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Enhanced Expression of the Inorganic Phosphate Transporter Pit-1 IsInvolved in BMP-2Induced Matrix Mineralization in

Osteoblast-Like Cells

Atsushi Suzuki,1 Chafik Ghayor,2 Jrome Guicheux,3 David Magne,3 Sophie Quillard,3 Ayako Kakita,4 YasunagaOno,1 Yoshitaka Miura,4 Yutaka Oiso,4 Mitsuyasu Itoh,1 and Joseph Caverzasio2

ABSTRACT: Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport inBMP-2induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells.Experimental analysis suggest that this response is required for bone matrix calcification.

Introduction:Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an importantrole in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and itstransport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study,we investigated the role of Pi transport in BMP-2induced matrix mineralization.Materials and Methods: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time

periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression byNorthern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarinred staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy,electron diffraction analysis, and Fourier transformed infrared resolution (FTIR).Results: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells byincreasing theVmaxof the transport system. This effect was preceded by an increase in mRNA encoding Pit-1but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted byeither phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineraliza-tion induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway.Conclusions: Results presented in this study indicate that, in addition to its well-known effect on severalmarkers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through aselective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated bythe JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.J Bone Miner Res 2006;21:674683. Published online on February 20, 2006; doi: 10.1359/JBMR.020603

Key words: Pit-1 transporter, BMP-2, c-Jun-N-terminal kinase, osteoblast, mineralization

INTRODUCTION

INORGANIC PHOSPHATE(Pi) transport represents a particu-lar function of bone-forming cells for extracellular matrixmineralization.(1,2) As in all eukaryotic cells, osteogeniccells (i.e., osteoblasts and chondrocytes) are endowed withNa-dependent Pi transport systems driven by an inwardlydirected Na gradient.(3) The type III Pi transport systems,namely Pit-1 and Pit-2, are involved in Pi handling by os-teogenic cells.(4,5) In situ hybridization analysis revealed ex-pression of the type III transporter Pit-1 (Glvr-1) in a sub-population of hypertrophic chondrocytes duringendochondral bone formation, suggesting a potential role

for this Pi carrier in primary events of extracellular matrix

calcification.(6) Enhanced expression and a role of this Pitransporter in pathological calcification have also been re-cently suggested.(7,8) Pi transport in osteogenic cells notonly provides sufficient Pi for metabolic processes but alsoserves a specialized function in matrix vesicles that are mi-crostructures playing a critical role in initial events of bonematrix calcification.(9) Studies have shown that Pi transportactivity is low in matrix vesicles released from chondrocytes

and osteoblasts during the proliferative phase and increasesduring their differentiation.(4,10) The increase in Pi trans-port activity in matrix vesicles released during the forma-tion of the collagenous matrix is likely explained by anenrichment of Pi carriers during the differentiation process.In line with this concept, several in vitro studies have al-The authors state that they have no conflicts of interest.

1Division of Endocrinology, Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Japan; 2Serviceof Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, Geneva, Switzerland; 3INSERM EM99-03, School of Dental Surgery, Nantes, France; 4Department of Metabolic Diseases, Nagoya University Graduate School of Medicine,Nagoya, Japan.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 21, Number 5, 2006

Published online on February 20, 2006; doi: 10.1359/JBMR.020603 2006 American Society for Bone and Mineral Research

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ready shown a stimulation of Pi transport by well-knownosteotropic factors such as PTH and IGF-I in either osteo-blast-like cells(5,11,12) or chondrocytes,(10) as well as basicfibroblast growth factor in osteoblastic cells.(13) Bone mor-phogenetic proteins (BMPs) are members of the TGF-superfamily and exert a wide range of biological effects indifferent tissues. In particular, they contribute to the for-

