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Endocrinology Vol. 148, No. 5 2553-2562
Copyright © 2007 by The Endocrine Society

Mechanical Loading Down-Regulates Peroxisome Proliferator-Activated Receptor {gamma} in Bone Marrow Stromal Cells and Favors Osteoblastogenesis at the Expense of Adipogenesis

Valentin David, Aline Martin, Marie-Hélène Lafage-Proust, Luc Malaval, Sylvie Peyroche, David B. Jones, Laurence Vico and Alain Guignandon

Institut National de la Santé et de la Recherche Médicale Unité 890 and Université Jean Monnet (V.D., A.M., M.-H.L.-P., L.M., S.P., L.V., A.G.), F-42023 St-Etienne, France; and Experimental Orthopedics and Biomechanics (D.B.J.), Philipps University, Baldingerst, D-35033 Marburg, Germany

Address all correspondence and requests for reprints to: Alain Guignandon, Institut National de la Santé et de la Recherche Médicale Unité 890, Faculté de Médecine 15 rue Ambroise Paré, F-42023 Saint-Etienne Cedex 2, France. E-mail: Alain.Guignandon{at}univ-st-etienne.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because a lack of mechanical information favors the development of adipocytes at the expense of osteoblasts, we hypothesized that the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})-dependent balance between osteoblasts and adipocytes is affected by mechanical stimuli. We tested the robustness of this hypothesis in in vivo rodent osteogenic exercise, in vitro cyclic loading of cancellous haversian bone samples, and cyclic stretching of primary stromal and C3H10T1/2 cells. We found that running rats exhibit a decreased marrow fat volume associated with an increased bone formation, presumably through recruitment of osteoprogenitors. In the tissue culture model and primary stromal cells, cyclic loading induced higher Runx2 and lower PPAR{gamma}2 protein levels. Given the proadipocytic and antiosteoblastic activities of PPAR{gamma}, we studied the effects of cyclic stretching in C3H10T1/2 cells, treated either with the PPAR{gamma} activator, Rosiglitazone, or with GW9662, a potent antagonist of PPAR{gamma}. We found, through both cytochemistry and analysis of lineage marker expression, that under Roziglitazone cyclic stretch partially overcomes the induction of adipogenesis and is still able to favor osteoblast differentiation. Conversely, cyclic stretch has additive effects with GW9662 in inducing osteoblastogenesis. In conclusion, we provide evidence that mechanical stimuli are potential PPAR{gamma} modulators counteracting adipocyte differentiation and inhibition of osteoblastogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DIFFERENTIATION of multipotent stem-cells of mesodermal origin results in the formation of adipocytes, chondrocytes, osteoblasts and myoblasts (1, 2, 3). A shift in differentiation and survival rates from osteoblastic to adipocytic lineage could lead to an altered ratio of fat to bone cells that may, eventually, alter bone mass (4). In humans, osteoporosis and age-related osteopenia were shown to be associated with an increase in marrow fat tissue (5, 6). In some of these studies, osteoblast numbers correlated negatively with the number of adipocytes (5, 7, 8), suggesting that adipocytes are generated at the expense of osteoblasts. This hypothesis is supported by the isolation from the bone marrow of single cell clones that can differentiate in vitro into either lineage (9).

Essential to cellular commitment to a differentiation lineage is the activation of defined transcription factors (10, 11, 12). Osteoblastic differentiation is driven by runx2, followed by osterix, and then characterized by the expression of alkaline phosphatase, osteocalcin, and eventually by the mineralization of the extracellular matrix. Differentiation of adipocytes is initiated through C/EBP{alpha} and C/EBPß that activate expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear hormone receptor family (13, 14). PPAR{gamma} regulates adipocyte-specific gene expression and is critical for the formation of mature lipid-filled adipose cells from pluripotent stem cells (15); it has also a central role in other processes such as, for example, inflammation and macrophage formation (16, 17). A recent study has demonstrated that use of the PPAR{gamma} ligands, thiazolidinediones (TZDs) induces changes in bone mineral density in elderly patients with type 2 diabetes (18), confirming the effect of TZDs (19) reported from animal models.

Among the various osteopenic animal models in which an inverse relationship was previously reported between the amount of bone marrow fat tissue and trabecular bone density are ovariectomy (20), glucocorticoid treatment (21), and also immobilization (7). Focusing on the latter case, we hypothesized that, if lack of mechanical stimuli favors the development of adipocytes at the expense of osteoblasts, the opposite might happen when external mechanical stimuli are applied. We first studied whether an osteogenic physical exercise is able to reduce bone marrow adiposity in rats. We then tested our hypothesis on lamellar bone and away from the potential confounding influence of systemic factors, by culturing bovine sternum samples in a recently developed bioreactor, the ZetOS, which allows ex vivo long-term compression loading and mechanical testing of perfused samples (22). It has been recently shown that manipulating cell tension regulates the commitment of human mesenchymal stem cells to adipocyte or osteoblast fate (23); we thus applied mechanical stretch known to alter cell tension to stromal cells extracted from the marrow of bovine bone cores and we compared their responses to the well-characterized pluripotent mesenchymal stem cell line C3H10T1/2.

