Pierre J. Marie, INSERM U606, Hopital Lariboisiere, 2 rue Ambroise Pare, 75475 Paris Cedex 10, France. Tel.: +33 1 49 95 63 89; fax: +33 1 49 95 84 52; e-mail: firstname.lastname@example.org
With aging, bone marrow mesenchymal stromal cell (MSC) osteoblast differentiation decreases whereas MSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. Here, we investigated whether activation of cell signaling by strontium ranelate (SrRan) can reverse the excessive adipogenic differentiation associated with aging. In murine MSC cultures, SrRan increased Runx2 expression and matrix mineralization and decreased PPARγ2 expression and adipogenesis. This effect was associated with increased expression of the Wnt noncanonical representative Wnt5a and adipogenic modulator Maf and was abrogated by Wnt- and nuclear factor of activated T-cells (NFAT)c antagonists, implying a role for Wnt and NFATc/Maf signaling in the switch in osteoblastogenesis to adipogenesis induced by SrRan. To confirm this finding, we investigated the effect of SrRan in SAMP6 senescent mice, which exhibit decreased osteoblastogenesis, increased adipogenesis, and osteopenia. SrRan administration at a clinically relevant dose level increased bone mineral density, bone volume, trabecular thickness and number, as shown by densitometric, microscanning, and histomorphometric analyses in long bones and vertebrae. This attenuation of bone loss was related to increased osteoblast surface and bone formation rate and decreased bone marrow adipocyte volume and size. The restoration of osteoblast and adipocyte balance induced by SrRan was linked to increased Wnt5a and Maf expression in the bone marrow. The results indicate that SrRan acts on lineage allocation of MSCs by antagonizing the age-related switch in osteoblast to adipocyte differentiation via mechanisms involving NFATc/Maf and Wnt signaling, resulting in increased bone formation and attenuation of bone loss in senescent osteopenic mice.
Age-related bone loss is associated with a slow and continuous decrease in bone formation (Khosla & Riggs, 2005). The decreased bone formation that occurs with aging results from decreased osteoblast number and function (Manolagas, 1998) as a result of extrinsic and intrinsic mechanisms (Marie & Kassem, 2011). In the mature skeleton, osteoblasts originate from the differentiation of mesenchymal stromal cells (MSCs), which have the capacity to differentiate into adipocytes in the bone marrow (Beresford et al., 1992). An inverse relationship between bone marrow fat and bone formation suggests that MSC differentiation into adipocytes proceeds at the expense of osteogenesis (Nuttall & Gimble, 2004). Adipocyte differentiation of MSCs is governed by the adipocyte transcription factor peroxisome proliferator-activated receptor-gamma-2 (PPARγ2) (Rosen & Spiegelman, 2001), whereas osteoblast differentiation is linked to Runx2 expression (Yang & Karsenty, 2002; Lian & Stein, 2003). Inhibition of PPARγ2 reduces adipocyte differentiation (Parhami et al., 1999; Cho et al., 2011) and increases osteoblast differentiation, whereas overexpression or activation of PPARγ2 promotes adipogenesis and reduces osteoblast differentiation (Lecka-Czernik, 2006), indicating that the adipocyte and osteoblast differentiation programs in MSCs are controlled in a reciprocal manner (Nuttall & Gimble, 2004; Lecka-Czernik, 2006; Marie & Kaabeche, 2006).
Aging is associated with a reciprocal relationship between osteogenesis and adipogenesis in the bone marrow. With advancing age, the number and size of bone marrow adipocytes increase and the increased bone marrow adipogenesis during aging is associated with bone loss (Kirkland et al., 2002; Rosen et al., 2009). Age-related bone loss is linked to decreased expression of Runx2 and increased PPARγ levels, resulting in decreased commitment of MSCs to the osteoblast lineage and increased commitment to the adipocyte lineage (Moerman et al., 2004). Reduced PPARγ2 expression can counteract the increased adipogenesis and the decreased osteoblastogenesis associated with aging (Akune et al., 2004). Although Runx2 and PPARγ play essential roles in the determination of MSC differentiation associated with aging, the molecular mechanisms underlying the switch in osteoblast/adipocyte differentiation of MSCs during aging remains poorly understood.
