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Phosphate Stimulates Matrix Gla Protein Expression in Chondrocytes through the Extracellular Signal Regulated Kinase Signaling Pathway

INSERM (Institut National de la Santé et de la Recherche Médicale), Unité (U) Mixte de Recherche 791 (M.J., M.M., P.W., J.G.), Université de Nantes, Faculté de chirurgie dentaire, LIOAD (Laboratoire d’ingénierie Ostéo-articulaire et dentaire), Nantes F-44042, France; LBCM (Laboratoire de Biologie Cellulaire et Moléculaire) (D.M.), Université du Littoral Côte d’Opale, Boulogne-sur-mer F-62327, France; INSERM U533 (M.R.-D., C.C.-T.), Institut du Thorax, Nantes F-44035, France; INSERM U443 (O.C.), Biomatériaux et Réparation Tissulaire, Bordeaux F-33076, France; and INRA (Institut National de la Recherche Agronomique) UMR703 (Y.C.), Ecole Nationale Vétérinaire, Développement et pathologie du tissu musculaire, Nantes F-44307, France

Address all correspondence and requests for reprints to: J. Guicheux, Institut National de la Santé et de la Recherche Médicale Unité 791, Laboratory of Osteoarticular and Dental Tissue Engineering, University of Nantes, 1 Place Alexis Ricordeau, 44042 Nantes cedex 1, France. E-mail: Jerome.guicheux{at}nantes.inserm.fr.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Whereas increasing evidence suggests that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting, respectively. The activation and role of MAPKs were, respectively, determined by Western blotting and the use of specific inhibitors. Immunohistological detection of ERK1/2 was performed in rib organ cultures from newborn mice. The results indicate that Pi markedly stimulates expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways reveals that Pi activates ERK1/2 in a cell-specific manner, because the stimulation was observed in ATDC5 and primary chondrocytes, MC3T3-E1 osteoblasts, and ST2 stromal cells, but not in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocks Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, mainly, the effects of Pi. These data demonstrate, for the first time, that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.

	Introduction

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LONGITUDINAL BONE GROWTH occurs by endochondral ossification. During this process, a growth plate cartilage template is replaced by bone in a temporally and spatially coordinated manner (1). Chondrogenesis is initiated with condensation of mesenchymal precursor cells, which is followed by their differentiation into chondrocytes. Growth plate chondrocytes subsequently enter a phase of proliferation before differentiating into hypertrophic chondrocytes. These chondrocytes mineralize their extracellular matrix, undergo apoptosis, and are finally replaced by osteoblasts that produce a bony matrix (2). Many extracellular factors, including growth factors and hormones, are involved in the regulation of chondrogenesis, chondrocyte proliferation, and differentiation (3). Among these numerous factors, extracellular inorganic phosphate (Pi) has been proposed as a regulator of growth plate chondrocyte differentiation, apoptosis, and extracellular matrix mineralization (4, 5, 6). Phosphate homeostasis in mammals is mainly regulated by three organs: the kidney, intestine, and bone. Deregulation of the proteins or hormones that control phosphatemia can lead to hypophosphatemic rickets characterized by defective cartilage and bone formation (7, 8). Conversely, hyperphosphatemia, which is associated with chronic renal disease, may induce vascular calcification (9). Interestingly, vascular calcification probably arises through Pi-induced transdifferentiation of vascular smooth muscle cells (VSMC) toward bone-like cells (9). Although the mechanisms by which phosphate induces vascular calcification are not yet fully elucidated, the reported down-regulation of matrix Gla protein (MGP) expression in phosphate-rich conditions may be involved (10).

MGP is mainly expressed in VSMC and chondrocytes (11). MGP-deficient mice develop severe calcification of arteries and cartilage, thus indicating a role for MGP as a calcification inhibitor (12). Overexpression of MGP in the chick limb bud has also shown that MGP can inhibit both cartilage mineralization and endochondral ossification (13). An in vitro study in the ATDC5 mouse chondrogenic cell line showed that MGP is expressed in late hypertrophic cells and controls both apoptosis and mineralization (14), therefore confirming a role for MGP in regulating mineralization by chondrocytes. Because Pi has been suggested to be a regulator of this late differentiation stage of growth plate chondrocytes (6), we speculated that Pi modulates MGP expression in growth plate chondrocytes.