We show that PPAR{gamma}2 activity is modulated by mechanical conditions, strongly enough to be still responsive to mechanical stimuli even when the cells are treated by agonist or antagonist compounds. We also demonstrate that osteo/adipogenesis control by mechanical stimuli is not restricted to a particular cell line, a unique mechanical regimen, or specific experimental conditions, because our results comprise cells of bovine and murine origin, primary and immortalized, in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture products
Insulin, all trans-retinoic acid, 4',6-diamidino-2-phenylindole, oil red O, L-ascorbic acid 2-phosphate, trypsin-EDTA reagent, clostridium histolyticum neutral collagenase, and p-nitrophenyl phosphate (PNP-p) were purchased from Sigma Aldrich (Lyon, France). DMEM-Ham’s F-12 (DMEM/F12, 1:1, vol/vol), {alpha} MEM, and DMEM was purchased from Eurobio (Courtaboeuf, France). Rosiglitazone and GW9662 were purchased from Interchim (Montluçon, France). Qiashedder and RNeasy mini kits were purchased from Qiagen (Courtaboeuf, France). First-strand cDNA synthesis kit for RT-PCR [avian myeloblastosis virus (AMV)], Light cycler-FastStart DNA Master, SYBR Green I, and Light Cycler Instrument were purchased from Roche Diagnostics (Meylan, France). Protein assay kit (bicinchoninic acid) was obtained from Interchim.

Mesenchymal precursor cell isolation
Bovine mesenchymal stem cells (bMSC) were isolated from the sternums of young males (6–8 months) and collected in sterile conditions at a local slaughterhouse immediately after sacrifice. We received permission from our local ethics committee. Briefly, after removing soft tissues, sternums were reduced to 5-mm-thick fragments. The marrow was then flushed and submitted to a 15-min enzymatic digestion with 1 mg/ml clostridium histolyticum neutral collagenase at 37 C in {alpha} MEM medium. Collagenase was neutralized with medium supplemented with 15% fetal calf serum (FCS). After neutralization with 15% FCS, the marrow was resuspended in Eagle’s medium supplemented with 10% FCS (Sigma Aldrich), 2 mM L-glutamine, and 1% antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) and plated at 5000 cells/cm2. The medium was changed after the first 24 h to remove nonadherent cells.

Cell culture
Cells were grown in tissue culture T75-flask (Elvetec, Venissieux, France) in 5% CO2 humidified atmosphere at 37 C. The mouse pluripotent mesenchymal stem cell line C3H10T1/2 (clone-8; American Type Culture Collection, LGC Promochem, Molsheim, France) was cultured in {alpha} MEM, whereas bMSC were cultured in DMEM, supplemented with 10% FCS (PromoCell GMBH, Heidelberg, Germany), L-glutamine, and antibiotics as above. After reaching a subconfluent state, cells were trypsinized with 1x trypsin-EDTA and plated onto flexible type I collagen-coated, silicon-bottom, six-well culture plates (Bioflex; Flexcell Corp., McKeesport, PA) at 2500 cell/cm2 for C3H10T1/2 and 5000 cell/cm2 for bMSC, and the medium was changed every other day.

Mechanical stretching
Starting 72 h after seeding (referred to as d 0), cells were subjected to daily mechanical deformation during 2 wk. Mechanical deformation was induced with a Flexcell Strain Unit Fx-3000 (Flexcell Corp., Hillsborough, NC) (24), which consists of a vacuum manifold regulated by solenoid valves that are controlled by a computer timer program. Each plate is inserted over six buttons in the Bioflex loading station. Application, through an air pump, of a negative pressure of 80 kPa stretches horizontally the bottom of the culture plate over the plastic button. Thus, 85% of the surface of the flexible wells is submitted to a known percentage of uniform elongation. The membranes are then released to their original conformation (24). The experimental regimen used in this study delivered 4000 µ{epsilon} elongation at 1 Hz frequency (triangular signal) during 300 cycles/d. Stretched cells remained adherent, and the deformation of the membrane was directly transmitted to the cells. Unstretched cells grown on Bioflex plates were used as controls. Starting from d 0, media were supplemented with 10% FCS (PromoCell GMBH), 50 µg/ml ascorbic acid, 10–6 M ß-glycerophosphate, 10–8 M all trans-retinoic acid, 10–8 M dexamethasone, 1% insulin, and 5 x 10–5 M 3-isobutyl-1-methylxanthine to create conditions inducing both osteoblastic and adipogenic cells. The differentiation into various cell lineages is regulated by factors such as cytokines and growth hormones, cAMP-elevating agents, and ligands for members of the steroid/thyroid receptor-gene family of transcription factors (25, 26). Among these factors, all trans-retinoic acid has been reported to increase the expression of osteoblastic-related cell markers such as alkaline phosphatase (27, 28).