Wnt signaling is an important pathway controlling bone formation and adipogenesis (Bodine & Komm, 2006). Wnt signaling promotes osteoblast differentiation in part through up-regulation of Runx2 (Gaur et al., 2005). Wnt signaling also inhibits PPARγ expression and adipogenic differentiation (Ross et al., 2000; Bennett et al., 2002). PPARγ inhibition leads to activation of Wnt signaling, indicating the existence of important regulatory links between adipogenesis and Wnt signaling (Takada et al., 2009). Wnt signaling is activated in osteoblast-committed MSCs and is reduced in adipocyte-committed MSCs, further indicating that this pathway controls the balance between osteoblastogenesis and adipogenesis in the bone marrow (Taipaleenmaki et al., 2011). Moreover, aging is associated with increased levels of natural PPARγ activators such as oxidized fatty acids, causing oxidative stress and subsequent attenuation of Wnt signaling (Almeida et al., 2007). These lines of evidence suggest that activation of Wnt signaling may prevent the increased adipogenesis associated with the decreased osteogenesis in the aging skeleton.
Strontium ranelate (SrRan) is a therapeutic agent used in the treatment of postmenopausal osteoporosis (Meunier et al., 2004). In vitro studies have shown that SrRan acts on osteoprogenitor cells and osteoblasts to favor osteoblastogenesis by activating multiple signaling pathways (Marie et al., 2011). We recently showed that SrRan promotes osteoblast differentiation in part via activation of canonical and noncanonical Wnt pathways and nuclear factor of activated T-cells (NFAT)c signaling (Fromigué et al., 2010). Whether SrRan may act on lineage allocation of aged MSCs to counteract the age-related switch in osteoblast/adipocyte MSC differentiation in the aging skeleton has not been investigated. In this study, we investigated whether SrRan modulates MSC osteoblast/adipocyte differentiation in vitro via Wnt signaling. We also investigated whether SrRan might reverse the impaired bone formation and increased adipogenesis in SAMP6 mice, an animal model of senile osteoporosis (Jilka et al., 1996; Kajkenova et al., 1997). Furthermore, we determined the implication of Wnt signaling in SrRan actions in vitro and in senescent osteopenic mice.
Osteoblastic/adipogenic differentiation is regulated by SrRan in MSCs
We first assessed the effect of SrRan on MSC differentiation by analyzing the changes in Runx2 and PPARγ2 expression, which typify osteoblast and adipocyte differentiation pathways, respectively. We found that SrRan concentration dependently increased Runx2 expression (Fig. 1a) and increased in vitro extracellular matrix mineralization, a hallmark of osteoblast function, as shown by alizarin red staining and calcium content in murine MSCs (Fig. 1). In addition to this positive effect on MSC osteoblast differentiation, SrRan decreased PPARγ2 expression in murine MSCs (Fig. 1d) and reduced adipogenic formation, as shown by oil red staining and quantification (Fig. 1). Similar effects were found in primary human MSCs (data not shown). These in vitro results indicate that SrRan enhances osteogenic differentiation and concomitantly decreases adipogenic differentiation of MSCs. Role of Wnt and NFATc signaling in SrRan-induced decreased MSC adipocyte differentiation
Fromigué et al. (2010), Rybchyn et al. (2011), and Yang et al. (2011) recently showed that strontium activates canonical Wnt signaling. We thus hypothesized that SrRan may act on MSC adipogenic differentiation by modulating Wnt expression. To test this hypothesis, we analyzed the effect of SrRan on the expression of Wnt partners, which are known to regulate adipogenesis and osteoblastogenesis (Bennett et al., 2002; Stevens et al., 2010). We focused on Wnt5a instead of canonical Wnt because of the important role of Wnt5a in the control of adipogenesis (Takada et al., 2009; Bilkovski et al., 2010). As shown in Fig. 2a, treatment with SrRan increased Wnt5a mRNA levels in murine MSCs. In contrast, Wnt3a mRNA expression remained unchanged and Wnt10b mRNA expression was not significantly increased in these cells (data not shown). To confirm the implication of Wnt5a in the effect of SrRan, murine MSCs were treated with SrRan (3 mm) in the presence of Dickkopf-1 (DKK1) and secreted Frizzled-related protein-1 (sFRP1), which are known Wnt signaling inhibitors. As shown in Fig. 2, DKK1 or sFRP1 abolished SrRan-induced modulation of Runx2 and PPARγ2 expression in murine MSCs, suggesting that Wnt signaling is implicated in the induction of osteoblastogenesis and reduction in adipogenesis by SrRan in MSCs.
