Phosphate Is a Specific Signal for ATDC5 Chondrocyte Maturation and Apoptosis-Associated Mineralization: Possible Implication of Apoptosis in the Regulation of Endochondral Ossification

Authors


  • The authors have no conflict of interest

Abstract

Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification.

Introduction: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered.

Materials and Methods: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-1 (EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis).

Results: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces π-induced mineralization.

Conclusions: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

INTRODUCTION

Vertebrate long bones form through a process called endochondral ossification, in which a cartilage template is replaced by a bony matrix. This process begins with the condensation of undifferentiated mesenchyme, from the core of which cells differentiate into chondrocytes, whereas cells at the periphery form the perichondrium. After a phase of proliferation, chondrocytes differentiate, expressing type II, type IX, and type XI collagen and sulfated glycosaminoglycans. Then chondrocytes further differentiate and become hypertrophic, expressing mainly type X collagen, and mineralize the extracellular matrix (ECM), by mechanisms that could involve matrix vesicles (MV). The fate of hypertrophic chondrocytes is still a matter of debate. The first hypothesis is that chondrocytes are converted into bone cells through a process called trans-differentiation.(1,2) However, there is growing evidence that terminally differentiated chondrocytes undergo programmed cell death in mammals.(3) In situ investigation of the cartilage growth plate revealed that hypertrophic chondrocytes are characterized by a loss of mitochondrial membrane potential,(4) a decrease in Bcl-2/Bax protein ratio,(5) and are TUNEL positive.(6–8) In addition, deletion of the gene encoding bcl-2 in mice leads to accelerated maturation of chondrocytes and shortening of long bones.(5)

Among the factors modulating chondrocyte maturation, much attention has been paid to the parathyroid hormone-related peptide/indian hedgehog axis,(9) bone morphogenic proteins,(10) and oxygen supply.(8) Surprisingly, whereas inorganic phosphate (Pi) levels strongly increase both in the ECM and in the cells from the proliferative to the hypertrophic region of the growth plate,(11–15) the possible role of Pi on chondrocyte maturation and endochondral ossification has not been fully investigated. Disorders in Pi homeostasis lead to abnormal endochondral ossification. On one hand, inefficient reabsorption of phosphate by kidney leading to hypophosphatemia is associated with defective mineralization of the skeleton,(16) which manifests as rickets and osteomalacia.(17) On the other hand, phosphate retention or hyperphosphatemia, accompanying chronic renal disease, is associated with soft tissue calcification and abnormal bone metabolism.(18) In addition, Pi has been suggested to be rate-limiting for cartilage mineralization,(19) and Pi or Pi released from β-glycerophosphate induces or stimulates mineralization in several models of chondrocyte culture.(20,21) Recently, Pi has also been reported to modulate cell differentiation(22–24) and apoptosis.(25,26)

Despite this large body of evidence indicating that Pi stimulates chondrocyte differentiation, mineralization, and apoptosis, no study has precisely explored the relationships between these effects, and the role of Pi during endochondral ossification remains unclear. In an elegant paper, Proudfoot et al.(27) suggested that in hyperphosphatemic conditions, Pi-induced mineralization of vascular smooth muscle cells through apoptotic-dependent mechanisms. Relationships between apoptosis and mineralization, both in physiological and pathological situations, have already been pointed out.(28) Although matrix vesicles and apoptotic bodies are ultrastructurally quite different, it was reported that MV share several characteristics with apoptotic bodies formed in some pathological conditions.(25,28,29) In this context, the aims of this study were to investigate the role of Pi on chondrocyte maturation during endochondral ossification and explore the possible relationships between differentiation, apoptosis, and mineralization using the ATDC5 cell line. This cell line, established by Atsumi et al.(30) from the mouse teratocarcinoma cells AT805, mimics many of the events described for differentiation of epiphyseal chondrocytes and calcifies the ECM without addition of an exogenous phosphate source.(31) This work is an effort to provide new insight in cartilage physiopathology and understand the role of apoptosis during organogenesis of mineralized tissues.

MATERIALS AND METHODS

Materials

Cell culture plasticware was purchased from Falcon (Becton-Dickinson, Franklin Lakes, NJ, USA) and Corning-Costar (Integra Biosciences, Wallisellen, Switzerland). Fetal calf serum (FCS) was obtained from Dominique Dutscher (Brumath, France). α-MEM, glutamine, antibiotics, and trypsin/EDTA were obtained from Life Technologies (Paisley, UK). A 1:1 mixture of DMEM and Ham's F12 medium (DMEM/F12) was provided by ICN Biochemicals (Orsay, France). Phosphonoformic acid (PFA), cycloheximide, ascorbic acid, calcium chloride, dimethyl sulfoxide (DMSO), bovine insulin, transferrin, and sodium selenite were purchased from Sigma (St Louis, MO, USA). ZVAD-fmk was obtained from R&D Systems (Abingdon, UK). Cycloheximide and ZVAD-fmk were dissolved as concentrated solutions in DMSO.