PPAR{gamma} induction and inhibition
To evaluate the involvement of PPAR{gamma} in the response to mechanical stretching, cells were treated during the culture period with 1 µM (EC50; Kd 43 Nm) of a powerful agonist of PPAR{gamma}, BRL49653, or Rosiglitazone or DMSO as a vehicle. PPAR{gamma} activation was inhibited with 1 µM (EC50) of an antagonist of PPAR{gamma}, GW9662. The compounds were added to the culture medium at d 0, and renewed every 2 d. Untreated cells received equal volumes of vehicle.

Histochemical staining
After 2% formaldehyde and rinsing, the activity of the plasma membrane-associated alkaline phosphatase was detected using an Alkaline Phosphatase Leukocyte Staining Kit (Sigma Aldrich), according to the manufacturer’s protocol. The cultures were then rinsed three times for 5 min in deionized water and cytoplasmic triglyceride droplets were stained with oil red O (29). Nuclei were stained with 4',6-diamidino-2-phenylindole. The percent of alkaline phosphatase and oil-red-O-positive cells was determined by counting cells in 30 contiguous fields per well after random starts.

Protein extraction
Total proteins were extracted in 2 ml lysis buffer per well containing 10 ml/liter Nonidet 40, 1.8 g/liter iodoacetamide, 3.5 ml/liter proteases inhibition mixture (Sigma Aldrich), and 2 µl/liter ß-mercaptoethanol. After centrifugation (5 min, 5000 rpm, 4 C), supernatants were stored at –20 C. Cytoplasmic and nuclear protein fractions were separated using a nuclear extraction kit (Active Motif, Rixensart, Belgium). Briefly, cells were scrapped—collected in 3 ml ice-cold PBS-phosphatase inhibitors mixture; the material was kept at 4 C thereafter. The cell suspension was spun for 5 min at 500 rpm. The pellet was resuspended in 500 µl hypotonic buffer and incubated for 15 min on ice. Cell membranes were lysed with 25 µl detergent. The cytoplasmic protein fraction was collected after a 30-sec spin at 14,000 x g. Nuclear pellets were resuspended in 50 µl lysis buffer then incubated for 30 min on ice on a rocking platform at 150 rpm. The suspension was then spun for 10 min at 14,000 x g and the nuclear fraction (supernatant) was collected in microcentrifuge tube. Aliquots were store at –80 C. Protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce, Perbio Science France SAS, Brebières, France).

Alkaline phosphatase assay
Alkaline phosphatase activity (ALP) was measured by assessing the hydrolysis of PNP-p in inorganic phosphate at 37 C. Briefly, the assay mixture consisted of 100 µl cell homogenate and 900 µl reaction mixture (2 mM PNP-p, 2 mM MgCl2, 2-amino-2-methyl-1-propanol 95%, pH 10.5). The reaction was initiated by the addition of the cell extract and product amounts were read after 50 min at 412 nm on a spectrophotometer. ALP was expressed as nanomoles of inorganic phosphate per milligram of protein per minute.

Sandwich ELISA
Sandwich ELISAs were designed in our laboratory to quantify PPAR{gamma}2 and Runx2 in protein extracts. A capture antibody [runx2; rabbit antihuman (CBFA11-A, 4ADI, TEBU) and PPAR{gamma}; rabbit IgG antimouse (PA1–824, ABR, TEBU)] was first coated on each well in 0.1 M bicarbonate buffer, pH 9.2, overnight at 4 C. The wells were then blocked for 60 min at room temperature in 100 µl of 100 mM phosphate buffer, pH 7.2, 1% BSA, and 0.5% Tween 20. After three washes in wash buffer (100 mM phosphate buffer, 150 mM NaCl, 0.2% BSA, and 0.05% Tween 20), samples or standards were added to the plates in 100 µl per well. Plates were then incubated at room temperature for 1 h then overnight at 4 C. After wash, 100 µl of the second antibody [runx2; goat antihuman (sc-12488, TEBU) and PPAR{gamma}; goat antihuman (sc-6284, TEBU)] were added to each well and incubated at room temperature for 4 h. The plates were washed, and an ALP-labeled secondary antibody [rabbit IgG antigoat (Cliniscience, Montrouge, France)] was added to each well and incubated at room temperature for 4 h. After wash, fast pNP enzyme substrate (Sigma Aldrich) was added to the wells and incubated for 30 min. Color intensities were measured at 412 nm with a spectrophotometer, using a blank reference. Assay specificity was ascertained in competition experiments against matching and mutated (negative controls) PPAR{gamma} and RUNX2 peptides (Abcam, Cambridge, UK; results not shown). Serial dilutions of a cell extract calibrated with specific peptides were used as assay standards.