We then tested whether SrRan may act on MSC adipogenic differentiation by modulating NFATc signaling, which we previously found to be activated by SrRan (Fromigué et al., 2010). To this goal, murine MSCs were treated with SrRan in the presence of the calcineurin inhibitors cyclosporin A (CSA) and FK506. As shown in Fig. 2, CSA or FK506 abolished the SrRan-induced modulation of Runx2 and PPARγ2 in MSCs, indicating that NFATc signaling is implicated in the induction of osteoblastogenesis and reduction in adipogenesis by SrRan in MSCs.
Recently, new important players involved in the bifurcation of the MSC lineage into osteoblasts or adipocytes have emerged. Notably, Maf, a basic leucine zipper transcription factor, which promotes osteoblast differentiation by regulating the activity of Runx2 and inhibits adipocyte differentiation through down-regulation of PPARγ expression (Nishikawa et al., 2010). Because Maf is regulated by Ca2 + /NFATc signaling (Tanaka et al., 2005), we tested whether SrRan that activates NFATc signaling (Fromigué et al., 2010) may increase Maf expression in MSCs. As shown in Fig. 2f, SrRan increased Maf mRNA levels in murine MSCs, suggesting that Maf is a target of SrRan in these cells. We also determined the implication of the retinoblastoma 1 (Rb1) tumor suppressor, which was recently found to be involved in lineage commitment of MSCs (Calo et al., 2010) and to play a role in osteoblast differentiation (Berman et al., 2008) by interacting with Runx2 (Thomas et al., 2001). Rb1 also interacts with E2F, suppressing PPARγ and adipocyte differentiation (Fajas et al., 2002). As shown in Fig. S1a, SrRan increased Rb1 mRNA levels in MSCs, suggesting a potential role of Rb1 in the switch in adipogenic and osteoblast differentiation induced by SrRan in MSCs.
SrRan attenuates age-related bone loss in senescent mice
Based on the above findings, we determined the efficacy of SrRan administration in SAMP6 mice, an animal model of senile osteoporosis, which exhibits impaired bone formation and increased adipogenesis associated with osteopenia (Jilka et al., 1996; Kajkenova et al., 1997). The animals were treated at 9 weeks of age at a time when animals show marked bone loss (Clement-Lacroix et al. 2005). The dose level of SrRan administered (1800 mg kg−1) was optimal for the induction of an increase in bone mass in mice. Plasma strontium levels in treated animals were close to those in postmenopausal women treated with SrRan for 3 years (Meunier et al., 2004) (Table 1). At this dose level, SrRan administration increased plasma and bone Sr content (Tables 1 and 2) and had no effect on body weight (data not shown) or plasma calcium levels (Table 1). As shown in Fig. 3a, SrRan increased BMD in the whole skeleton, proximal femur (Fig. 3b), and vertebrae (Fig. 3c). Microcomputer tomography (μCT) analysis of the distal tibia metaphysis showed that SrRan administration in senescent mice increased bone microarchitecture (Fig. 3d), increased trabecular number and thickness and decreased trabecular separation, resulting in increased bone volume (Fig. 3). Histomorphometric analysis of the femur metaphysis showed that SrRan increased trabecular bone mass by 33% (Fig. 4) and trabecular thickness in senescent mice treated with SrRan (Fig. 4c), confirming the results of the microCT analysis. These results indicate that SrRan improved bone microarchitecture and attenuated bone loss in senescent mice.
Table 1. Plasma concentrations in SAMP6 senescent mice treated with SrRan or the vehicle
*P < 0.05 vs. Control.
Mean ± SD (mg L−1)
0.047 ± 0.012
90.8 ± 5.9
18.01 ± 5.4*
88.0 ± 4.7
Table 2. Bone strontium content in SAMP6 senescent mice treated with SrRan or the vehicle
Mean ± SD Sr/(Sr + Ca) mol/mol %
*P < 0.05 vs. Control.
0.0083 ± 0.0004
1.8115 ± 0.2298*
SrRan increases bone formation and decreases adipogenesis in senescent mice
To determine the mechanisms by which SrRan attenuated bone loss in senescent mice, we examined bone formation and resorption parameters. Histology of the femur metaphysis revealed that SrRan administration increased the mineral apposition rate and bone formation rate (Fig. 4). Moreover, SrRan increased the osteoblast surface (Fig. 4g). Similar effects were found in the vertebrae, indicating a general effect on the skeleton (Fig. S2). Additionally, the trabecular separation (Fig. 3h), TRAP+ osteoclast number (Fig. 4h), and osteoclast surface (data not shown) were reduced in the femur metaphysis at the end of SrRan administration, suggesting decreased bone resorption.