DNAse I and TaqDNA polymerase were obtained from Life Technologies. Avian myeloblastosis virus-reverse transcriptase (AMV-RT), random hexamers, and recombinant ribonuclease inhibitor were purchased from Promega (Madison, WI, USA). Trizol reagent was from Life technologies.

Cell tracker green (CTG) and ethidium homodimer 1 (EthD-1) were purchased from Molecular Probes (Leiden, The Netherlands). The in situ cell death detection kit, based on the TUNEL, obtained from Roche Molecular Biochemicals (Meylan, France), was generously provided by Dr Dominique Heymann. Antibodies (BD PharMingen) were kindly provided by Dr Lisa Valette. All other chemicals were from standard laboratory suppliers and were of the highest purity available.

Cell and culture conditions

ATDC5 cells, derived from mouse teratocarcinoma stem cells, differentiate into chondrocytes in the presence of insulin.(30) In this study, ATDC5 cells (used between the second and the fourth passage) were routinely grown in a maintenance medium consisting of DMEM/F12 (1:1) containing 5% FCS, 10 μg/ml human transferrin (T), 3 × 10−8 M sodium selenite (S), 1% antibiotics, and 1% glutamine. Cells were subcultured once a week using trypsin/EDTA and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. To induce chondrogenesis and cartilage nodule formation, ATDC5 cells (1.5 × 104/cm2) were seeded in a differentiation medium consisting of maintenance medium supplemented with 10 μg/ml of bovine insulin (I) for the first 3 weeks. When indicated, DMEM/F12 was replaced on day 21 by α-MEM containing 5% FCS and the ITS supplement at 37°C in a humidified atmosphere of 3% CO2 in air. Medium was replaced every second day. In these conditions, mineralization begins at day 29. DMEM/F12 and α-MEM contain 0.9 and 1 mM Pi, respectively. Pi concentrations of 2, 4, 7, and 10 mM were obtained by addition of a mixture of NaH2PO4 and Na2HPO4 (pH 7.3). DMEM/F12 and α-MEM contain 1 and 2.4 mM Ca, respectively; higher Ca concentrations were obtained by adding calcium chloride. To reduce the nonspecific effects of agonists present in culture medium, cells were incubated in low-serum (0.5%) and insulin-free medium for 24 h before stimulation with Pi (in all experiments excepted those in which cells were cultured for 7 days). All inhibitors were added 30 minutes before treatments with agonists.

RNA isolation

Cells were seeded in 25-cm2 flasks for RNA isolation. After indicated times, media were removed, and cell layers were rinsed with RNase-free PBS and stored at −80°C until total RNA was extracted using the Trizol reagent according to the manufacturer's instructions. Briefly, lysis of the cells in Trizol was followed by centrifugation at 10,000g, 4°C, for 15 minutes in the presence of chloroform. The upper aqueous phase was collected, and the RNA was precipitated by addition of isopropanol and centrifugation at 7500g, 4°C, for 5 minutes. RNA pellets were washed with cold 75% ethanol, dried, reconstituted with sterile water, and quantified by spectrometry.

Reverse transcription and polymerase chain reaction analysis

After DNAse I digestion, RNA samples (2 μg) were reverse transcribed using AMV-RT and random hexamer primers in a total volume of 30 μl. Template cDNAs (5 μl) were then amplified in a typical 50 μl polymerase chain reaction (PCR) reaction containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1 μM of the respective primers, 200 μM dNTP, and 2.5 U of TaqDNA polymerase. The magnesium chloride concentration was 1.5 mM. The absence of DNA contamination in RNA preparations was tested by including RNA samples that had not been reverse-transcribed. Primers sequences and annealing temperatures are detailed in Table 1. Amplifications were carried out in an Eppendorf master cycler (Dr Vaudaux AG, Schonenbuch, Switzerland) under the following conditions: denaturation for 3 minutes at 94°C followed by cycles of 30 s denaturation at 94°C, 30 s annealing at the primer specific temperature, and 45 s elongation at 72°C. All PCR results show amplification products obtained in the linear range of amplification. Semiquantitative analysis of RNA levels and normalization to glyceraldehyde phosphate dehydrogenase (GAPDH) levels were realized by densitometry (Q500; Leica, Heidelberg, Germany).

Table Table 1. Primers Used for RT-PCR Analysis (Sequences, Annealing Temperature [T], Amplification Size, and Reference)
original image

Confocal microscopy

Imaging was performed using a confocal laser-scanning inverted microscope (Leica TCS SP1) equipped with an argon/krypton laser. Cells were visualized using either a ×63/1.4 APO or a ×16/0.5 immersion oil objective lens. The data were collected with a simultaneous dual-channel detector and visualized with a 24-bit imaging system including Leica TCS NT software.

Briefly, for CTG and EthD-1 staining, cells were treated with 5 μM CTG for 30 minutes at 37°C, for 30 minutes in FCS containing medium, and finally for 30 minutes in 1 μM EthD-1 at room temperature. The fluorescence of CTG incorporated into living cells was detected using an isothiocyanate (FITC)-fluorescent set : λex = 488 nm; λem collected = 490–560 nm. The red EthD-1 emission was viewed using the confocal microscope (λex = 568 nm; λem collected = 580–620 nm).