PPAR{gamma} activity measurement
DNA binding PPAR{gamma} activity was determined using the ELISA-based PPAR{gamma} activation TransAM kit (Active Motif). PPAR{gamma} contained in nuclear extracts bind specifically to an oligonucleotide containing the peroxisome proliferator response element (PPRE, 5'-AACTAGGTCAAAGGTCA-3') and are detected with an anti-PPAR{gamma} antibody. A secondary antibody conjugated to horseradish peroxidase provides a sensitive colorimetric readout that is quantified by spectrophotometry at 405 nm.

RNA extraction and RT and real-time PCR
RNA extraction was performed on cells at various time points up to 14 d after the beginning of stimulation. Total RNA was isolated by guanidium isothiocyanate extraction using the RNeasy mini kit according to manufacturer’s instruction. Briefly, the samples were disrupted in lysis buffer containing guanidium isothiocyanate and homogenized using Qiashedder. The samples were then applied to the RNeasy spin column and total RNAs bound to the membrane were eluted in water. Integrity of RNA was checked by electrophoresis, after ethidium bromide staining. RNAs were reverse-transcribed into single-stranded cDNA using first-strand cDNA synthesis kit for RT-PCR (AMV) from 2 µg total RNA in a 20-µl reaction mix containing 2 µl of 10x reaction, 5 mM MgCl2, 20 mmol each of dNTP, 50 pmol oligo-p(dT)15 primer, 50 U RNase inhibitor, and 20 U AMV reverse transcriptase. The reaction was incubated for 60 min at 42 C. The single-strand cDNA was diluted 1:10, and 8 µl were amplified with a LightCycler (Roche Diagnostics) in 20 µl PCR mixture containing 2 µl of Light cycler-FastStart DNA Master SYBR Green I, 3 mM MgCl2, 0.5 µM of 5' and 3' oligo primers, and water. A typical protocol included a denaturation step at 95 C for 10 min followed by 40 cycles with 95 C for 1 sec, Tm°C annealing for A s, and Te°C extension for M s. The fluorescence product was detected at the end of the extension period after 60 sec at 60 C. {theta}m, A, Tm, D, M, X, E, primers, and product length are summarized in Table 1Go. Quantified data were analyzed with the Light-Cycler analysis software. Serial dilution of total RNA was performed from 16–0.25 ng and used as standards. For real-time PCR assay, 2–4 ng of input RNA was used. Results were analyzed following the manufacturer’s instructions: 1) checking the PCR products specificity and 2) calculating the variation in PCR products concentration between experimental groups, expressed as percentage of mean control values.


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  		TABLE 1. Primers and conditions for real-time PCR

 
Ex vivo studies
Cancellous bovine bone was used because it presented similar architecture as human trabecular bone and has been successfully used as xenografts to repair bony defects (30). Cancellous bovine bone was isolated from sternum of young males (6–8 months), machined with high precision to cylindrical cores (10 mm diameter, 5 mm height) under sterile conditions, and inserted into the loading chambers of a Zetos bone perfusion system (22). Each core was maintained at 37 C and perfused with 5 ml DMEM/F12 (1:1), recirculating at a rate of 6 ml/h. Half of the bone samples were subjected to 300 cycles daily mechanical compression at 1 Hz, 4000 µ{epsilon} amplitude (similar to FlexerCell protocol), the other half were unloaded controls. Five loaded and five unloaded bones were collected at d 7, 14, and 21, and the protein fraction was collected (see protein extraction section).

In vivo study
Nine-week young adult male Wistar rats were used for the experiment. Animals were kept in the laboratory for 1 wk before the experiment to allow acclimatization to the diet and new environment. The light/dark cycle was 12 h with lights on from 0700–1900 h. The rats were allowed free access to water and chow diet. The rats were trained on a treadmill at 60% of maximal O2 consumption, 5 d per week. VO2max was determined on the open-flow system apparatus as described in Bourrin et al. (31). Briefly, the rats ran on a treadmill placed in a closed Plexiglas chamber with a controlled and measured air flow. After acclimatization to the new environment, the rats were trained to obtain a maximal exercise. The O2 and CO2 expired were measured and recorded every 3 min while the animal is exercising. On the first day, rats of the exercise group ran 15 min at a speed of 20 m/min on the treadmill being maintained horizontal. Thereafter, the duration of each training session was progressively increased until the animals ran 1 h and 30 min/d at a speed of 20 m/min after 1 wk of training. By the fifth week of training, rats ran 1 h and 30 min/d at a speed of 30 m/min on the level. At the end of the experiment, rats were injected with fluorochromes twice (6 d apart) to measure the dynamic parameters of bone formation. The bone effects of this training program were published elsewhere (31). Tibiae from 20 male Wistar rats, 10 sedentary control rats, and 10 running animals from the study of Bourrin et al. (31) were analyzed by histology. The proximal tibia metaphysis were fixed in 4% formaldehyde solution, dehydrated in acetone, and embedded in methylmethacrylate. Longitudinal frontal slices were cut from the embedded bones with a Jung Model K microtome (Carl Zeiss, Heidelberg, Germany). Six nonserial sections, 8 µm thick, were used for modified Goldner staining. The relative volume of fat in the marrow cavity (Ad.V/MV) was measured on Goldner sections using a manual counter and a hundred-point grid according to Ref. 32 .