We next determined whether the increased bone formation induced by SrRan was associated with decreased adipogenesis. Histological analysis showed that SrRan administration reduced adipogenesis in the femur marrow stroma of senescent mice (Fig. 5). Quantitative analysis showed that the size of bone marrow adipocytes in the femur was decreased (Fig. 5c) while adipocyte number showed a trend for a decrease after SrRan administration (Fig. 5d). In the vertebrae, both the size and number of adipocytes were decreased after SrRan treatment, resulting in decreased bone marrow adipogenesis (Fig. S3). These results show that the increased bone formation induced by SrRan is concomitantly associated with a decrease in adipogenesis in the bone marrow of senescent mice.
SrRan increases Wnt5a, Maf, and Rb1 expression in vivo
The above in vitro data suggest the implication of Wnt signaling in the effect of SrRan on the switch in MSC adipocyte to osteoblast differentiation. To assess the implication of Wnt signaling in the observed attenuation of adipogenesis by SrRan in senescent mice, we determined the expression of Wnt5a and Maf, which were found to promote osteoblast differentiation in mice in long bone marrow stroma. As shown in Fig. 6a, Wnt5a mRNA expression level was increased in the bone marrow stroma of mice treated with SrRan, supporting a role of Wnt5a in the switch between adipogenic and osteoblastogenic differentiation in these mice. SrRan treatment also increased Maf expression in the bone marrow stroma of senescent mice, suggestive of a role of Maf in MSC differentiation into osteoblasts rather than adipocytes (Fig. 6b). Recent data indicate that loss of Rb1 favors adipogenesis and reduces osteogenesis in vivo (Calo et al., 2010). Interestingly, we found that SrRan increased Rb1 mRNA levels in the bone marrow stroma of senescent mice, suggesting that Rb1 may also be involved in the effects of SrRan on MSC adipocyte to osteoblast differentiation (Fig. S1b). Taken together, our data reveal that both the excessive adipogenic differentiation and reduced bone formation in senescent mice are reversed by SrRan administration, resulting in attenuation of bone loss and improved bone microarchitecture. The results also suggest that Wnt signaling and mechanisms involving NFATc/Maf and Rb1 that control MSC fate and differentiation contribute to the increase in osteoblastogenesis and decreased adipogenesis induced by SrRan in senescent osteopenic mice (Fig. 6c).
The continuous decrease in bone-forming activity in the aging skeleton is associated with preferential differentiation of MSCs into adipocytes and increased adipogenesis in the bone marrow (Moerman et al., 2004). Identifying the mechanisms that regulate the shift from osteoblast to adipocyte differentiation is thus of major importance for developing therapeutic strategies preventing age-related bone loss (Rosen et al., 2009). Here, we present evidence that activation of signaling pathways by SrRan in MSC counteracts the switch from osteoblastogenesis to adipogenesis, resulting in attenuation of bone loss in senescent osteopenic mice.
Our in vitro results indicate that SrRan acts on lineage allocation of MSCs and that both osteoblast and adipocyte differentiation of MSCs are modulated by SrRan. Our finding that the increased Runx2 expression induced by SrRan in MSCs was associated with decreased PPARγ2 expression and adipogenesis indicates that SrRan favors MSC differentiation into osteoblasts rather than adipocytes. These results extend our previous findings showing that SrRan promotes osteoblastic cell proliferation and differentiation via activation of canonical and noncanonical Wnt signaling and indicate that SrRan acts on lineage commitment of MSCs toward osteoblasts and adipocytes via activation of this pathway, thereby promoting osteoblastogenesis.
Based on this in vitro evidence, we investigated the effects of SrRan administration in SAMP6 mice, an animal model of senile osteoporosis characterized by decreased osteoblastogenesis and increased adipogenesis (Jilka et al., 1996; Kajkenova et al., 1997), which are hallmarks of skeletal aging (Rosen et al., 2009). Remarkably, we found that administration of SrRan at a dose providing clinically relevant plasma levels (Meunier et al., 2004) attenuated the increased adipogenesis while promoting osteoblast number and bone formation in senescent mice. These results support our in vitro data indicating that SrRan modulates the age-related MSC bifurcation into osteoblasts and adipocytes. The observed switch in adipocyte to osteoblast differentiation of MSCs induced by SrRan has important functional implications because this effect translated into attenuation of bone loss at both peripheral and axial skeletal sites in senescent mice.