Despite a large body of evidence indicating that Pi is a specific signal for differentiation of chondrocytes (6), osteoblasts (15), and VSMC (9), the intracellular signaling pathways activated by Pi are poorly elucidated. Only one recent study indicated that Pi modulates osteopontin gene expression in osteoblastic cells through a well-defined member of the MAPKs (16). MAPKs are members of the family of serine/threonine kinases. All MAPK pathways consist of cascades of phosphorylation in which MAPK-kinase-kinases first activate downstream MAPK kinases, which then phosphorylate MAPK. Targets of MAPK include cytoplasmic proteins and transcription factors (17). Three major MAPK-dependent signaling cascades have been identified in mammalian cells: ERK1/2, p38 kinases, and c-Jun-N terminal kinases (JNK1/2). The role of MAPK signaling pathways in regulating chondrocyte proliferation and differentiation has been widely investigated (18, 19, 20). Surprisingly, and despite growing evidence indicating a role for MAPK and Pi in chondrocyte differentiation, the effect of Pi on signaling pathways in growth plate chondrocytes has not yet been investigated.

In view of the previously mentioned data and to better understand the molecular mechanisms induced by Pi in chondrocytes, we sought to investigate the effects of Pi on MGP expression and MAPK activation in ATDC5 cells and primary mouse chondrocytes. Here, we demonstrate, for the first time, that Pi stimulates expression of MGP in growth plate chondrocytes mainly through the ERK1/2 signaling pathway.

	Materials and Methods

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Materials
Cell culture plastic ware was purchased from Corning-Costar (Corning BV Life Sciences, Schiphol-Rijk, The Netherlands). Fetal calf serum (FCS) was obtained from D. Dutscher (Brumath, France). A 1:1 mixture of DMEM and Ham’s F12 medium (DMEM/F12) was provided by MP Biomedicals (Strasbourg, France). -MEM, MEM, DMEM, L-glutamine, penicillin and streptomycin (P/S), trypsin/EDTA, TRIzol reagent, deoxyribonuclease, deoxyribonucleoside triphosphates, TaqDNA polymerase, NuPAGE 4–12% Bis-Tris gel, and polyvinylidene difluoride. Invitrolon membrane was obtained from Invitrogen Corp. (Paisley, UK). UO126 was purchased from CalBiochem (Merck Eurolab, Darmstadt, Germany). Avian myeloblastosis virus-reverse transcriptase, random primers, and recombinant ribonuclease inhibitor (RNAsin) were purchased from Promega (Charbonnières, France). SyBr Green detection reagents were obtained from Molecular Probes Inc. (Leiden, The Netherlands) and Titanium Taq DNA polymerase was from Clontech (Saint Quentin Yvelines, France).

Protein content was determined using the Pierce Coomassie Plus assay (Pierce, Rockford, IL). The anti-phospho-ERK1/2 (9101), phospho-JNK1/2 (9251), phospho-p38 (9211), ERK1/2 (9102), p38 (9212), JNK1/2 (9252), and antirabbit IgG horseradish peroxidase-linked (7074) and antimouse IgG horseradish peroxidase-linked (7076) antibodies were purchased from Cell Signaling Inc. (Beverly, MA). The anti-MGP antibody was purchased from Alexis Corp. (Lausen, Switzerland). The antiactin antibody was obtained from Chemicon International, Ltd. (Hampshire, UK). The Western blotting detection system was obtained from Amersham Biosciences. Goat serum (X0907) was obtained from Dako (Trappes, France). Immunoreactivity was detected with a streptavidin-biotin-peroxidase technique (P0397; Dako), and 3,3'-diaminobenzidine was used as a chromogen (K3465; Dako). All other chemicals were from standard laboratory suppliers and were of the highest purity available.