For anti-active caspase-3 immunostaining, cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature, permeabilized for 5 minutes on ice, and treated with a rabbit anti-active caspase 3 antibody for 30 minutes at room temperature. After washing the cells, an anti-rabbit FITC-conjugated antibody was added for 30 minutes in obscurity. For negative control, the anti-active caspase 3 antibody was omitted. The fluorescence was detected using an isothiocyanate (FITC)-fluorescent set: λex = 488 nm; λem collected = 490–560 nm. Before incubation with antibodies, some coverslips were stained with EthD-1 as described above.

For DNA fragmentation staining, cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized for 5 minutes on ice, and treated by the TUNEL reagent for 1 h at 37°C. All coverslips were mounted with Antifade kit (Molecular Probes). The green TUNEL emission was analyzed using an isothiocyanate (FITC)-fluorescent set as described above.

Fourier transform infrared microspectroscopy

ATDC5 nodules (six multiwell plates) were fixed in 70% ethanol and placed on a barium fluoride (BaF2) disk. The crystallinity was analyzed from the periphery to the center of the nodules to study the maturation of the mineral phase. In parallel, growth plate cartilage was studied as a control. Briefly, 1-week-old mice were killed, and their tibias were fixed in 70% ethanol, dehydrated, and embedded in glycolmethylmethacrylate. Sections (2 μm thick) were cut with a Supercut 2050 microtome (Reichert-Jung, Heidelberg, Germany) and placed on a BaF2 disk. Spectra were recorded with a Magna-IR 550 spectrometer (Nicolet, Trappes, France) equipped with an IR-plan Advantage microscope (Spectra-Tech, Shelton, CT; X15 Reflachromat lens) fitted with a high-sensitivity mercury cadmium tellurite (MCT) detector. Sample positioning was realized with a motorized x-y stage under computer control. Fourier transform infrared microspectroscopy (FTIR) data were acquired with the spatial resolution provided by a 20 × 20-μm2 aperture to prevent diffraction artifacts and maximize the signal/noise ratio. Infrared spectra were recorded at 4 cm−1 resolution with 512 or 1024 interferograms co-added and Happ-Genzel apodization. Omnic software (Nicolet, Trappes, France) was used for data analysis. Residual H2O and CO2 absorptions were automatically subtracted. Before mineral analysis by deconvolution of the ν1ν3 PO4 domain (k = 2.3 and σ = 22.5 cm−1), collagen and proteoglycan absorptions were minimized by subtracting the spectrum of non-mineralized regions.

Transmission electron microscopy

ATDC5 nodules (six multiwell plates) were fixed in cacodylate buffered 4% glutaraldehyde for 15 minutes at 4°C, washed, and post-fixed in cacodylate buffered 2% osmium tetroxide for 20 minutes at 4°C. Nodules were dehydrated in successive dilutions of ethanol and embedded overnight in Epon at 37°C and for 2 other days at 55°C. Sections (80 nm thick) were cut with an Ultracut E ultramicrotome (Reichert-Jung), mounted on copper grids, stained with uranyle acetate, and observed on a Jeol 1010 electron microscope at a voltage of 100 kV.

Analytical methods

ECM mineralization was measured as the amount (μg) of total Ca present in cell layers (12 multiwell plates) determined by atomic absorption spectrometry at 422.7 nm (Unicam 989 AA spectrometer; SOLAR) after extraction of the cells with 4 M HCl containing 1% LaCl3. Protein content (mg) was determined with the Pierce Coomassie Plus assay reagent (Pierce, Rockford, IL). Cell survival (12 multiwell plates) was determined by measuring at 490 nm the formation of formazan from MTS tetrazolium with the CellTiter 96 Aqueus Non-radioactive cell proliferation assay (Promega, Wallisellen, Switzerland). Results are expressed as relative MTS activity and compared with untreated cells. Each experiment was repeated at least once with similar results. Results are expressed as mean ± SE of triplicate determinations. Comparative studies of means were performed 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

As previously described,(30) ATDC5 cells proliferate to reach confluence between days 5 and 7 (Fig. 1A). In the presence of insulin, they begin to form nodules (Fig. 1B) in which they differentiate,(31) expressing type II collagen, link-protein, and sulfated glycosaminoglycans. From days 12 to 15, cells in the nodules express type X collagen, become hypertrophic, and from day 29, mineralize their ECM (Fig. 1C). In this study, effects of Pi were investigated on day 21 in differentiated cells, at a stage when mineralization has not yet started.

Figure FIG. 1..

ATDC5 cell differentiation. ATDC5 cells (1.5 104 cells/cm2) were cultured for (A) 7 days, (B)21 days, and (C) 35 days in differentiation medium containing insulin as indicated in the Materials and Methods section. Standard phase contrast photomicrographs (magnification ×40) illustrate the various phases of cell differentiation: (A) proliferation, (B) nodule formation and cellular hypertrophy (*), and (C) extracellular matrix mineralization (arrow), as evidenced by the black deposits observed within nodules.