mation of bone and connective tissues(14) by inducing thedifferentiation of mesenchymal cells into bone-formingcells.(15) This factor is one of the most potent stimulators ofosteoblastic cell differentiation mainly characterized by ex-pression of alkaline phosphatase (ALP), type I collagen,the production of osteocalcin, and bone matrix mineraliza-tion.(1618) Whether BMP-2 influences Pi transport in os-teogenic cells for inducing matrix mineralization is notknown and was investigated in this study. Results obtainedindicate that BMP-2 selectively enhances Pi transport ac-tivity in MC3T3-E1 osteoblast-like cells. This effect ismainly caused by enhanced expression of Pit-1 by a tran-scriptional process that probably involves the c-Jun-N-terminal kinase (JNK) pathway for its activation. Biochemi-cal and molecular experimental evidence suggests thatenhanced expression of this Pi transport system contributesto matrix mineralization induced by BMP-2 in osteoblasticcells.

MATERIALS AND METHODS

Chemicals

FCS, glutamine, antibiotics, and trypsin/EDTA were ob-tained from Gibco (Life Technologies, Basel, Switzerland).-MEM was purchased from Amimed (Bioconcept, All-schwill, Switzerland). Phosphonoformic acid (PFA), actino-mycin D, and cycloheximide were obtained from Sigma (St

Louis, MO, USA). SB203580, SP600125, and U0126 com-pounds were obtained from Calbiochem-Novabiochem(San Diego, CA, USA). Recombinant human BMP-2 waskindly provided by Genetics Institute (Cambridge, MA,USA). H3[

32P]O4and L-[3H]alanine were from DuPont de

Nemours (Brussel, Belgium). All other chemicals werefrom standard laboratory suppliers and were of the highestpurity available.

Cell culture and transfection

Murine calvaria-derived MC3T3-E1 osteoblast-like cells(ATCC, LGC Promochem, France) were grown in -MEMmedium containing 10% FCS, 1% (vol/vol) nonessentialamino acids, 100 IU/ml penicillin, and 100 g/ml strepto-

mycin. Subcultures were obtained once a week by removingthe cells from the dish using 0.1% collagenase and 0.25%trypsin in a Ca- and Mg-free Earles salt solution containing0.02% EDTA. Cultures were maintained at 37C in a hu-midified atmosphere of 5% CO2-95% air and medium waschanged every 23 days. Cells were seeded into 24-wellplates (40 103 cells/well) for transport and 6-well plates(150 103 cells/well) for matrix mineralization studies.

The Pit-1 antisense oligonucleotide (AS-Pit-1; ODN se-quence: TATTATTTAAAGAACCACTACACTAGAG-AATGGAATCTACTGTGGCAACGATTACTAGTA-CCCTAGCTGC) was synthesized by MWG-Biotech

GmbH (Ebersberg, Germany) and inserted into pcDNA3vector. MC3T3-E1 cells were transfected with either theAS-Pit-1 or empty vector (pcDNA3) using superfect re-agent following manufacturers instructions (QIAGEN,Venlo, The Netherlands). Transfected cells were selectedwith G418 sulphate (Calbiochem-Novabiochem) at 0.8, 0.4,and 0.2 mg/ml during the first, second, and third week, re-

spectively, after plasmid transfection. Selected cell lineswere cultured in the presence of 0.02 mg/ml G418 sulphate.G418 is an aminoglycoside similar in structure to gentami-cin B1. G418 blocks polypeptide synthesis by inhibiting theelongation step in both prokaryotic and eukaryotic cells.Resistance to G418 is conferred by the neo gene from Tn5encoding an aminoglycoside 3-phosphotransferase. G418 isthus used for selection of mammalian cells expressing a neocassette in a vector like pcDNA3.

