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Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells
BBRC Biochemical and Biophysical Research Communications 308 (2003) 458–462 www.elsevier.com/locate/ybbrc
Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells Chunli Song,a Zhaoqing Guo,a Qingjun Ma,a Zhongqiang Chen,a Zhongjun Liu,a Hongti Jia,b and Gengting Danga,* b
a Department of Orthopedics, Peking University Third Hospital, Beijing 100083, PR China Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Peking University, Beijing 100083, PR China
Received 30 June 2003
Abstract To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p < 0:05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p < 0:05). Lastly, we found that simvastatin induces high expression of BMP2 in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP2 expression in BMSCs. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Simvastatin; Bone marrow stromal cells; Osteoblast; Adipocyte; Cell differentiation; Bone morphogenetic protein 2
The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by an increase in marrow adipose tissue as seen following ovariectomy [1], immobilization [2], treatment with glucocorticoids [3], aging [4], and other conditions that lead to bone loss. Osteogenic and adipogenic cells arise from a common multipotential precursor, bone marrow stromal cells (BMSCs), and a reciprocal relationship exists between adipogenesis and osteogenesis [5–7]. It is possible, therefore, that the inhibition of marrow adipogenesis with a concomitant increase in osteoblastogenesis could provide a therapeutic target with which to either prevent further increase in adipocyte formation or divert existing adipocytes to become more osteoblastic with a resulting increase functional bone cells [8]. It has been shown that statins promote osteoblasts to synthesize bone morphogenic protein 2 (BMP2 ) [9], a * Corresponding author. Fax: +86-10-6201-7700. E-mail address: [email protected] (G. Dang).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01408-6
growth factor that not only causes osteoblasts to differentiate, proliferate, mature, and form new bone in vitro and in vivo, but also inhibits adipocyte differentiation [6,10]. Specifically, lovastatin, an early developed statin, has been shown to inhibit the preadipocytic cells (3T3-L1) to differentiate into adipocytes [11]. Here, we investigated the effect of simvastatin on osteoblastic and adipocytic differentiation, and BMP2 expression in BMSCs in vitro. The information is helpful to our understanding of the stimulatory effect of simvastatin on bone formation.
Materials and methods Cell cultures. Culture medium was DMEM, supplemented with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 10 mM b-glycerophosphate sodium, and 50 lg/mL ascorbate acid sodium (Sigma). Female BALB-C mice (8 weeks) were killed by cervical dislocation, metaphyses of femurs and tibias were cut aseptically, diaphysis cavities were repeatedly flushed with culture medium, and bone marrow cells were collected and plated. Culture medium was replaced every 2–3 days, red blood cells and non-adherent cells were
C. Song et al. / Biochemical and Biophysical Research Communications 308 (2003) 458–462 removed. For adipocyte-like cell induced culture, after 80% confluence, BMSCs were treated with adipogenetic agonist [12] composed of 0.5 lM hydrocortisone and 60 lM indomethacin (HI, Sigma) dissolved in 0.1% DMSO. RNA purification and gene expression analysis by RT-PCR. After BMSCs were treated with HI and simvastatin (Ruibang Pharm, Zhejiang, China) or simvastatin alone for 72 h, total cellular RNA was isolated using Trizol (Gibco-BRL, Life Technologies). Reverse transcription was carried out with the Superscript first-strand synthesis system (Gibco, Life Technologies). Primers for osteocalcin (OCN), lipoprotein lipase (LPL), and b-actin were synthesized based on the reported sequences. OCN (199 bp): forward: 50 -TCTGACAAAGCCT TCATGTCC-30 , reverse: 50 -AAATAGTGATACCGTAGATGCG-30 [13]; LPL (557 bp): forward: 50 -ACTCATCTCCGCCATGCC-30 , reverse: 50 -CCAGCTTTCTCCTAGCAAGG-30 [14]; and b-actin (275 bp): forward: 50 -CAGGAGATGGCCACTGCCGCA-30 , reverse: 50 -TCCTTCTGCATCCTGTCAGCA-30 [15]. Reaction mixtures (50 lL) contained 2 lL cDNA, 50 pmol of each primer, 0.2 lL of 10 mM