because of the potential effects of lovastatin in enhancing the differentiation of progenitor cells toward the osteoblastic phenotype, it is possible that this or other statins, e.g., simvastatin [37], cerivastatin [41], and pravastatin [37], may have a beneficial effect in the prevention or treatment of osteoporosis. But whether the inhibitory effect on HMG Co-A reductase activity correlates with the effect of statins on bone formation is not clear. An inhibitory effect on bone resorption has been found to correlate directly with the inhibition of HMG Co-A reductase activity [42]. A comparison between different statins and the effect on transcription factors is necessary to establish the relationship between the inhibition of HMG Co-A reductase activity and enhanced bone formation. Our results indicate that lovastatin acts on precursor cells in bone marrow stroma to modulate their differentiation by enhancing osteoblast differentiation, acting at the level of commitment, through increased expression of the Cbfa1/ Runx2 gene and also by increasing activity of the osteocalcin promoter. Concomitantly, lovastatin inhibits adipocyte differentiation, apparently by acting on the expression of fat cell specific genes PPAR␥2 and 422aP2, and subsequent maturation. Thus, our evidence suggests that lovastatin moves uncommitted precursor cells in marrow stroma from the adipocytic to the osteoblastic phenotype.

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