Statistical analysis
Statistical analysis was performed using the STATISTICA software (StatSoft Inc., Tulsa, OK). One- or two-way ANOVA was performed on protein and RNA data. When F values for a given variable were found to be significant, the sequentially rejecting Bonferroni-Holm test (33) was subsequently performed using the Holm’s adjusted P values taken from the t table. Results were considered to be significantly different at P < 0.05. The Mann-Whitney U test was used to compare histomorphometry data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Osteogenic physical exercise reduces marrow fat in male rat tibia metaphysis in vivo
Over 5 wk of training, treadmill-running rats at 60% of their maximal O2 consumption display a 33% increase of bone formation rate (Fig. 1AGo) and an 18% decrease of bone resorption (data not shown, see Ref. 31). The mineral apposition rate relative to osteoblastic activity is unaltered (data not shown), suggesting that osteoblastic recruitment is stimulated (31). Mechanical stimulation also decreases adipocyte volume (Ad.Ar/T.Ar) by 39% in running rats (Fig. 1BGo).


Figure 1
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  		FIG. 1. Effects of physical exercise on bone formation rate (BFR) (A) and marrow adipocyte volume (Ad.Ar/T.Ar) (B) in the proximal tibia metaphysis of 5-wk treadmill-running trained rats. Animals were either sedentary controls (open boxes) or running (striped boxes) in a treadmill. Values are box plots of 10 samples per group. *, P < 0.05 vs. sedentary, Mann-Whitney U test.

 
Cyclic mechanical compression increases Runx2 and decreases PPAR{gamma}2 protein levels in bovine cancellous bone cores cultivated ex vivo
We evaluated the effects of a loading regimen on Runx2 and PPAR{gamma} expression in sternum bovine cylindrical bone cores submitted to cyclic compression. We used an accurate mechanical loading system combined with a trabecular bone culture-loading chamber, the Zetos (22), which provides the ability to study trabecular bone under controlled culture and loading conditions over 3 wk. We have shown previously that daily cyclic compression of cancellous bone in this device results in increased bone formation rate, leading to thicker trabeculae and higher Young’s Modulus (David, V., A. Guignandon, A. Martin, L. Malaval, B. Noble, D. Jones, and L. Vico, submitted for publication). Here we show that daily cyclic mechanical compression increases Runx2 protein levels after 7 and 14 d (Fig. 2AGo). In contrast, PPAR{gamma}2 levels decrease after 21 d in loaded samples compared with baseline values (Fig. 2BGo). In unloaded control samples, PPAR{gamma}2 protein expression remain stable over the 21-d culture period.


Figure 2
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  		FIG. 2. Effects of cyclic mechanical compression of sternum bone cores in the Zetos organotypic system on Runx2 (A) and PPAR{gamma}2 (B) protein contents. Samples were either unloaded (open bars) or loaded once a day for 5 min (striped bars). Values are mean ± SEM of five samples (bone cores) per group expressed as a percentage of unloaded control at d 7. *, P < 0.05 vs. matched unloaded; #, P < 0.05 vs. unloaded at d 7, two-way ANOVA with post hoc Holm test (see Materials and Methods for details).

 
In vitro, mechanical stretching of multipotent mesenchymal progenitors results in more osteoblasts and less adipocytes
When grown under static conditions, in permissive osteoblastic/adipocytic media, 45% of C3H10T1/2 cells become alkaline phosphatase positive (34) and 27% oil red O positive after 14 d, whereas the figures are 34 and 10%, respectively, in bMSC grown for 21 d.

In bMSC, at d 21, mechanical stretching increases the percent of alkaline phosphatase-positive cells (38 ± 2.8 vs. 33 ± 3.1%, P < 0.05) and decreases the proportion of oil-red-O-positive cells (6 ± 3 vs. 10 ± 2.5%, P < 0.05). Furthermore, at d 14, alkaline phosphatase activity is greatly increased in stretched bMSC cells, compared with unstretched controls (Fig. 3AGo). Daily mechanical stretching induces a strong increase in Runx2 protein amounts at d 7 and of osteocalcin protein content at d 14 (Fig. 3BGo). Consistent with these findings, runx2 and osterix transcripts are greatly increased at d 7 and 14 in stretched cells, as well as osteocalcin mRNA levels at d 14 (Fig. 3CGo). In contrast, PPAR{gamma}2 protein levels (Fig. 3BGo, d 14), mRNA (d 7), as well as aP2 mRNA (d 14) decrease in stretched cultures (Fig. 3CGo).