Cells and culture conditions
ATDC5 cells (21) were used after a low number of passages and were routinely grown in maintenance medium consisting of DMEM/F12 (1:1) containing 5% FCS, 1% P/S, and 1% L-glutamine. Cells were subcultured once a week using trypsin/EDTA and maintained at 37 C in a humidified atmosphere with 5% CO₂ in air. To induce chondrogenesis and nodule formation, ATDC5 cells were seeded at 1.5 x 10⁴/cm² in a differentiation medium consisting of maintenance medium supplemented with 10 µg/ml bovine insulin (I), 10 µg/ml human transferrin (T), and 3 x 10^–8 M sodium selenite (S) and cultured for 21 d. The medium was replaced every 2 d. To reduce the nonspecific effects of agonists present in the culture medium, cells were incubated in ITS-free MEM with 0.5% FCS for 24 h before stimulation with Pi. Pi was used as a mixture of NaH₂PO₄ and Na₂HPO₄ (pH 7.2). When UO126 was added, an equivalent amount of dimethyl sulfoxide was used as a control.

Low-passage MC3T3-E1 cells (22) were cultured for 8–10 d (10,000 cells/cm²) in -MEM containing 10% FCS, 1% P/S, and 1% L-glutamine. Cells were cultured at 37 C in a humidified atmosphere with 5% CO₂ in air, and the medium was replaced every 2 d. Pi was added 24 h after incubation in MEM containing low serum levels (0.5%). The fibroblastic L929 cell line (23), the myogenic C2C12 cell line (24), and the stromal ST2 cell line (25) were grown in DMEM supplemented with 10% FCS, 1% L-glutamine, and 1% P/S. Cells were cultured until confluence at 37 C in a humidified atmosphere with 5% CO₂ in air, and the medium was replaced every 2 d. Pi was added 24 h after incubation in medium containing low serum levels (0.5%).

Primary chondrocytes were prepared from ventral rib cages of 1- to 3-d-old mice as previously described (26). Briefly, chondrocytes were isolated from rib cages by digestion of cartilage with bacterial collagenase after complete elimination of soft tissue by preliminary digestions with pronase and bacterial collagenase. Cells were then plated at a density of 50,000 cells/cm² and cultured in DMEM containing 10% FCS, 1% P/S, and 1% L-glutamine for 10 d at 37 C in a humidified atmosphere with 5% CO₂ in air. The medium was replaced every 2 d. These cells were previously shown to express the major chondrocytic markers, including type II collagen and type X collagen (26, 27). Pi was added 24 h after incubation in medium containing low serum levels (0.5%).

RNA isolation
Cells were seeded in 25-cm² flasks for RNA isolation. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. Briefly, lysis of the cells in TRIzol was followed by centrifugation at 10,000 x g for 15 min at 4 C in the presence of chloroform. The upper aqueous phase was collected, and the RNA was precipitated by addition of isopropanol and centrifugation at 7500 x g for 5 min at 4 C. RNA pellets were washed with cold 75% ethanol, dried, reconstituted in sterile water, and quantified by spectrometry.

RT- and real-time quantitative PCR analysis
After deoxyribonuclease I digestion, RNA samples (2.5 µg) were reverse transcribed using avian myeloblastosis virus-reverse transcriptase and random primers in a total volume of 30 µl. Real-time quantitative PCR was performed in the iCycleriQDetection System (Bio-Rad Laboratories, Hercules, CA) using SyBr Green detection and Titanium Taq DNA polymerase according to the manufacturer’s recommendations. The following temperature profile was used: 40 cycles of 15 sec at 95 C and 1 min at 60 C. Cycle numbers obtained at the log-linear phase of the reaction were plotted against a standard curve prepared with serially diluted cDNA samples. Expression of the target gene was normalized to glyceraldehyde-3-phosphate dehydrogenase levels. The sequences of primers for mouse MGP cDNA were 5'-TCAACAGGAGAAATGCCAACAC-3' (forward) and 5'-CGGTTGTAGGCAGCGTTGT-3' (reverse). The sequences of primers for mouse glyceraldehyde-3-phosphate dehydrogenase cDNA were 5'-GAAGGTCGGTGTGAACGGAT-3' (forward) and 5'-CGTTGAATTTGCCGTGAGTG-3' (reverse). PCR primers were synthesized by MWG Biotech (Ebersberg, Germany). The Ct (cycle threshold) method was used to calculate relative expression levels as previously described (28). Results are reported as fold change in gene expression relative to control conditions (untreated cultures).