dNTP, and 1 U Taq-DNA polymerase (TaKaRa Biotechnology). Amplification conditions were as follows: 30 cycles of 94 °C for 45 s; 55 °C for 30 s; and 72 °C for 1 min, followed by a 72 °C incubation for 7 min. The PCR products were detected by 2.0% agarose gel electrophoresis and photographed. Oil Red O staining. When BMSCs reached 80% confluence, cells were treated for 12 days with one of the following: vehicle (DMSO, normal control), HI and rhBMP2 , HI and simvastatin, or HI alone. Adipocyte formation was monitored by Oil Red O staining as described previously [16]. Fluorescence activated cell sorting. BMSCs were treated as above for 12 days. Fluorescence activated cell sorting (FACS) was performed as described previously [10]. Briefly, the monolayer of BMSCs was resuspended in 0.25% trypsin, 0.02% EDTA, and 0.2% collagenase (Sigma) and then fixed with 0.5% paraformaldehyde buffer solution (pH 7.2). Neutral lipids of adipocytes were stained with Nile red at a final concentration of 1 lg/mL. Cells were analyzed on FACScan (Becton– Dickinson, San Jose, CA) and fluorescence emissions were detected between 564 and 604 nm using 582/42 bandpass filter [10,17,18]. Western blot. To detect OCN and BMP2 in supernatants, conditioned medium was collected and concentrated by freeze-dry vacuum centrifugation. After centrifugation for 10 min at 10,000g, 4 °C, the supernatants were collected. Equal amounts of protein were electrophoresed in a 10% SDS–PAGE and then electrotransfered onto a nitrocellulose membrane. After blocking, the membrane was immunoblotted with OCN (a generous gift from L.W. Fisher, Matrix Biochemistry Unit, NIH, USA) or BMP2 antibodies (Santa Cruz Biotechnology), followed by conjugation with horseradish peroxidaseconjugated secondary antibody. Signals were visualized with diaminobenzidine (DAB). To detect osteopontin (OPN) in BMSCs, the cells were lysed in 200 lL of Nonidet P-40 (NP-40) lysis buffer (25 mmol/L Tris, pH 7.4, 10% glycerol, 1% NP-40, 50 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], and 10 mg/mL aprotinin, leupeptin, and pepstatin A). The homogenates were collected and clarified by centrifugation at 14,000 rpm for 5 min at 4 °C, and then analyzed by Western Blot as described above except for the use of anti-mouse OPN (1:100) as primary antibody (Santa Cruz Biotechnology). Alkaline phosphatase (ALP) activity assay. Cells were seeded in 12well plates. After BMSCs were treated with simvastatin or rhBMP2 (Biolink Biological Technology, Beijing, China) for 72 h, BMSCs were collected and sonicated in 400 lL of 10 mM Tris–HCl (pH 7.5) containing 0.1% Triton X-100 for 30 s with a sonifier cell disrupter at 4 °C. Sonicates were centrifuged at 4 °C for 10 min at 10,000g and the supernatants were assayed for ALP activity using an ALP assay kit (Zhongsheng Biochemical, Beijing). Enzyme activity was normalized against the protein concentration and expressed as U/g/L. Statistical analysis. Data were expressed as means SD. Statistical differences among treatment groups were evaluated with analysis of
459
variance (ANOVA), while the difference between values at each point was assessed by post hoc test using Dunnet multiple comparisons. A value of p < 0:05 was considered significant.
Results Effect of simvastatin on osteoblastogenesis of BMSCs After BMSCs were treated with simvastatin for 72 h, expression level of mRNA for OCN (Fig. 1A) and protein for OCN and OPN increased in a concentrationdependent manner (Fig. 2). BMSCs exhibited increased ALP activity in response to simvastatin; ALP activity increased 2–3-fold above basal levels (Fig. 3). Taken together, results showed that treatment of BMSCs with simvastatin induces osteoblastogenesis of BMSCs. Effect of simvastatin on adipogenesis of BMSCs Results showed that treatment of simvastatin inhibits adipocytic differentiation of BMSCs induced by HI in a concentration-dependent manner, while adipocyte decreased by about 51% in the group treated with 1.0 lM
Fig. 1. Effect of simvastatin on osteocalcin (OCN) and lipoprotein lipase (LPL) mRNA expressions in mouse bone marrow stromal cells. OCN and LPL mRNA levels were measured by RT-PCR after cells were induced with simvastatin (0, 0.1, 0.2, 0.5, 1.0, or 2.0 lM) for 72 h with (B) or without (A) adipogenic agonists (0.5 lM hydrocortisone, 60 lM indomethacin: HI).