Figure 3
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  		FIG. 3. Effects of cyclic mechanical stretching of bMSC. A, bMSC alkaline phosphatase activity. Values are expressed as the percentage of unstretched cells at d 14. B, Runx2, osteocalcin (OC), and PPAR{gamma}2 protein levels at the indicated days. Values are expressed as the percentage of unstretched cells. Values are mean ± SEM of six wells per group. *, P < 0.05 vs. unstretched, Mann-Whitney U test. C, Runx2, osterix (osx), osteocalcin (oc), PPAR{gamma}2, and ap2 gene expression at 7 and 14 d of stretch. Values are mean ± SEM of six wells per group; *, P < 0.05 vs. unstretched at d 7, Mann-Whitney U test

 
PPAR{gamma} is involved in the mechanically regulated balance between osteoblasts and adipocytes
We evaluated the role of PPAR{gamma} in mechanically stretched C3H10T1/2 cells. Unstretched and stretched cells were treated either with Rosiglitazone, a potent PPAR{gamma} agonist (35), or with GW9662, a selective PPAR{gamma} antagonist. As expected (36), Rosiglitazone greatly increased and GW9662 reduced the number of differentiated adipocytes (oil-red-O-positive cells) after 14 d (Fig. 4AGo). Fourteen days of mechanical stretching increase osteoblast differentiation as assessed by the percent of alkaline phosphatase-positive cells (Fig. 4BGo). Conversely, adipogenic differentiation is decreased in stretched cultures (Fig. 4CGo). Interestingly, in GW9662-treated cells, stretching increases the number of alkaline phosphatase-positive cells (Fig. 4BGo), and is still able to decrease adipocyte numbers under rosiglitazone treatment (Fig. 4CGo). This is reflected in marker expression. Mechanical stretch increases gene expression of Runx2 at d 3 (data not shown), 7, and 14 (Fig. 5AGo). Rosiglitazone and GW9662 treatment decrease the expression of Runx2 markers in unstretched conditions. GW9662 at d 7 and 14 and rosiglitazone at d 7 do not impair stretch-induced Runx2 stimulation (Fig. 5AGo). Rosiglitazone treatment increases gene expression of adipogenic markers such as ADD1/Srebp1 at d 7 (data not shown), PPAR{gamma}2 at d 14 (Fig. 5BGo), and aP2 at d 14 (data not shown). As expected, expression of these genes is inhibited under GW9662 treatment. Interestingly, mechanical stretch is still able to reduce PPAR{gamma}2 mRNA expression (Fig. 5BGo), as well as ADD1/Srebp1, aP2, and adipsin expression (data not shown), in both agonist and antagonist conditions.


Figure 4
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  		FIG. 4. Effects of cyclic mechanical stretching, Rosiglitazone, or GW9662 treatment on the differentiation of C3H10T1/2 cells. A, Representative photomicrographs (x20) of C3H10T1/2 cells stained for alkaline phosphatase (elongated cells, white arrows) and oil red O (round cells with droplets, black arrows). Do note that, in nontreated conditions, alkaline phosphatase-positive cells are abundant only in stretched cultures; in Rosiglitazone-treated cells, lipid droplets are much more abundant, especially in unstretched cultures; under GW9662 treatment very few lipid droplets are seen. Number of alkaline phosphatase-positive cells (B) and number of oil red O-positive cells (C) at d 14 in unstretched (open bars) or stretched (striped bars) cultures, treated or not with either Rosiglitazone or GW9662. Values are mean ± SEM of six wells per group expressed as percentage of positive cell per well. *, P < 0.05 vs. matched unstretched cells; #, P < 0.05 vs. untreated and unstretched cells.

 

Figure 5
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  		FIG. 5. Effects of cyclic mechanical stretching under Rosiglitazone or GW9662 treatment of C3H10T1/2 on Runx2 and PPAR{gamma}2 mRNA levels. Cells were unstretched (open bars) or stretched (striped bars) and treated or not with either Rosiglitazone or GW9662. Changes are expressed as percentage of unstretched control expression at d 3. Expression of Runx2 (A) and expression of PPAR{gamma}2 (B), at d 7 and 14 of the culture. Values are mean ± SEM of six wells per group. *, P < 0.05 vs. matched unstretched cells; #, P < 0.05 vs. untreated and unstretched cells (two-way ANOVA with post hoc Holm test).