We also determined the molecular mechanisms that are involved in the switch between osteoblast and adipocyte differentiation induced by SrRan. Strontium was shown to activate Wnt signaling in osteoblastic cells (Fromigué et al., 2010; Rybchyn et al., 2011; Yang et al., 2011), and Wnt signaling is known to reduce adipocyte differentiation (Takada et al., 2009). Our finding that inhibition of Wnt signaling blunted the reduction in PPARγ2 induced by SrRan indicates that Wnt signaling contributes in part to the SrRan-mediated reduction in adipogenic differentiation. We investigated the changes in Wnt5a, which was shown to enhance osteoblast differentiation and to impair PPARγ-induced adipogenesis in MSCs (Takada et al., 2009; Bilkovski et al., 2010). Interestingly, we found that Wnt5a expression was up-regulated by SrRan in MSCs. Consistently, our gene expression analysis in SAMP6 mice revealed that SrRan increased the expression of Wnt5a in the bone marrow stroma. These results support a role for the noncanonical Wnt5a ligand induced by SrRan in favoring bone marrow MSC differentiation into osteoblasts instead of adipocytes in vivo.
The noncanonical, β-catenin-independent pathway activated by Wnt5a involves several cascades for signal transduction including NFATc (Dejmek et al., 2006). In bone, NFATc signaling has emerged as an important modulator of bone mass (Takayanagi, 2007). Recent studies indicate that NFATc1 positively controls bone formation through increased osteoblast replication and function (Koga et al., 2005; Winslow et al., 2006). Interestingly, the transcription factor Maf is regulated by NFATc signaling (Tanaka et al., 2005) and plays an essential role in the bifurcation between osteoblasts and adipocytes by regulating Runx2 activity and inhibiting PPARγ expression (Nishikawa et al., 2010). Recently, Maf levels were found to decrease with aging, which may contribute to the age-related switch in MSC differentiation into adipocytes rather than osteoblasts (Nishikawa et al., 2010). We therefore investigated whether these genes may be implicated in the positive effect of SrRan on lineage allocation of aged MSCs and osteoblastogenesis. We found that SrRan increased Maf expression in MSCs in vitro and in bone marrow stromal cells in vivo, indicating that NFATc/Maf signaling contributes to the effect of SrRan on the switch from adipogenesis to osteoblastogenesis in senescent mice. Although we identified Wnt5a and Maf as potential mediators of the effect of SrRan on osteoblast/adipocyte differentiation in senescent mice, other molecules may also be involved. One candidate is Rb1 that binds to Runx2 and acts with E2F to suppress PPARγ2 (Thomas et al., 2001; Fajas et al., 2002), thereby favoring osteogenesis over adipogenesis (Calo et al., 2010). Consistent with this concept, Rb1-/- calvarial cells showed reduced osteoblast differentiation and increased adipogenic ability (Gutierrez et al., 2008). Here, we found that SrRan increased the expression of Rb1 in cultured MSCs and in bone marrow stroma in vivo, suggesting a possible role of Rb1 in the switch from adipocyte to osteoblast differentiation induced by SrRan in senescent mice.
In conclusion, this study reports a previously unrecognized positive effect of SrRan on lineage allocation of aged MSCs and osteoblastogenesis in senescent mice by antagonizing the excessive adipogenic differentiation in the bone marrow. Our data suggest that SrRan counteracts the age-related switch in bone marrow stromal cell osteoblast to adipocyte differentiation via mechanisms involving at least two pathways, Wnt5a and NFATc/Maf signaling, resulting in attenuation of bone loss in senescent mice (Fig. 6c). These results provide novel insights into the effects of the anti-osteoporotic drug SrRan on MSC adipogenic/osteoblastic fate in vivo and suggest an efficient therapeutic modulation of the altered adipocyte/osteoblast differentiation in the bone marrow stroma during senescence.
Cell cultures and treatments
Murine C3H10T1/2 mesenchymal cells (ATCC, Manassas, VA, USA) and primary human MSCs (Promocell, Heidelberg, Germany) were used in this study. Cells were routinely cultured in Dubelcc o’s Modified Eagles Medium (DMEM; Invitrogen Corporation, Paisley, Scotland) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1%l-glutamine and penicillin/streptomycin (10 000 U mL−1 and 10 000 μg mL−1, respectively). Murine and human cells were treated with strontium chloride and sodium ranelate (Servier Laboratories, Courbevoie, France) leading to strontium concentration of 0, 1, 3 mm. To determine whether the changes in gene expression are dependent on canonical or noncanonical Wnt signaling, murine cells were treated with SrRan in the presence of DKK1 (50 ng mL−1) or sFRP1 (250 ng mL−1) (Sigma, St Louis, USA), and gene expression was determined. To determine the role of NFATc in the effect of SrRan, murine cells were treated with SrRan in the presence of CSA (100 ng mL−1) or FK506 (10 ng mL−1) (Fromigué et al., 2010), and gene expression was determined as described below.