Fig. 2. Effect of simvastatin on expression of osteocalcin (OCN), osteopontin (OPN), and BMP2 in mouse bone marrow stromal cells. After cells were treated with simvastatin (0, 0.1, 0.2, 0.5, 1.0, or 2.0 lM) for 72 h, supernatants (for determination of OCN and BMP2 ) or cellular homogenate (for determination of OPN) was analyzed by Western blot.
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Fig. 3. Effect of simvastatin or rhBMP2 on ALP activity changes of mouse bone marrow stromal cells. Cells were induced with simvastatin (0, 0.1, 0.2, 0.5, or 1.0 lM) or rhBMP2 (100 ng/mL) for 72 h, ALP activity was assayed. Values are means SD of three determinations from six separate experiments. *p < 0:05, compared with the control group (simvastatin 0 lM).
simvastatin compared to controls that did not receive simvastatin (Figs. 4 and 5). Morphological observation showed that adipocytes existed in clusters, lipid droplets that appeared in the cytoplasm of adipocytes were positive for Oil Red O stain, and there were less lipid droplets seen in simvastatin-treated groups (Fig. 4). After induction with HI and simvastatin for 72 h, the adipocytic phenotype, LPL mRNA level decreased in a concentration-dependent manner (Fig. 1B), further confirming a decrease of adipocyte differentiation. Effects of simvastatin on BMP2 expression in BMSCs Following treatment on BMSCs with simvastatin for 72 h, Western blot revealed that expression of BMP2
Fig. 5. Effect of simvastatin or rhBMP2 on adipogenesis of bone marrow stromal cells. Cultures were induced with vehicle (DMSO, normal control), or adipogenetic agonists (0.5 lM hydrocortisone, 60 lM indomethacin: HI) and rhBMP2 100 ng/mL, or HI and simvastatin, or HI alone for 12 days, adipocyte percentage was determined by FACS. Each point is the mean SD from three separate experiments. Compared with the negative control group (HI + simvastatin 0 lM), *p < 0:05; ***p < 0:001.
increased in a concentration-dependent manner (Fig. 3), suggesting that simvastatin causes high expression of BMP2 in BMSCs as well as osteoblasts.
Discussion Studies have demonstrated that BMSCs can differentiate into multiple cell types, including osteoblasts, myoblasts, chondrocytes, and adipocytes [19,20]. Adipocytic and osteogenic cells are reciprocal cell types that are dominant in marrow. It is thought that changes in the ratios of these cells are involved in bone volume decreases
Fig. 4. Oil Red O staining and fluorescence activated cell sorting (FACS) for adipogenesis of bone marrow stromal cells affected by simvastatin and rhBMP2. Cultures were treated with vehicle (DMSO, normal control), or adipogenetic agonists (0.5 lM hydrocortisone, 60 lM indomethacin: HI) and rhBMP2 100 ng/mL, or HI and simvastatin, or HI alone for 12 days. Adipocyte formation was monitored by Oil Red O staining (upper panel, original magnification: 100) and quantified by FACS (lower panel).
C. Song et al. / Biochemical and Biophysical Research Communications 308 (2003) 458–462
associated with osteoporosis, such as that seen in cases of ovariectomy [1], immobilization [2], treatment with glucocorticoids [3], and age-related osteopenia [4]. One mechanism that may account for the reciprocal relationship between decreased bone density and increased fat formation is an imbalance in the production of osteogenesis and adipogenesis cells in the bone marrow cavity, and an increase in the number of adipocytes occurs at the expense of osteoblasts in osteopenic disorders. Furthermore, mature mammary adipocytes inhibited proliferation of human osteoblastic cells in culture [21]. Inhibition of marrow adipocyte differentiation and a concomitant enhancement of osteoblastogenesis may provide a novel strategy for the treatment of osteoporosis [8]. Mundy et al. [9] discovered the bone-forming potential of statins in the course of screening thousands of chemicals for the ability to synthesize BMP2 in osteoblast. Most recently, Sugiyama et al. [22] and Ohnaka et al. [23] also proved that statins enhance high expression of BMP2 in bone cells. We reasoned that, as osteoblasts arise from BMSCs, simvastatin might also promote the synthesis of BMP2 in BMSCs as well as bone cells, thus enhancing osteoblast differentiation and inhibiting adipocyte differentiation. Although the precise physiological role of ALP in bone is unknown, mature osteoblasts are characterized by high ALP activity, making this a marker of osteoblast differentiation [6,24]. Therefore, we examined the changes of ALP activity in BMSCs exposed to simvastatin and found that it increased significantly. Similarly, two other osteoblastic phenotypes, osteocalcin and osteopontin, also increased