Pi stimulates ECM mineralization

We first asked whether Pi could initiate mineralization by hypertrophic cells as early as on day 21. ATDC5 cells were grown for the first 21 days in DMEM/F12 (which contains 1 mM Ca and no ascorbic acid) and were treated on day 21 in α-MEM (which contains 2.4 mM Ca and 50 μg/ml ascorbic acid), as initially described for ATDC5 differentiation.(31) Mineralization was quantified by measurement of Ca content by atomic absorption spectrometry and expressed as micrograms Ca per milligrams protein in cell layers. In these conditions, Pi dose-dependently stimulated Ca deposition in 7 days (Fig. 2A). The time course of Ca deposition indicated that mineralization with 4 mM Pi began within 8 h and strongly increased after 24 h and until 48 h (Fig. 2B). Ca deposition induced by 4 mM Pi seemed to be dependent on Pi entry into cells because phosphonoformic acid (PFA), an inhibitor of phosphate transporters,(18,35) blocked mineralization (Fig. 2C). Then, we questioned whether Pi could initiate mineralization in DMEM/F12, which contains no ascorbic acid and only 1 mM Ca. In these conditions, while ascorbic acid was not necessary (data not shown), Pi-induced mineralization required 2.5 mM Ca, which comes close to Ca levels present in α-MEM (Fig. 2D). Finally, to determine whether mineralization induced by 4 mM Pi and 2.4 mM Ca was dependent on chondrocyte differentiation, ATDC5 cells grown without insulin were treated with 4 mM Pi in α-MEM containing 2.4 mM Ca (Fig. 2E). In these conditions, the absence of calcium deposition indicates that mineralization was dependent on chondrocyte differentiation and not merely caused by physico-chemical precipitation of calcium phosphate.

Figure FIG. 2..

Pi-stimulated calcium deposition in cell layers. Cells were cultured in DMEM/F12 (1 mM Pi, 1 mM Ca) for 21 days before treatment. Calcium deposition in cell layers was determined by spectrometry as detailed in the Materials and Methods section. (A) Calcium deposition in cell layers after 7 days of treatment with indicated Pi concentrations in α-MEM (1 mM Pi, 2.4 mM Ca). (B) Time course of calcium deposition after treatment with 4 mM Pi in α-MEM. (C) Effects of 0.5 mM PFA on calcium deposition by cells treated with 4 mM Pi for 24 h in α-MEM. PFA was added 30 minutes before Pi treatment. (D) Effects of calcium concentration in DMEM/F12 on calcium deposition after 24-h treatment. (E) Effects of insulin (10 μg/ml) on 4 mM π-stimulated mineralization in α-MEM (2.4 mM Ca) after 7 days of treatment. *p < 0.05 compared with control without Pi and Ca supplementation. **p < 0.05 compared with 4 mM Pi-treated cells. NS, not significant.

Pi-stimulated calcification is similar to in vivo mineralization

Mineralization at the different Pi concentrations was analyzed by transmission electron microscopy (TEM) after 8 days of treatment in α-MEM (Fig. 3). With 4 mM Pi, crystals were observed in the ECM (Fig. 3A), in association with collagen fibrils (Fig. 3B). TEM micrographs showed thin and relatively long crystals associated parallel to the long axis of collagen fibrils, consistent with physiological mineralization (Fig. 3C). In contrast, with higher Pi concentrations (10 mM), nonphysiologic crystal deposition was observed, probably caused by a mere physicochemical precipitation (Fig. 3D). X-ray diffraction analyses of the crystals formed with 10 mM Pi and 2.4 mM calcium revealed that they consisted in apatite (data not shown). Thus, crystal composition and maturation in vitro with 4 mM Pi (Fig. 4A) were investigated by Fourier transform infrared microspectroscopy (FTIR-M) and compared with those of crystals in murine growth plate cartilage in vivo (Fig. 4B). After deconvolution between 1200 and 900 cm−1 of the ν1ν3 PO4 domain (which gives information on crystal composition and organization), the high intensity of the bands at 1145, 1125, and 1112 cm−1 indicated that crystals both in vitro and in vivo contain large amounts of HPO42− ions and nonapatitic PO43− ions (Figs. 4C and 4D). This suggests that crystals formed in vitro and in vivo are poorly crystalline and may contain weakly organized regions. As a control, deconvoluted spectrum of the most mature regions from dentin slices (Fig. 4E) indicates the presence of a more organized apatite (week intensity for the peaks at 1145, 1125, and 1112 cm−1).(36) Moreover, crystals from in vitro and in vivo cartilage have also a very poor if not nil carbonate content as revealed by the weak intensity of the ν2CO3 domain (data not shown). During maturation, crystals in vitro and in vivo evolved similarly toward a greater crystallinity with a loss of HPO42− (1145 cm−1) and nonapatitic PO43− ions (1125 and 1112 cm−1).

Figure FIG. 3..

Pi-stimulated ECM mineralization. ATDC5 cells were cultured for 21 days in DMEM/F12 and treated for 7 days with (A-C) 4 mM Pi or (D) 10 mM Pi in α-MEM (2.4 mM Ca). Calcified nodule sections were observed by TEM, as detailed in the Materials and Methods section. (A; ×5000) Note the localization of crystals in ECM (arrows). (B; ×6000) Mineralized areas join together along collagen fibrils. (C; ×25,000) Crystals are associated with collagen fibrils (arrow). (D; ×30,000) With 10 mM Pi, crystal formation occurs independently of collagen fibrils.