 
Effect of mechanical stretch on PPAR{gamma} activity
To better characterize the effect of mechanical stretch on PPAR{gamma} activity, we quantified nuclear Runx2 and PPAR{gamma} as well as PPAR{gamma} DNA-binding activity. Fourteen days of mechanical stretch increase nuclear Runx2 protein content in C3H10T1/2 cultures (Fig. 6AGo). Rosiglitazone and GW9662 treatment do not affect nuclear Runx2, but Rosiglitazone abolishes the effect of stretching, whereas GW9662 induces a 75% increase in Runx2 content above stretched controls (Fig. 6AGo). Mechanical stretch decreases nuclear PPAR{gamma}2 protein content (Fig. 6BGo) and PPAR{gamma} DNA binding (Fig. 6CGo) after 7 d. As expected, 7 d of Rosiglitazone treatment alone increase PPAR{gamma}2 protein expression (Fig. 6BGo) and PPAR{gamma} DNA binding activity (Fig. 6CGo). Interestingly, in Rosiglitazone-treated cells stretching decreases nuclear PPAR{gamma} amounts (Fig. 6BGo) and significantly reduces PPAR{gamma} DNA binding (Fig. 6CGo). Seven days of GW9662 treatment decrease PPAR{gamma}2 protein content in the nucleus (Fig. 6BGo) as well as PPAR{gamma} DNA binding activity (Fig. 6CGo). Stretching of GW9662-treated cells induces a further decrease of PPAR{gamma}2 nuclear protein content (Fig. 6BGo) whereas no additive effect of stretch is detected on the residual level of PPAR{gamma} DNA binding (Fig. 6CGo).


Figure 6
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  		FIG. 6. Effects of cyclic mechanical stretching under Rosiglitazone or GW9662 treatment of C3H10T1/2 cells on Runx2 and PPAR{gamma}2 activity at d 7. Cells were unstretched (open bars) or stretched (striped bars) and treated or not with either Rosiglitazone or GW9662. Values are expressed as percentage of values for unstretched untreated cells (U). The graphs present the protein amounts of nuclear Runx2 (A), nuclear PPAR{gamma}2 (B), and PPAR{gamma} DNA (C) binding activity. Values are mean ± SEM of six wells per group. *, P < 0.05 vs. matched unstretched cells; #, P < 0.05 vs. untreated and unstretched cells (two-way ANOVA with post hoc Holm test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Five weeks of osteogenic treadmill running induce in rats a higher bone formation resulting from increased mineralizing surfaces (i.e. active osteoblasts), and mirrored by lower adipocyte numbers in the bone marrow. Similarly, in a bone core explant dynamic culture system, we found that Runx2 protein levels are enhanced in compression-loaded samples, whereas PPAR{gamma}2 protein levels are decreased. Those results obtained on different species, with lamellar (bovine) and nonlamellar (rodent) bone and for different mechanical activities emphasize the fact that local mechanical signals are strong actors of the osteoblast/adipocyte balance. Very few studies have investigated the effects of mechanical stretch on uncommitted cells, whereas it has been shown that mechanical loading triggers an increase in intramedullary pressure as well as streaming potentials (37), therefore, providing mechanical signals for multipotent progenitor in vivo (38).

Our in vitro results show that mechanical stretch results in more osteoblasts both in primary bMSC and in the pluripotent mesenchymal stem cell line C3H10T1/2 grown under media permissive for both osteoblast and adipocyte differentiation. Up-regulation of protein and mRNA levels of Runx2 was seen in both models and Runx2 was also elevated in strained bovine cores. Runx2 was already reported to be stimulated by mechanical stress in several models such as human spinal ligament cells (39) and human preosteoblasts (40). The regulation of this transcription factor, expressed by mesenchymal stem cells before cell differentiation, preosteoblasts, and prehypertrophic chondrocytes (41) occurs as early as the third day of stretching, suggesting that early stages of osteoblast differentiation might be also responsive to mechanical stimuli. Alkaline phosphatase activity, an early marker of the osteoblastic lineage, osterix, which is expressed later, and osteocalcin, a marker of mature osteoblasts, were all stimulated by mechanical stretching in a time course that matches osteoblastic differentiation kinetics. Furthermore, osteoblast numbers were higher in stretched than in static conditions.