in a concentration-dependent manner following the addition of simvastatin. These suggest that simvastatin stimulates osteoblastic differentiation of BMSCs. To determine whether simvastatin might have a corresponding ability to decrease adipocyte formation of bone marrow stromal cells, we detected the adipocyte differentiation using FACS, Oil Red O staining, and changes of the LPL mRNA expression level. We found that simvastatin inhibited BMSCs to differentiate into adipocyte and decrease the expression level of mRNA for LPL, an adipocyte marker in a dose-dependent manner. Gimble et al. [10] reported that rhBMP2 100 ng/ mL could inhibit adipocytic differentiation, but this was not significantly observed in our study. In their study, after bone marrow stromal cell line, BMS2 were treated with adipocyte inducing agents and rhBMP2 for 72 h, fresh medium without adipocyte inducing agents was used, and rhBMP2 was maintained at their initial concentrations, cells were examined for adipocyte differentiation 6 days after the induction of adipocyte differentiation. While in our study, we used primary cultured BMSCs and the adipocyte inducer was used throughout the 12 days. The quality and activity of rhBMP2 may be another reason.
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It has been shown that BMP2 promotes osteoblastic differentiation and inhibits adipogenetic differentiation of bone marrow stromal cells [6,10]. Our results showed that simvastatin induces high expression of BMP2 in BMSCs, indicating that the modulating effect of simvastatin on osteoblastic and adipocytic differentiation of BMSCs may be mediated by up-regulation of BMP2 expression. In summary, we demonstrated that simvastatin promotes osteoblastic differentiation of BMSCs, inhibits adipocytic differentiation, and causes high expression of BMP2 in BMSCs. These may be the mechanisms of the stimulatory effect of simvastatin on bone formation and this effect is at least partially mediated by inducing BMP2 expression in BMSCs.
Acknowledgments The authors thank Professor L.W. Fisher for generous gift of primary antibody. We also thank Dr. Ming Gong and Dr. Shuyan Li for their technical assistance on molecular biology.
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Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells Chunli Song,a Zhaoqing Guo,a Qingjun Ma,a Zhongqiang Chen,a Zhongjun Liu,a Hongti Jia,b and Gengting Danga,* b
a Department of Orthopedics, Peking University Third Hospital, Beijing 100083, PR China Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Peking University, Beijing 100083, PR China
Received 30 June 2003
Abstract To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p < 0:05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p < 0:05). Lastly, we found that simvastatin induces high expression of BMP2 in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP2 expression in BMSCs. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Simvastatin; Bone marrow stromal cells; Osteoblast; Adipocyte; Cell differentiation; Bone morphogenetic protein 2
The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by an increase in marrow adipose tissue as seen following ovariectomy [1], immobilization [2], treatment with glucocorticoids [3], aging [4], and other conditions that lead to bone loss. Osteogenic and adipogenic cells arise from a common multipotential precursor, bone marrow stromal cells (BMSCs), and a reciprocal relationship exists between adipogenesis and osteogenesis [5–7]. It is possible, therefore, that the inhibition of marrow adipogenesis with a concomitant increase in osteoblastogenesis could provide a therapeutic target with which to either prevent further increase in adipocyte formation or divert existing adipocytes to become more osteoblastic with a resulting increase functional bone cells [8]. It has been shown that statins promote osteoblasts to synthesize bone morphogenic protein 2 (BMP2 ) [9], a * Corresponding author. Fax: +86-10-6201-7700. E-mail address: [email protected] (G. Dang).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01408-6
growth factor that not only causes osteoblasts to differentiate, proliferate, mature, and form new bone in vitro and in vivo, but also inhibits adipocyte differentiation [6,10]. Specifically, lovastatin, an early developed statin, has been shown to inhibit the preadipocytic cells (3T3-L1) to differentiate into adipocytes [11]. Here, we investigated the effect of simvastatin on osteoblastic and adipocytic differentiation, and BMP2 expression in BMSCs in vitro. The information is helpful to our understanding of the stimulatory effect of simvastatin on bone formation.