Western blotting
After treatment, cells were rapidly frozen in liquid nitrogen before lysis at 4 C and were conserved at –80 C until use. Cells were lysed by addition of a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM potassium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 20 mM ß-glycerophosphate, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride , and 1 mM NaF. The insoluble material was pelleted at 12,000 x g for 10 min at 4 C. The protein concentration of cell lysates was determined with a Pierce Coomassie-Plus-protein assay. Twenty micrograms of total protein were resolved by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoridemembrane according to the manufacturer’s protocol (Invitrogen). Membranes for JNK1/2 and phospho-JNK1/2 were blocked in 5% nonfat dry milk in PBS/Tween 20 and probed in 5% BSA in PBS/Tween 20 (1/1000). For all other antibodies, membranes were blocked and probed in 5% nonfat dry milk in PBS/Tween 20. The ERK1/2, phospho-ERK1/2, p38, and phospho-p38 antibodies were diluted 1/1000, the MGP antibody 1/500, and the actin antibody 1/2000. Primary antibodies were detected using antirabbit or antimouse horseradish peroxidase-conjugated secondary antibodies diluted in 5% nonfat dry milk in PBS/Tween 20 (1/2000). The blots were visualized by enhanced chemiluminescence development using a Western blotting detection system. Semiquantitative analysis of protein level and normalization to actin levels were realized by densitometry (Q550 IW; Leica, Wetzlar, Germany).

Growth plate organ culture
Skeletal preparations were performed as follows. One- to 3-d-old mice were killed, skinned, and eviscerated. Ribs were collected in ice-cold PBS solution containing antibiotics. Each rib was incubated in a well containing 2 ml FCS-free DMEM supplemented with antibiotics. Explants were then treated with 10 mM Pi for 15 min at 37 C in a humidified atmosphere with 5% CO₂ in air. After the incubation period, explants were immediately immersed in 4% phosphate-buffered formalin.

Immunohistochemistry
Ribs were fixed in 4% phosphate-buffered formalin for 24 h, dehydrated in graded ethanol, and embedded in paraffin. Longitudinal sections (5 µm) were collected on polylysine-coated slides. Sections were deparaffinized in xylene and rehydrated. For unmasking antigen, sections were heated in 10 mM sodium citrate buffer (pH 6.0) for 10 min. Sections were then incubated in 3% H₂O₂ for 10 min to inhibit endogenous peroxidase followed by three washes with PBS. The sections were then blocked in goat serum diluted 1:5 in PBS to block nonspecific binding sites for 1 h at room temperature. Sections were incubated overnight at 4 C with a rabbit polyclonal primary antibody directed against mouse phospho-ERK1/2 or mouse ERK1/2 diluted 1:100 in blocking solution. After three washes in PBS, the secondary antibody diluted in blocking solution (1:300; goat antirabbit antibody conjugated with biotin) was added and incubated for 30 min at room temperature. Immunoreactivity was detected with a Dako streptavidin-biotin-peroxidase kit using the manufacturer’s instructions. Sections were counterstained for 30 sec in hematoxylin, dehydrated, and mounted with permanent mounting fluid. Negative controls were performed by substituting the primary antibody with PBS. Positive cells were stained brown.

Statistical analysis
Each experiment was repeated at least three times with similar results. Results are expressed as mean ± SEM of triplicate determinations. Comparative studies of means were performed by using one-way ANOVA followed by a post hoc test (Fisher’s projected least significant difference) with a statistical significance at P < 0.05.

	Results

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Effect of Pi on MGP expression
To determine whether Pi plays a role in regulating expression of MGP in growth plate chondrocytes, we first tested the effect of Pi on d 21 in ATDC5 cells at a stage when MGP is expressed (14). ATDC5 cells were treated with 10 mM Pi for up to 32 h, and MGP expression was analyzed by real-time PCR (Fig. 1A). The results indicate that MGP expression was already significantly increased after only 4 h of Pi treatment in comparison to the control. After 24 h, the steady-state level of mRNA encoding MGP was stimulated by more than 3-fold compared with the control. Between 24 and 32 h, the stimulation of MGP expression decreased (Fig. 1A). To confirm the stimulatory effect of Pi in a nontransformed chondrocyte model, we used primary growth plate chondrocytes isolated from ribs of newborn mice. A treatment time of 24 h, corresponding to the maximal stimulation of MGP mRNA level in ATDC5 cells, was chosen to test the effect of Pi in primary chondrocytes. A 24-h treatment with Pi stimulated the steady-state level of mRNA encoding MGP. Indeed, the analysis by real-time RT-PCR indicated a significant threefold increase in MGP expression compared with the control (Fig. 1B).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Effect of Pi on MGP mRNA expression in chondrocytes. ATDC5 cells (A) and primary rib-derived chondrocytes (B) were cultured as described in Materials and Methods before treatment with 10 mM Pi for the indicated times (A) or for 24 h (B). The effect of 10 mM Pi on MGP mRNA levels was assessed by real-time PCR as described in Materials and Methods. Results are reported as fold increase in gene expression. Data are representative of experiments with similar results.*, P < 0.05 compared with untreated cells.