Cyclic stretch has been recently shown to reduce adipocyte differentiation in the mouse preadipocyte 3T3-L1 cell line (42), providing the first evidence for a direct effect of mechanical stimuli on fat cells. Interestingly, this effect was the result of the down-regulation of PPAR{gamma}2 by stretched-induced ERK activation. We (43) and others (44) previously showed that mechanical strain exerts its stimulating effects on osteoblasts through ERK activation. Thus, the MAPK signaling pathway appears as one of the potential molecular links modulating the osteoblast/adipocyte balance. We showed both in vivo and in vitro that lipid droplets, the hallmark of the adipocyte phenotype are decreased by mechanical stretch. The control of adipogenesis involves the interaction of a number of intracellular signaling pathways and the activation of numerous transcription factors (45, 46), particularly PPAR{gamma} (26). Mounting evidence indicates an important role of PPAR{gamma} in skeletal metabolism. Specifically, PPAR{gamma} haploinsufficient mice exhibit increased bone mass associated with increased osteoblastogenesis and decreased adipogenesis (36). Our experiments, both in vitro and ex vivo, indicate that inhibition of PPAR{gamma}2—the most potent adipogenic isoform in vitro (47)—is part of the mechanism whereby mechanical stretch inhibits adipogenesis and stimulates osteoblastogenesis. In bMSC cultures, mechanical stretch reduced PPAR{gamma}2 and protein levels. This suggests that mechanical stretch acts as a PPAR{gamma} antagonist. Mechanical stretch-induced PPAR{gamma}2 inhibition was followed by decreased expression of aP2, a late marker of adipocyte differentiation. Similar effects were found in C3H10T1/2 cells. In addition, we found that the reduction in PPAR{gamma}2 amounts in nuclear fraction was paralleled by a reduction in PPAR{gamma} nuclear activity demonstrating the inhibitory effect of mechanical stimulation on PPAR{gamma} transcriptional activity. That the expression of ADD1/SREBP1 was decreased in stretched conditions provides a plausible explanation for PPAR{gamma} loss of activity, as transactivation of the PPAR{gamma} promoter depends on transcription factors such as add1/serbp1 (48).

TZDs, a novel class of antidiabetic agents that acts as insulin sensitizers in vivo, bind PPAR{gamma} with high affinity. PPAR{gamma} regulates target gene transcription as an heterodimer with the retinoid X receptor, and this heterodimeric complex has been shown to be activated synergistically by TZDs and RXR-specific ligands (49). TZDs enhance adipogenesis in stromal cells (50). Activation of PPAR{gamma} by Rosiglitazone has been shown to stimulate adipogenesis and inhibits osteoblastogenesis in murine bone marrow-derived clonal cell line (51) and in mice, with an associated bone loss (52, 19), an action that we confirm in the murine C3H10T1/2 cell line. Mechanical stretch applied to Rosiglitazone-treated cultures was able to counteract the increase of PPAR{gamma} expression and activity. Moreover, in Rosiglitazone-treated cells, mechanical stretch was still efficient in promoting osteoblastogenesis. On the other hand, combining GW9662 treatment and mechanical stretching had additive effects on osteoblast numbers and Runx2 expression. These results emphasize the power of mechanical stretch in promoting osteoblastogenesis. Thus, mechanical signals are potential PPAR{gamma} modulators counteracting adipocyte overdifferentiation and osteoblastogenesis inhibition, as summarized in Fig. 7Go.


Figure 7
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  		FIG. 7. The effect of mechanical stretching on PPAR{gamma}: proposed mechanism. Mechanical stretching promotes osteoblastogenesis by both up-regulating Runx2 and down-regulating PPAR{gamma}. Rosiglitazone-induced PPAR{gamma} activation promotes adipogenesis and decreases osteoblastogenesis whereas GW9662, a potent antagonist of PPAR{gamma}, induces the opposite. Mechanical stretching reduces the stimulation of adipogenesis and the inhibition of osteoblastogenesis induced by Rosiglitazone while increasing the osteoblastic stimulation induced by GW9662.

 
Conclusions
Overall, our findings show that mechanical stimuli have a pivotal role in modifying the osteoblastogenesis/adipogenesis balance in different species, at the cell, tissue, and organism levels, by challenging two key transcription factors, Runx2 and PPAR{gamma}, which are strongly interdependent in serving osteoblastogenesis or adipogenesis. These results provide new insights into a physiological mechanism by which physical exercise might promote bone formation. Controversial duality of PPAR{gamma} as a therapeutic target for obesity-associated insulin resistance on the one hand, and as an adipogenic determination factor that might lead to osteopenia on the other hand, has to be clarified. Nevertheless, our data suggest that osteoblastogenesis, when inhibited secondary to TZD treatment, could be restored in part by a cyclic mechanical regimen.


   Acknowledgments
 
We thank Dr. Christian Dani (Unité Mixte de Recherche 6543 Centre National de la Recherche Scientifique/Université de Nice Sophia-Antipolis) for helpful discussion.


   Footnotes
 
This study was supported by the European Space Agency: European Research in Space and Terrestrial Osteoporosis Contract No. 14232/NL/SH (CCN3) and Microgravity Program AO-99-122 Contract No. 14426, and by the Institut National de la Santé et de la Recherche Médicale within the "ATC vieillissement 2002" program.

Present address for V.D. and A.M.: The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160.

Disclosure Summary: The authors have nothing to disclose.

First Published Online February 22, 2007

Abbreviations: ALP, Alkaline phosphatase activity; AMV, avian myeloblastosis virus; bMSC, bovine mesenchymal stem cell; FCS, fetal calf serum; PNP-p, p-nitrophenyl phosphate; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; TZD, thiazolidinedione.

Received December 18, 2006.

Accepted for publication February 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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