Materials and methods Cell cultures. Culture medium was DMEM, supplemented with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 10 mM b-glycerophosphate sodium, and 50 lg/mL ascorbate acid sodium (Sigma). Female BALB-C mice (8 weeks) were killed by cervical dislocation, metaphyses of femurs and tibias were cut aseptically, diaphysis cavities were repeatedly flushed with culture medium, and bone marrow cells were collected and plated. Culture medium was replaced every 2–3 days, red blood cells and non-adherent cells were
C. Song et al. / Biochemical and Biophysical Research Communications 308 (2003) 458–462 removed. For adipocyte-like cell induced culture, after 80% confluence, BMSCs were treated with adipogenetic agonist [12] composed of 0.5 lM hydrocortisone and 60 lM indomethacin (HI, Sigma) dissolved in 0.1% DMSO. RNA purification and gene expression analysis by RT-PCR. After BMSCs were treated with HI and simvastatin (Ruibang Pharm, Zhejiang, China) or simvastatin alone for 72 h, total cellular RNA was isolated using Trizol (Gibco-BRL, Life Technologies). Reverse transcription was carried out with the Superscript first-strand synthesis system (Gibco, Life Technologies). Primers for osteocalcin (OCN), lipoprotein lipase (LPL), and b-actin were synthesized based on the reported sequences. OCN (199 bp): forward: 50 -TCTGACAAAGCCT TCATGTCC-30 , reverse: 50 -AAATAGTGATACCGTAGATGCG-30 [13]; LPL (557 bp): forward: 50 -ACTCATCTCCGCCATGCC-30 , reverse: 50 -CCAGCTTTCTCCTAGCAAGG-30 [14]; and b-actin (275 bp): forward: 50 -CAGGAGATGGCCACTGCCGCA-30 , reverse: 50 -TCCTTCTGCATCCTGTCAGCA-30 [15]. Reaction mixtures (50 lL) contained 2 lL cDNA, 50 pmol of each primer, 0.2 lL of 10 mM dNTP, and 1 U Taq-DNA polymerase (TaKaRa Biotechnology). Amplification conditions were as follows: 30 cycles of 94 °C for 45 s; 55 °C for 30 s; and 72 °C for 1 min, followed by a 72 °C incubation for 7 min. The PCR products were detected by 2.0% agarose gel electrophoresis and photographed. Oil Red O staining. When BMSCs reached 80% confluence, cells were treated for 12 days with one of the following: vehicle (DMSO, normal control), HI and rhBMP2 , HI and simvastatin, or HI alone. Adipocyte formation was monitored by Oil Red O staining as described previously [16]. Fluorescence activated cell sorting. BMSCs were treated as above for 12 days. Fluorescence activated cell sorting (FACS) was performed as described previously [10]. Briefly, the monolayer of BMSCs was resuspended in 0.25% trypsin, 0.02% EDTA, and 0.2% collagenase (Sigma) and then fixed with 0.5% paraformaldehyde buffer solution (pH 7.2). Neutral lipids of adipocytes were stained with Nile red at a final concentration of 1 lg/mL. Cells were analyzed on FACScan (Becton– Dickinson, San Jose, CA) and fluorescence emissions were detected between 564 and 604 nm using 582/42 bandpass filter [10,17,18]. Western blot. To detect OCN and BMP2 in supernatants, conditioned medium was collected and concentrated by freeze-dry vacuum centrifugation. After centrifugation for 10 min at 10,000g, 4 °C, the supernatants were collected. Equal amounts of protein were electrophoresed in a 10% SDS–PAGE and then electrotransfered onto a nitrocellulose membrane. After blocking, the membrane was immunoblotted with OCN (a generous gift from L.W. Fisher, Matrix Biochemistry Unit, NIH, USA) or BMP2 antibodies (Santa Cruz Biotechnology), followed by conjugation with horseradish peroxidaseconjugated secondary antibody. Signals were visualized with diaminobenzidine (DAB). To detect osteopontin (OPN) in BMSCs, the cells were lysed in 200 lL of Nonidet P-40 (NP-40) lysis buffer (25 mmol/L Tris, pH 7.4, 10% glycerol, 1% NP-40, 50 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], and 10 mg/mL aprotinin, leupeptin, and pepstatin A). The homogenates were collected and clarified by centrifugation at 14,000 rpm for 5 min at 4 °C, and then analyzed by Western Blot as described above except for the use of anti-mouse OPN (1:100) as primary antibody (Santa Cruz Biotechnology). Alkaline phosphatase (ALP) activity assay. Cells were seeded in 12well plates. After BMSCs were treated with simvastatin or rhBMP2 (Biolink Biological Technology, Beijing, China) for 72 h, BMSCs were collected and sonicated in 400 lL of 10 mM Tris–HCl (pH 7.5) containing 0.1% Triton X-100 for 30 s with a sonifier cell disrupter at 4 °C. Sonicates were centrifuged at 4 °C for 10 min at 10,000g and the supernatants were assayed for ALP activity using an ALP assay kit (Zhongsheng Biochemical, Beijing). Enzyme activity was normalized against the protein concentration and expressed as U/g/L. Statistical analysis. Data were expressed as means SD. Statistical differences among treatment groups were evaluated with analysis of
459
variance (ANOVA), while the difference between values at each point was assessed by post hoc test using Dunnet multiple comparisons. A value of p < 0:05 was considered significant.