Figure FIG. 4..

Crystal composition and maturation in vitro and ex vivo. (A) Nodules in ATDC5 cell culture grown for 21 days in DMEM/F12 before treatement with 4 mM Pi in α-MEM for 7 days (×40). (B) Murine cartilage growth plate stained with Movat's pentachrome (×200). Crystal maturation, that is, evolution of crystal composition during aging, from the periphery to the center of a mineralizing nodule in culture (A; arrrow indicates tissue maturation) and from the mineralizing septae to the junction with newly formed bone in unstained cartilage growth plate (B; arrow indicates tissue maturation), was monitored by FTIR microspectroscopy (C and D, respectively). (E) FTIR microspectroscopic spectrum of the most mature region from dentin. Spectra in the ν1ν3PO4 domain between 1200 and 900 cm−1 were deconvoluted as described in the Materials and Methods section.

Consequently, because TEM and FTIR-M experiments suggest that a 4 mM Pi concentration reproduces in vitro the mineralization observed in cartilage growth plate, subsequent experiments were conducted using this concentration.

Pi stimulates ATDC5 cell differentiation

The effects of Pi on ATDC5 differentiation were investigated by RT-PCR (Fig. 5). Cells were incubated on day 20 in DMEM/F12 without insulin containing 0.5% FCS and treated on day 21 with 4 mM Pi. Results show that Pi increased type X collagen mRNA levels, whereas it did not increase mRNA levels for osteopontin (OPN; Fig. 5A). The effect of Pi on type X collagen expression was inhibited by 0.5 mM PFA, suggesting that Pi enters cells to increase type X collagen mRNA levels (Fig. 5B). To investigate whether Pi-stimulated type X collagen production was involved in mediating the effect of Pi on mineralization, we measured Ca deposition after treatment with Pi for 48 h in the presence of the translation inhibitor cycloheximide. In these conditions, cycloheximide not only failed to inhibit mineralization but even enhanced Ca deposition induced by 4 mM Pi and 2.4 mM Ca (Fig. 5C). Because cycloheximide is also an apoptogen, we next investigated the effects of Pi on apoptosis.

Figure FIG. 5..

Pi-stimulated collagen X mRNA expression and effect of cycloheximide on ECM mineralization. ATDC5 cells were cultured for 21 days in DMEM/F12 before treatment. (A) The time course effect of 4 mM Pi on collagen X (Coll X) and osteopontin (OPN) mRNA levels was assessed by RT-PCR, as described in the Materials and Methods section. A representative agarose gel is shown. (B) The effect of the Pi transport inhibitor PFA (0.5 mM) on collagen X mRNA expression induced by 4 mM Pi was assessed by RT-PCR. PFA was added 30 minutes before cells were treated with Pi for 2 h. A representative agarose gel is shown. MW, molecular weight marker; RT, control without reverse transcriptase. (C) Effects of the protein translation inhibitor, cycloheximide (CHX, 5 μM) on Pi-induced mineralization. CHX was added 30 minutes before treatment with Pi and Ca for 48 h. Calcium deposition in cell layers was determined by spectrometry as detailed in the Materials and Methods section. *p < 0.05 compared with ion pair-treated cells.

Pi stimulates ATDC5 cell apoptosis

The effect of Pi on cell viability was assessed by measuring the mitochondrial conversion of MTS into formazan. Figure 5 shows that, if Pi alone had no effect on cell viability on day 21, it dramatically reduced MTS activity when added together with 2.4 mM Ca (Fig. 6A). Cycloheximide used alone only slightly decreased cell viability but increased the effects of Pi and Ca on ATDC5 cell death. Inhibition of cellular Pi uptake with 0.5 mM PFA blocked cell death induced by Pi and Ca, suggesting that cell death is dependent on Pi entry into ATDC5 cells (Fig. 6B). Similar experiments realized with undifferentiated ATDC5 cells, cultured for only 7 days, indicated that in the absence of nodules, Pi and Ca failed to stimulate cell death (data not shown). Confocal microscopy was used to localize living and dead cells within the nodules after 48 h treatment with 4 mM Pi and 2.4 mM Ca. Cultures were treated with CTG and EthD-1, which, respectively, stain living cells in green and dead cells in red. Pictures were collected from the top to the bottom of the nodules (Fig. 6C). In both treated and untreated cultures, dead cells were found essentially in the center of the nodule (differentiated cells), whereas viable cells were mostly observed in the nodule periphery (undifferentiated cells). The proportion of dead cells in nodules was markedly increased in cells treated with 4 mM Pi compared with untreated cells (data not shown).

Figure FIG. 6..