Animals and treatment
SAMP6 mice, a murine model of aging in the P6 strain of senescence-accelerated mice, were obtained at 7 weeks (Harlan laboratories, Blackthorn, UK) and the treatment started at 9 weeks of age. These animals show decreased bone mass and bone formation as early as 3–4 months of age (Jilka et al., 1996; Clement-Lacroix et al. 2005). The animals were fed with mouse standard diet (A04 rat/mouse diet, SAFE, Augy, France) containing calcium (0.84%), phosphorus (0.57%), and vitamin D (1000 IU kg−1) and weighed once a week. Mice (20 mice per group) were administered with either a strontium ranelate suspension (1800 mg kg−1) (Servier Laboratories, Courbevoie, France) or vehicle, 5 days per week for 10 weeks. To label bone mineralization fronts, control and treated mice were given tetracycline (20 mg kg−1; Sigma, St Quentin, France) and calcein (10 mg kg−1) by s.c. injection, respectively at days 8 and 3 before sacrifice.
Bone mineral density, microarchitecture, and histomorphometry
After 10 weeks of treatment, the animals were sacrificed, and the right femurs were embedded in methylmethacrylate (Haÿet al., 2009). Five-micrometer sections were stained with aniline blue to analyze structural parameters (bone volume, trabecular number and thickness) and toluidine blue to analyze cellular parameters (adipocyte and osteoblast numbers). TRAP+ staining was carried out to evaluate osteoclast numbers. Eight-μm unstained sections were used to assess dynamic parameters. The right tibia (distal metaphysis) were scanned using a high-resolution micro computed tomography (Micro CT) system (Scanco Medical AG micro CT-40) and analyzed using the 3-D morphometry evaluation program (software version μCT Version 6.0; Charles River Laboratories Preclinical Services, Sonneville, Canada).
Ex vivo mRNA analysis
Bone marrow cells were harvested from left tibiae in control and treated mice after 10 weeks of treatment, as described by Haÿet al. (2009). The bone marrow cavities of tibiae were flushed, and total RNA was extracted from the bone marrow and converted to cDNA using standard methods. Real-time PCRs were used to compare changes in expression of osteoblast (Runx2) and adipocyte (PPARγ2) markers using 18S as internal control.
Osteogenic and adipogenic assays
For osteogenic differentiation, cell culture medium was supplemented with 50 μm ascorbic acid and 3 mm inorganic phosphate (NaH2PO4) from day 1 to 21 of culture to allow matrix synthesis and mineralization. At 21 days of culture, cells were fixed in 4% paraformaldehyde in phosphate buffered saline. Matrix mineralization was evaluated by alizarin red staining (Miraoui et al., 2009) and microphotographed using an Olympus microsocope (Japan). For adipogenesis, cells were treated with a PPARγ agonist (linoleic acid) from day 1 to 21 of culture and the accumulation of lipid droplets was detected by oil red staining and quantified spectrophotometrically at 510 nm.
Quantitative PCR analysis
Total RNA was extracted using Trizol reagent (InVitrogen, Paisley, UK). One microgram of total RNA from each sample was reverse-transcribed using Applied Biosystems kit. The relative mRNA levels for adipogenic (PPARγ2) and osteoblastic (Runx2) differentiation markers were evaluated by quantitative PCR analysis (LightCycler; Roche Applied Science, Indianapolis, OH, USA) using a SYBR Green PCR kit (ABGen, Courtabœuf, France) and specific primers (Miraoui et al., 2009). Signals were normalized to 18S as internal control. The expression of Wnt markers (Wnt3a, Wnt5a, Wnt10b), Maf, and Rb1 was determined using specific primers (Data S1).
Values are presented as the mean ± SEM and are representative of at least three experiments. Data were analyzed with the unpaired two-tailed Student’s t-test. A P value < 0.05 was considered statistically significant.
Financial support was provided in part by Institut National de la Santé et de la Recherche Médicale (Inserm) and Institut de Recherches Servier (Courbevoie, France) which was not involved in data analysis or manuscript writing.
ZS and PJM designed in vitro and in vivo experiments, analyzed the data, and wrote the paper. EH, CM, and AB performed and analyzed in vivo experiments.