Results Effect of simvastatin on osteoblastogenesis of BMSCs After BMSCs were treated with simvastatin for 72 h, expression level of mRNA for OCN (Fig. 1A) and protein for OCN and OPN increased in a concentrationdependent manner (Fig. 2). BMSCs exhibited increased ALP activity in response to simvastatin; ALP activity increased 2–3-fold above basal levels (Fig. 3). Taken together, results showed that treatment of BMSCs with simvastatin induces osteoblastogenesis of BMSCs. Effect of simvastatin on adipogenesis of BMSCs Results showed that treatment of simvastatin inhibits adipocytic differentiation of BMSCs induced by HI in a concentration-dependent manner, while adipocyte decreased by about 51% in the group treated with 1.0 lM
Fig. 1. Effect of simvastatin on osteocalcin (OCN) and lipoprotein lipase (LPL) mRNA expressions in mouse bone marrow stromal cells. OCN and LPL mRNA levels were measured by RT-PCR after cells were induced with simvastatin (0, 0.1, 0.2, 0.5, 1.0, or 2.0 lM) for 72 h with (B) or without (A) adipogenic agonists (0.5 lM hydrocortisone, 60 lM indomethacin: HI).
Fig. 2. Effect of simvastatin on expression of osteocalcin (OCN), osteopontin (OPN), and BMP2 in mouse bone marrow stromal cells. After cells were treated with simvastatin (0, 0.1, 0.2, 0.5, 1.0, or 2.0 lM) for 72 h, supernatants (for determination of OCN and BMP2 ) or cellular homogenate (for determination of OPN) was analyzed by Western blot.
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Fig. 3. Effect of simvastatin or rhBMP2 on ALP activity changes of mouse bone marrow stromal cells. Cells were induced with simvastatin (0, 0.1, 0.2, 0.5, or 1.0 lM) or rhBMP2 (100 ng/mL) for 72 h, ALP activity was assayed. Values are means SD of three determinations from six separate experiments. *p < 0:05, compared with the control group (simvastatin 0 lM).
simvastatin compared to controls that did not receive simvastatin (Figs. 4 and 5). Morphological observation showed that adipocytes existed in clusters, lipid droplets that appeared in the cytoplasm of adipocytes were positive for Oil Red O stain, and there were less lipid droplets seen in simvastatin-treated groups (Fig. 4). After induction with HI and simvastatin for 72 h, the adipocytic phenotype, LPL mRNA level decreased in a concentration-dependent manner (Fig. 1B), further confirming a decrease of adipocyte differentiation. Effects of simvastatin on BMP2 expression in BMSCs Following treatment on BMSCs with simvastatin for 72 h, Western blot revealed that expression of BMP2
Fig. 5. Effect of simvastatin or rhBMP2 on adipogenesis of bone marrow stromal cells. Cultures were induced with vehicle (DMSO, normal control), or adipogenetic agonists (0.5 lM hydrocortisone, 60 lM indomethacin: HI) and rhBMP2 100 ng/mL, or HI and simvastatin, or HI alone for 12 days, adipocyte percentage was determined by FACS. Each point is the mean SD from three separate experiments. Compared with the negative control group (HI + simvastatin 0 lM), *p < 0:05; ***p < 0:001.
increased in a concentration-dependent manner (Fig. 3), suggesting that simvastatin causes high expression of BMP2 in BMSCs as well as osteoblasts.