View larger version (39K): [in this window] [in a new window]   FIG. 2. Effect of Pi on MGP protein expression in ATDC5 cells. ATDC5 cells were cultured as described in Materials and Methods before treatment with 10 mM Pi for the indicated times. The effect of 10 mM Pi on MGP at the protein level was analyzed by Western blot (A) as described in Materials and Methods using specific antibodies against MGP and actin as indicated. Semiquantitative analysis of MGP protein level in response to Pi treatment was performed by densitometric analysis (B). A representative experiment is shown (n = 3).*, P < 0.05 compared with untreated cells.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effect of Pi on MAPK phosphorylation in ATDC5 cells. ATDC5 cells were cultured as described in Materials and Methods before treatment with 10 mM Pi for 15 min (A) or for the indicated times (B). At the end of the incubation period, cells were rapidly frozen in liquid nitrogen before lysis at 4 C. The resulting samples were analyzed by Western blotting using specific antibodies against phospho (P)-ERK1/2, P-p38, and P-JNK1/2 or antibodies against ERK1/2, p38, and JNK1/2 as indicated.

Effect of Pi in primary chondrocytes and growth plate organ cultures
To further assess the influence of Pi on MAPK, we next questioned whether Pi may affect ERK1/2, p38, and JNK1/2 phosphorylation in cultured primary chondrocytes. As illustrated in Fig. 4, Pi enhanced the phosphorylation of ERK1/2 in primary chondrocytes. In contrast, Pi failed to affect the phosphorylation level of p38 and JNK1/2 MAPK (Fig. 4).

View larger version (38K): [in this window] [in a new window]   FIG. 4. Effect of Pi on ERK1/2 phosphorylation in primary chondrocytes. Primary rib-derived chondrocytes were cultured as described in Materials and Methods and exposed to 10 mM Pi for 15 min. At the end of the incubation period, cells were rapidly frozen in liquid nitrogen before lysis at 4 C. The resulting samples were analyzed by Western blotting using specific antibodies against phospho (P)-ERK1/2, P-p38, and P-JNK1/2 or antibodies against ERK1/2, p38, and JNK1/2 as indicated.

View larger version (89K): [in this window] [in a new window]   FIG. 5. Effect of Pi on ERK1/2 phosphorylation in the growth plate. Nondecalcified sections of rib growth plates of 3-d-old mice were stained with a rabbit antibody against mouse phospho-ERK1/2 or ERK1/2 and detected by a goat antirabbit antibody conjugated with biotin. A, Hematoxylin-eosin staining of rib growth plate without treatment showing the different zones of the growth plate differentiation (magnification, x10). PZ, proliferative zone; HZ, hypertrophic zone; OZ, ossification zone. B, Negative controls for ERK1/2 and phospho-ERK1/2 in hypertrophic zone were performed by omitting primary antibodies. C, Immunohistochemical staining for phospho-ERK1/2 (P-ERK1/2) or ERK1/2 in PZ after 15 min treatment with 10 mM Pi. D, Immunohistochemical staining for phospho-ERK1/2 (P-ERK1/2) or ERK1/2 in HZ after 15 min treatment with 10 mM Pi. (B, C, and D, magnification, x40). Arrows indicate the presence of immunostained cells (brown staining). Data are representative of experiments with similar results.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Cellular specificity of Pi-induced phosphorylation of ERK1/2. Confluent L929, C2C12, ST2, and MC3T3-E1 cells were cultured as described in Materials and Methods and treated with 10 mM Pi for 30 min. At the end of the incubation period, cells were rapidly frozen in liquid nitrogen before lysis at 4 C. The resulting samples were analyzed by Western blotting using specific antibodies directed against P-ERK1/2 or ERK1/2. Data are representative of experiments with similar results.