Pi and alanine transport analysis

BMP-2 effect on Pi and alanine transport was analyzed inconfluent MC3T3-E1 cells cultured in -MEM mediumcontaining 2% FCS for 24 h. In some experiments, cellswere pretreated either with inhibitors or their vehicle for 1h before adding BMP-2 for 6 h. After treatment withagents, Pi and alanine transport activity was determined inEarles buffered salt solution (EBSS) containing either0.052.5 mM labeled H3[

32P]O4or 0.1 mM [3H]alanine, re-

spectively, as previously described.(13) Transport experi-ments started by rinsing three times the cell layer withEBSS. Then, 0.3 ml of EBSS containing labeled substrate (1Ci/ml) was added during 6 minutes for Pi and 2 minutesfor alanine transport analysis (corresponding to respectivetime-points of initial rate of transport activities(11)). Finally,the uptake solution was aspirated, and the cell layer was

rinsed three times with 0.3 ml of ice-cold EBSS. The celllayer was solubilized with 0.25 ml of 0.2N sodium hydrox-ide, and the radioactivity of a 200-l aliquot was counted bystandard liquid scintillation technique. As previously docu-mented, preliminary experiments indicated that the so-dium-independent component of Pi transport determinedin presence of 143 mM choline chloride was


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sion no. L20852).(22) The differences in sample loadingwere corrected by the expression of a housekeeping gene,eithercyclophilinorGAPDH. Probes were labeled with 50Ci [-32P] dCTP by random priming. Pre-hybridization(30 minutes) and hybridization (2 h) were performed inQuickhyb buffer (Stratagene, La Jolla, CA, USA), whichwas supplemented for the hybridization with 100 g/ml

salmon sperm DNA. Membranes were washed twice for 15minutes in 2 SSC, 0.1% SDS at 25C and for 1030 min-utes at 60C in 0.1 SSC, 0.1% SDS before exposition toKodak X-OMAT AR films for 1872 h at 80C.

Alizarin red staining

Confluent MC3T3-E1 cells (day 5) were treated every23 days with BMP-2 or its vehicle in -MEM culture me-dium containing 10% FCS and 10 mM -glycerophosphate.When indicated, cells were pretreated with agents for 1 hand stimulated by 1030 ng/ml of BMP-2 or its vehicle.After 6 weeks, mineralized nodules was determined byalizarin red staining. Calcified area was quantified using

NIH image analysis.

Mineral deposited in the organic matrix of

MC3T3-E1 cells

Cells were prepared as described above for alizarin redstaining. After 6-week treatment with either BMP-2 or ve-hicle, cells were fixed in cacodylate/glutaraldehyde (4%)buffer for 90 minutes at 4C and postfixed in cacodylatebuffer with 2% osmium tetroxide for 1 h at 4C. Sampleswere dehydrated with successive dilutions in ethanol andembedded in Epon overnight at 37C and during 2 addi-tional days at 55C. Sections (80 nm thick) were cut with anUltracut E ultramicrotome (Reichert-Jung, Heidelberg,Germany), mounted on copper grids, stained according toReynolds lead citrate stain method, and analyzed with aJeol 1010 electron microscope at 80 kV. The crystal struc-ture was determined by selected area diffraction patterns at100 kV and camera length of 50 cm.(23) The infrared spectrawere registered with a Magna IR 550 spectrometer (Nico-let, Trappes, France) coupled to an IR Plan Advantagemicroscope (15 reflachromat lens; spectra Tech, Shelton,CT, USA), equipped with a mercury-cadmium-telluride de-tector. The measurements were made on thin films (2 mthick) in the transmission configuration at 4 cm1 resolu-tion. Samples (with and without BMP-2 treatment) wereembedded in Epon, and sections were cut with an ultramicrotome and deposited on BaF2window for analysis. The

spectra were acquired with a spatial aperture of 40 40 m2

and corrected for residual H2O and CO2absorptions. Eponpeaks were also subtracted. For further detailed mineralanalysis, deconvolution of the n1n3 (PO4) was made withthe following typical parameters: k 2.3 and s 24.5cm1.

ALP activity

ALP activity was determined as previously described.(24)

Dose-dependent effect of BMP-2 on ALP activity was de-termined in culture medium containing 1% FCS after 48-hincubation. Cells were harvested in 0.2% Nonidet P-40 and

disrupted by sonication. The homogenate was centrifugedat 1500gfor 5 minutes, and ALP activity was determined inthe supernatant by the method of Lowry et al.(25)

Statistical analysis

Results are expressed as mean SE. A two-sided un-paired Studentst-test or ANOVA for multiple comparisonwas used for statistical analysis. A difference between ex-perimental groups was considered to be significant when

p< 0.05.