Discussion Studies have demonstrated that BMSCs can differentiate into multiple cell types, including osteoblasts, myoblasts, chondrocytes, and adipocytes [19,20]. Adipocytic and osteogenic cells are reciprocal cell types that are dominant in marrow. It is thought that changes in the ratios of these cells are involved in bone volume decreases
Fig. 4. Oil Red O staining and fluorescence activated cell sorting (FACS) for adipogenesis of bone marrow stromal cells affected by simvastatin and rhBMP2. Cultures were treated with vehicle (DMSO, normal control), or adipogenetic agonists (0.5 lM hydrocortisone, 60 lM indomethacin: HI) and rhBMP2 100 ng/mL, or HI and simvastatin, or HI alone for 12 days. Adipocyte formation was monitored by Oil Red O staining (upper panel, original magnification: 100) and quantified by FACS (lower panel).
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associated with osteoporosis, such as that seen in cases of ovariectomy [1], immobilization [2], treatment with glucocorticoids [3], and age-related osteopenia [4]. One mechanism that may account for the reciprocal relationship between decreased bone density and increased fat formation is an imbalance in the production of osteogenesis and adipogenesis cells in the bone marrow cavity, and an increase in the number of adipocytes occurs at the expense of osteoblasts in osteopenic disorders. Furthermore, mature mammary adipocytes inhibited proliferation of human osteoblastic cells in culture [21]. Inhibition of marrow adipocyte differentiation and a concomitant enhancement of osteoblastogenesis may provide a novel strategy for the treatment of osteoporosis [8]. Mundy et al. [9] discovered the bone-forming potential of statins in the course of screening thousands of chemicals for the ability to synthesize BMP2 in osteoblast. Most recently, Sugiyama et al. [22] and Ohnaka et al. [23] also proved that statins enhance high expression of BMP2 in bone cells. We reasoned that, as osteoblasts arise from BMSCs, simvastatin might also promote the synthesis of BMP2 in BMSCs as well as bone cells, thus enhancing osteoblast differentiation and inhibiting adipocyte differentiation. Although the precise physiological role of ALP in bone is unknown, mature osteoblasts are characterized by high ALP activity, making this a marker of osteoblast differentiation [6,24]. Therefore, we examined the changes of ALP activity in BMSCs exposed to simvastatin and found that it increased significantly. Similarly, two other osteoblastic phenotypes, osteocalcin and osteopontin, also increased in a concentration-dependent manner following the addition of simvastatin. These suggest that simvastatin stimulates osteoblastic differentiation of BMSCs. To determine whether simvastatin might have a corresponding ability to decrease adipocyte formation of bone marrow stromal cells, we detected the adipocyte differentiation using FACS, Oil Red O staining, and changes of the LPL mRNA expression level. We found that simvastatin inhibited BMSCs to differentiate into adipocyte and decrease the expression level of mRNA for LPL, an adipocyte marker in a dose-dependent manner. Gimble et al. [10] reported that rhBMP2 100 ng/ mL could inhibit adipocytic differentiation, but this was not significantly observed in our study. In their study, after bone marrow stromal cell line, BMS2 were treated with adipocyte inducing agents and rhBMP2 for 72 h, fresh medium without adipocyte inducing agents was used, and rhBMP2 was maintained at their initial concentrations, cells were examined for adipocyte differentiation 6 days after the induction of adipocyte differentiation. While in our study, we used primary cultured BMSCs and the adipocyte inducer was used throughout the 12 days. The quality and activity of rhBMP2 may be another reason.
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It has been shown that BMP2 promotes osteoblastic differentiation and inhibits adipogenetic differentiation of bone marrow stromal cells [6,10]. Our results showed that simvastatin induces high expression of BMP2 in BMSCs, indicating that the modulating effect of simvastatin on osteoblastic and adipocytic differentiation of BMSCs may be mediated by up-regulation of BMP2 expression. In summary, we demonstrated that simvastatin promotes osteoblastic differentiation of BMSCs, inhibits adipocytic differentiation, and causes high expression of BMP2 in BMSCs. These may be the mechanisms of the stimulatory effect of simvastatin on bone formation and this effect is at least partially mediated by inducing BMP2 expression in BMSCs.
Acknowledgments The authors thank Professor L.W. Fisher for generous gift of primary antibody. We also thank Dr. Ming Gong and Dr. Shuyan Li for their technical assistance on molecular biology.
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