Pi effects on ATDC5 cell viability. ATDC5 cells were cultured for 21 days in DMEM/F12 before treatment. (A) ATDC5 cell viability was assessed by MTS activity after 48-h treatment with either 4 mM Pi, 2.4 mM Ca, or both, in the presence or absence of 5 μM cycloheximide (CHX). (B) Effect of the Pi transport inhibitor PFA (0.5 mM) on MTS activity after treatment with 4 mM Pi and 2.4 mM Ca. PFA was added 30 minutes before treatment with 4 mM Pi and 2.4 mM Ca for 48 h. (C; ×160) Confocal microscopy analysis for localization of viable cells, stained with cell tracker green (CTG), and dead cells, stained with ethidium homodimer-1 (EthD-1), in a nodule after treatment with 4 mM Pi and 2.4 mM Ca for 48 h. Observations were performed using a ×16 immersion oil objective lens. *p < 0.05 compared with control. **p < 0.05 compared with ion pair-treated cells.

To examine whether cell death in response to Pi occurs through an apoptotic process, biochemical and morphological features of apoptosis were investigated. Treatment of ATDC5 cells for 24 h with Pi and/or Ca affected the expression levels of mRNAs encoding for bcl-2 and bax, which are respectively anti- and proapopotic proteins. Densitometric analysis of agarose bands indicated that the value of bcl-2/bax ratio was reduced after treatment with both Pi and Ca (Fig. 7, Panel 1). On the other hand, treatment with Pi or Ca alone had weak or no effects on the bcl-2/bax ratio.

Figure FIG. 7..

Apoptotic features of ion pair-induced cell death. ATDC5 cells were cultured for 21 days in DMEM/F12 before treatment. (Panel 1A) The effects of 24-h treatment with 4 mM Pi and/or 2.4 mM Ca on bcl-2 and bax mRNA expression were investigated by RT-PCR, as described in the Materials and Methods section. A representative agarose gel is shown. (Pane 1B) Densitometric representation after normalization to GAPDH levels (MW, molecular weight; RT, control without reverse transcriptase). (Panel 2, A-D) Effects of treatment with 4 mM Pi and 2.4 mM Ca for 48 h on ATDC5 DNA fragmentation as determined by TUNEL. (Panel 2A) Negative control (without enzyme; ×630). (Panel 2B) Positive control (DNase treatment; ×630). Nodule observation in (Panel 2C) light microscopy and (Panel 2D) fluorescence (×160). The dotted line indicates the nodule periphery. (Panel 2, E and F) ATDC5 cells were treated with 4 mM Pi for 7 days in α-MEM (2.4 mM Ca). The TEM micrographs show chromatin condensation (Panel 2E; arrow) and mineralized collagen fibrils (Panel 2F; arrow).

DNA fragmentation in Pi- and Ca-treated cells was investigated by the TUNEL method. Figure 7 (Panel 2) shows that only Pi- and Ca-treated cells in the center of the nodules were stained. The negative control without TUNEL enzyme was only faintly stained, and cells treated with DNase (positive control) stained positively, independent of their localization. Cell morphology was then assessed by TEM after 7 days of treatment with 4 mM Pi in α-MEM (Fig. 7, Panel 2). Only individual cells, surrounded by dense collagen framework or located in proximity of mineralized areas, exhibited typical apoptotic morphology with chromatin condensation at the periphery of the nucleus.

Pi-stimulated mineralization is related to caspase activation

Finally, implication of caspases in Pi-induced cell death was investigated by staining ATDC5 cells with an anti active caspase-3 antibody (Fig. 8). Positive staining was observed specifically within the nodules. Cells that stained positively for active caspase-3 (Fig. 8A) also stained positive for EthD-1 (Fig. 8B), indicating that they had already lost part of their viability. Overlay of the images shown in Figs. 8A and 8B showed condensed chromatin stained with EthD-1 and cytoplasmic localization of active caspase-3 (Fig. 8C). The negative control (without anti-active caspase 3 antibody) showed only a barely noticeable green staining (Fig. 8D). Involvement of caspases in Pi induced cell death was further suggested by the fact that ZVAD-fmk, a broad range caspase inhibitor, blocked the decrease in cell viability induced by Pi and Ca (Fig. 8E). Finally, we investigated whether modulating apoptosis by caspase inhibition might affect mineralization. Inhibition of apoptosis with ZVAD-fmk significantly decreased mineralization induced by Pi and Ca (Fig. 8F), suggesting that mineralization is related to caspase activity.

Figure FIG. 8..

Implication of caspases in ATDC5 cell apoptosis and ECM mineralization. ATDC5 cells were cultured for 21 days in DMEM/F12 before 24-h treatment with 4 mM Pi and 2.4 mM Ca. Confocal microscopy analysis performed with a ×63 objective lens of (A) anti-active caspase-3 immunostaining and (B) EThD-1 staining. A and B are overlays of 19 images collected from the top toward the bottom of a nodule. C is an overlay of A and B. (D) Negative control (without anti-active caspase 3 antibody). (E and F) Effect of 100 μM ZVAD-fmk on (E) MTS activity and (F) mineralization. ZVAD-fmk was added 30 minutes before treatment with ions. *p < 0.05 compared with ion pair-treated cells.