View larger version (26K): [in this window] [in a new window]   FIG. 7. Effect of UO126 on Pi-induced MGP expression in ATDC5 cells. ATDC5 cells were pretreated with UO126 (1, 10, or 30 µM) for 1 h followed by treatment with 10 mM Pi for 24 h (A) and for 15 min (B) as described in Materials and Methods. A, The effect of UO126 and 10 mM Pi on MGP mRNA levels was assessed by real-time PCR as described in Materials and Methods. Results are reported as fold increase in gene expression. Data are representative of experiments with similar results.*, P < 0.05 compared with untreated cells. B, The effect of UO126 on Pi-stimulated ERK1/2 phosphorylation was analyzed by Western blotting using specific antibodies against phospho-ERK1/2 or ERK1/2 as indicated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Despite the clinical impact of Pi homeostasis, the mechanisms through which Pi modulates cell differentiation remain poorly understood. Remarkably, a common effect of Pi on cell behavior is the induction of matrix calcification. Indeed, Pi stimulates mineralization not only in chondrocytes and osteoblasts, but also in VSMC (30). In VSMC, generation of Pi by ß-glycerophosphate correlates with mineralization and with a decrease in the levels of MGP, an inhibitor of the calcification process (10). The inhibitory role of MGP in mineralization has been demonstrated by the dramatic vascular calcification observed in MGP null mice (12). In light of these data, we questioned whether Pi could also modulate MGP expression in growth plate chondrocytes.

In this regard, the effects of Pi on MGP expression were first investigated in a chondrogenic in vitro model: the ATDC5 cell line. ATDC5 cells represent a well-characterized culture model for growth plate chondrocyte differentiation, because these cells display the stage of proliferating chondrocytes, nodule formation, and hypertrophic stages, ending with matrix mineralization (21). Furthermore, a previous in vitro study performed in ATDC5 cells showed that MGP displays a biphasic expression pattern during chondrocyte differentiation (14) similar to that observed in the growth plate in vivo (11). Interestingly, we found that, in concentrations that induce mineralization (6), Pi stimulates the steady-state level of mRNA encoding MGP both in ATDC5 cells and in primary chondrocytes after a 24-h treatment. This increase could be related to a transcriptional activation of the MGP gene or to an increase in MGP mRNA stability. The time course experiment performed in ATDC5 cells revealed an increase in mRNA level as soon as 4 h after treatment. Whereas this early response suggests that ERK1/2 directly activates MGP gene transcription, we cannot exclude that this activation could occur through an intermediate gene product. Of interest, the increase in MGP expression at the mRNA level is followed by an enhanced production of the corresponding protein.

Because crystal formation occurs in our conditions in the first 8 h (6), it is possible that the effect of Pi on MGP expression after a 24-h treatment is mediated by the formation of apatite crystals. Experiments aimed at blocking crystal formation or phosphate transport may help us determine whether these effects are the result of phosphate ions or apatitic crystals (31, 32). Nevertheless, the up-regulation of MGP synthesis by Pi may be explained by setting off a feedback mechanism to control Pi-induced mineralization. Likewise, the overexpression of MGP in late differentiating ATDC5 (14) and chick hypertrophic chondrocytes (13) was demonstrated to reduce matrix mineralization. These data strengthen the hypothesis of a MGP-dependent negative feedback loop controlling mineralization.

Despite the great number of studies dealing with the inhibitory effects of MGP on calcification, the mechanisms by which MGP inhibits mineralization remain unclear. At least two mechanisms could account for the inhibitory function of MGP on calcification. On the one hand, MGP is a member of the mineral-binding Gla protein family (33), which includes osteocalcin. MGP binds calcium ions and hydroxyapatite via its five -carboxylated glutamic acid (Gla) residues. Inhibition of the -carboxylation of Gla residues with warfarin both in cell culture and in vivo results in increased matrix mineralization, suggesting that the mineral-binding Gla residues are crucial for regulation of matrix mineralization (13, 34). The in vivo mutagenesis experiments that showed that the Gla residues are required for MGP antimineralization functions support this hypothesis (35). On the other hand, MGP has been shown to modulate the biological activity of the TGF-ß superfamily members such as bone morphogenetic proteins (BMPs) (36). It was thereafter reported that MGP modulates BMP activity in mesenchymal cell differentiation (37). More recently, MGP has been demonstrated to exert a dose-dependent inhibitory effect on osteoblastic differentiation through interference with binding of BMP-2 to its receptor (38). Viewed together, these data highlight the possibility that MGP inhibits mineralization at least through two concomitant mechanisms involving its calcium and BMP binding abilities.