DISCUSSION

In the cartilage growth plate, Pi levels increase considerably from the proliferative to the hypertrophic region, both in cells(13) and in the ECM.(15) Whether increase in intracellular Pi concentration results from an active Pi transport from the ECM or from intracellular Pi generation is not yet fully understood. On one hand, chondrocytes highly express sodium-dependent(37,38) and -independent(39) Pi transporters that could account for high intracellular Pi levels; on the other hand, this high Pi content and the low ATP/Pi ratio reported in vivo(13) could result from hypoxic injury as it was reported in other cell types.(40) In the poorly vascularized growth plate, the most hypoxic chondrocytes are localized in the hypertrophic zone.(8) In the present study, we sought to explore the relationship between chondrocyte maturation and cartilage mineralization induced by Pi using the ATDC5 in vitro model of endochondral ossification.(31) Among the reported models of chondrocyte differentiation, ATDC5 cells express many of the events described for differentiation of epiphyseal chondrocytes and the ability to mineralize spontaneously, although mineralization with low ion levels (1 mM Pi and 1 mM Ca) only begins after 4 weeks in culture. Moreover, these cells are a convenient tool to study Pi effects on chondrocyte biology because, like chondrocytes in vivo,(37) they express type III NaPiTs, including glvr-1 and/or ram, both in the cells and in matrix vesicles.(41) Finally, results of this study show that mineralization in ATDC5 culture seems identical to that in murine growth plate cartilage, indicating that this cell line is also suitable to study the mineralization process in vitro.

In in vitro models of chondrocyte cultures, an exogenous phosphate source, consisting of Pi(21) or β-glycerophosphate (β-GP) salts,(20,42) is frequently added to induce or stimulate mineralization. Although some studies have precisely investigated the composition of the mineral phase formed with varying concentrations of Pi or β-GP,(43) Pi or β-GP are often used at concentrations far beyond the physiological range. In the present study, for instance, if Pi dose- and time-dependently increased Ca deposition in ATDC5 cell culture from 1 to 10 mM, concentrations above 4 mM led to nonphysiologic calcifications, probably through calcium phosphate precipitation caused by an excessive Ca × Pi ion product. In contrast, the fact that 4 mM Pi and 2.4 mM Ca failed to induce mineralization in absence of insulin argues strongly that these concentrations induce crystal formation in differentiated cell culture through a cell-mediated process. Moreover, ultrastructural and spectroscopic analyses demonstrated that 4 mM Pi and 2.4 mM Ca in the ATDC5 culture medium induces a mineralization that seems identical to that in murine growth plate in vivo. The crystals in vitro are deposited in the ECM. They are long-shaped and(44) deposited parallel to the long axis of collagen fibrils. Moreover, this crystal formation may not be merely caused by Pi and Ca from the medium precipitating on collagen fibrils because inhibition of Pi transport with PFA blocks Ca deposition, suggesting that Pi needs to enter cells and/or matrix vesicles.(45) However, we cannot totally rule out that these effects are mediated by inhibition of crystal formation by PFA.(46) Crystal composition was analyzed by FTIR-M that associates FTIR spectroscopy to microscopy and allows crystal composition(47) to be studied as a function of location.(43) Both in vivo and in vitro crystals consist in poorly organized apatite, contain large amounts of HPO42− and very few carbonate ions and evolve toward a greater crystallinity with a loss of acidic phosphate groups. These findings are consistent with previously published data on cartilage mineralization(44,47) and suggest that 4 mM is an adequate concentration to study the in vitro effects of Pi. The same Pi concentration was reported to mimic in primary chondrocyte culture the in vivo mineralization.(43,48)

Pi reportedly induces or upregulates expression of genes exclusively expressed in mineralizing cell types.(22,23,42) For instance, Pi stimulates osteopontin gene transcription in osteoblastic MC3T3-E1 cells.(23) Moreover, it was recently shown that Pi induces the osteoblastic phenotype in culture of smooth muscle cells, with induction of Cbfa1 expression.(18,24) In ATDC5 cells, Pi specifically upregulates mRNA levels for the late chondrogenic marker type X collagen. Because type X collagen is a characteristic marker of hypertrophic chondrocytes, these results suggest that Pi accelerates terminal chondrocyte differentiation.(22,42) In cartilage growth plate, the increase in both intra- and extracellular compartments in Pi levels could thus play a key role during chondrocyte maturation. On the other hand, Pi does not seem to stimulate osteopontin transcription in ATDC5 cells as it was reported in osteoblastic MC3T3-E1 cells,(23) suggesting that Pi stimulation of type X collagen mRNA levels is specific and not merely caused by stimulation of the basal transcriptional activity of hypertrophic chondrocytes. The precise molecular mechanisms involved by Pi in gene expression remain to be elucidated. These effects may at least be dependent on Pi entry into cells because they are blocked by PFA,(18,23) which was demonstrated to inhibit Pi uptake in the concentration range used in the present study.(18,35) Today, specific signaling pathways potentially activated by Pi transport or by increasing intracellular Pi levels have not yet been reported and are under intense investigation.

Pi significantly stimulates apoptosis in the presence of 2.4 mM Ca, whereas separately Pi and Ca have no effect. Several studies reported that Pi induces chondrocyte or osteoblast apoptosis,(4,25) but a recent report indicated that Pi-stimulated cell death is dramatically modulated by Ca levels.(49) Pi-induced apoptosis in chondrocytes seems dependent on Pi entry into cells. On the other hand, it was reported that inhibitors of Ca transport fail to rescue cells from ion pair-treated cultures,(49) suggesting an implication of Ca sensing receptors in chondrocyte maturation.(50) It is, however, not unconceivable that ion entry into cells are provoked by membrane damage caused by Pi and Ca precipitates. It is unlikely that Pi- and Ca-induced cell death results from direct cellular toxicity, at the mitochondrial level for instance,(51) because it is maturation stage dependent.(25) ATDC5 apoptosis in our study was restricted to differentiated cells (within the nodules), and when Pi and Ca were added in the proliferation phase before the formation of nodules, there was no induction of apoptosis. The maturation stage dependency of Pi-induced apoptosis may be related to the fact that only hypertrophic chondrocytes contain large amounts of intracellular Ca,(11,12,52) particularly within their mitochondria.(53) An alternative possibility is that terminally differentiated chondrocytes undergo a maturation-dependent loss of mitochondrial function(4) independent of Pi levels that, together with a decrease in levels of the cell death inhibitor bcl-2,(5,54) would render cells more susceptible to Pi toxicity.(4) In the cartilage growth plate, there is a decrease in the Bcl-2/Bax protein ratio from the proliferative to the hypertrophic zone,(5) coinciding with the reported increase in Pi(13,14) and Ca(11,12,52) levels. In the present study, treatment with Pi and Ca led to a decrease in the Bcl-2/Bax ratio, which is believed to disrupt the mitochondrial membrane and promote release of mitochondrial components, irreversibly engaging the cell toward apoptosis. Among the mitochondrial components released during the apoptotic process, cytochrome c leads to subsequent activation of caspases.(55) Accordingly, Pi and Ca treatment led to caspase-3 activation in differentiated ATDC5 cells. Colocalization of cytoplasmic active caspase-3 and nuclear EthD-1 staining indicated that active caspase-3 positive cells were no more viable. Activation of caspases by Pi and Ca ions was further reinforced by the fact that a broad-range caspase inhibitor (ZVAD-fmk) completely rescued cells from death. Caspase-3 is known to cleave the inhibitor of caspase-3 activated DNase (CAD), leading to CAD release that is responsible for oligonucleosome fragmentation (DNA laddering). In the present study, confocal microscopic observations of ion pair-treated cells revealed that only the differentiated cells in nodules exhibited DNA fragmentation as determined by TUNEL, which was further confirmed by TEM observations that showed cells with characteristic apoptotic features, particularly chromatin condensation.

In the present ATDC5 in vitro model, Pi accelerates chondrocyte terminal differentiation, and together with Ca, induces apoptosis and ECM mineralization. Mineralization does not seem to require de novo protein synthesis, because blocking protein translation with cycloheximide did not prevent Ca deposition. However, it cannot be ruled out that enhanced calcium deposition with cycloheximide could result from the inhibition of the expression of an inhibitor of mineralization. Finally, results of the present study highlight the possible relationships between apoptosis and mineralization: Ca deposition is significantly enhanced by the apoptogen cycloheximide and reduced by the apoptosis inhibitor ZVAD-fmk. Moreover, TEM observation showed that apoptotic cells were almost always located in the vicinity of mineralized collagen matrix. Although a relationship between apoptosis and mineralization had already been observed in other tissues in pathological conditions, such as vascular calcifications,(27) this study is, to our knowledge, the first one to report relationship between mineralization and apoptosis in a model of endochondral ossification. Precise mechanisms of chondrocyte apoptosis induced by Pi and Ca and leading to mineralization remain largely unknown. It is likely that MV play an important role in epiphyseal calcification,(56) and some studies have implied that apoptotic bodies, similar to MV, could initiate pathological or physiological calcification.(27–29,56) Moreover, it was reported that mitochondrial Pi and Ca released during chondrocyte hypertrophy and/or apoptosis are included into nascent MV and/or apoptotic bodies and could participate to matrix mineralization.(11,53,57–59) ZVAD-fmk could thus inhibit mineralization by blocking the release of apoptotic bodies from cells.(60) Finally, it was reported that if Pi levels regularly increase from proliferative to hypertrophic regions of the growth plate,(14) Ca levels are relatively constant in all zones of the plate, increasing only in hypertrophic zones, both in extracellular(15) and intracellular compartments.(11) Our data indicating that Pi is able to increase type X collagen expression on its own, but requires Ca to induce apoptosis-associated mineralization, might thus be of physiological relevance.

In conclusion, results presented in this study suggest an important role of Pi during endochondral ossification. Increased Pi levels from proliferative to hypertrophic regions may accelerate chondrogenic differentiation, and together with increased Ca levels in the hypertrophic zone, induce apoptosis-associated mineralization. Moreover, if apoptosis is believed to play an important role during organogenesis, this study suggests that programmed cell death is an essential process regulating skeletal growth.

Acknowledgements

This study was supported by grants from INSERM 9903. DM received a fellowship from the French Ministry of Research and Technology. CF received a fellowship from “region pays de la Loire.”

Ancillary