To better understand the physiological effect of Pi on cell differentiation and MGP expression, we investigated the cellular mechanisms activated by Pi. We first found that Pi enhances ERK1/2 phosphorylation in a time-dependent manner in ATDC5 cells. We also confirmed that Pi activates ERK1/2 in primary chondrocytes. However, ATDC5 and primary chondrocyte cultures may contain cells at different stages of differentiation: undifferentiated cells around nodules and probably proliferative, prehypertrophic, and hypertrophic chondrocytes inside nodules. These models are therefore poorly adapted to the identification of cells in which ERK1/2 is activated in response to Pi. In this context, to clearly localize the cellular populations in which Pi induces ERK1/2 phosphorylation, we embarked on growth plate organ cultures. This model further allows maintaining the original spatial organization of chondrocytes within their extracellular matrix as found in intact growth plate. The effects of Pi found in this model could therefore mimic the endogenous activity of Pi in the growth plate. Our results indicate that Pi enhances ERK1/2 phosphorylation in hypertrophic and proliferative chondrocytes. In addition, the phosphorylation of ERK1/2 induced by Pi does not seem to be restricted to growth plate chondrocytes because in vitro, Pi also enhanced the ERK1/2 phosphorylation in osteoblastic MC3T3-E1 and bone marrow-derived stromal ST2 cells. Because stromal cells have the capacity to differentiate into bone-forming cells (39), one can assume that ST2 cells share a common feature with osteoblasts and chondrocytes that would allow stromal cells to trigger a signal for ERK1/2 activation.

We found that the stimulation of MGP expression by Pi was prevented by specific blockade of the ERK1/2 signaling pathway. With respect to the large variety of signaling pathways potentially involved in the regulation of chondrocyte differentiation, it seems reasonable to speculate that ERK1/2 is not the exclusive pathway activated by Pi. Although protein kinase C and proteasome have already been suggested to mediate the effects of Pi on gene expression in osteoblastic cells (16), it remains however to be determined whether these pathways play a role in Pi-induced MGP expression in chondrocytes. Finally, the molecular mechanisms by which ERK could mediate the effects of Pi on MGP expression are unknown. Downstream signaling elements from ERK involve transcription factors that regulate gene expression (17). Interestingly, it has been shown that the MGP gene is a specific target of the Fos-related antigen, which is associated with the regulation of bone mass through bone matrix production by osteoblasts and chondrocytes (40). The relationship between Fos-related antigen and Pi-induced MGP expression should be paid further attention.

Conclusion
This study demonstrates, for the first time, that Pi stimulates the expression of MGP in growth plate chondrocytes. In addition, we show that Pi is able to activate at least one member of the MAP kinase family, ERK1/2. Finally, our data indicate a role for ERK1/2 in the regulation of MGP. These findings provide new insights into the molecular mechanisms induced by Pi in growth plates.

	Acknowledgments

 
The authors gratefully acknowledge Lydie Guigand and Jérome Amiaud from the National Veterinary School of Nantes as well as Françoise Moreau from Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 791 for their technical assistance in histological investigation. We also thank Marie Thérèse Corvol for helpful comments (INSERM Unité 747).

	Footnotes

 
This work was partially supported by grants from INSERM, the French Society of Rheumatology, and the French Ministry of Research. M.J. received a fellowship from INSERM and Région des Pays de la Loire.

First Published Online October 26, 2006

Abbreviations: BMP, Bone morphogenetic protein; FCS, fetal calf serum; JNK, c-Jun-N terminal kinase; MGP, matrix Gla protein; Pi, inorganic phosphate; P/S, penicillin and streptomycin; VSMC, vascular smooth muscle cells.

Received June 7, 2006.

Accepted for publication October 16, 2006.

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