RESULTS

Characteristics of Pi transport stimulation by

BMP-2 in MC3T3-E1 cells

The effect of recombinant human BMP-2 was studied inMC3T3-E1 cells that responded quite well to BMP-2 forinducing expression of ALP (Fig. 1B). In confluent cells, wefound that this BMP dose-dependently and selectively in-creased Pi transport (Fig. 1A). A stimulatory effect was

detected at the dose of 1 ng/ml of BMP-2 with maximalstimulation at 30 ng/ml (Fig. 1A). Interestingly, the doseresponse effect on Pi uptake stimulation was different fromthat of ALP induced by BMP-2 (Figs. 1A and 1B). Thestimulation of Pi uptake induced by BMP-2 was detectedafter 3 h, further enhanced after 6 h, and reached a maximalvalue after 24-h incubation (Fig. 2). Alanine uptake was notsignificantly affected in the same conditions (Table 1). Be-cause alanine is transported into mammalian cells mainlythrough an Na co-transport system, this observation indi-cates that the effect of BMP-2 on Pi uptake is mediated bya selective cellular mechanism. Furthermore, the stimula-tory effect of BMP-2 on Pi uptake seemed to be dependenton RNA and protein synthesis, because it was completelyblocked in cells pretreated with either 2.5 g/ml actinomy-cin D or 5 M cycloheximide respectively (Table 2). Fi-nally, kinetic analysis indicated that the stimulatory effectof BMP-2 on Pi uptake is related to an increase in themaximal rate (Vmax) of the transport system, without achange in its apparent affinity constant (Km) for phosphate(Table 1).

Selective stimulation of Pit-1 by BMP-2 in

MC3T3-E1 cells

Northern blotting analysis showed that BMP-2 increasedPit-1 but not Pit-2 mRNA expression in MC3T3-E1 cellswith a time-course response similar to the time stimulation

of Pi uptake. An increase in Pit-1 mRNA was indeed de-tectable after 2 h and was nearly maximal after 24-h incu-bation (Fig. 3A). Expression of Pit-2 and of the housekeep-ing genecyclophilinwas not significantly affected by BMP-2. Preincubation of the cells with actinomycin D, whichmarkedly reduced the abundance of Pit-1 mRNA in ve-hicle-treated cells, completely blocked the induction ofPit-1 by BMP-2, further suggesting that this response isdependent on transcription (Fig. 3B). Inhibition of proteinsynthesis by preincubation with cycloheximide slightly re-duced the stimulation of Pit-1 mRNA expression inducedby BMP-2 without, however, blocking this response, sug-

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gesting that this effect of BMP-2 is, at least in part, inde-pendent of new protein synthesis (Fig. 3B).

Expression of Pit-1 and matrix mineralization

induced by BMP-2

Matrix mineralization of MC3T3-E1 cells was deter-mined by alizarin red staining and quantitative computer

FIG. 1. Dose-dependent effect of BMP-2 on Pi transport andALP activity in MC3T3-E1 osteoblast-like cells (A) For Pi trans-port (Pi TRANSP) analysis, cells were cultured for 4 days andswitched to-MEM containing 2% FCS for 24 h. Then, they wereincubated for 6 h with various concentrations of BMP-2 or itsvehicle. Pi uptake was determined in EBSS containing 0.1 mM32PO4. (B) For ALP analysis, cells were cultured for 5 days andswitched to culture medium containing 2% FCS for 24 h. Then,they were exposed to various concentrations of BMP-2 or its ve-hicle for 24 h. ALP activity and the amount of proteins weredetermined. Each value represents mean SE of four to six de-terminations from a representative experiment. *p < 0.05 com-pared with vehicle.

FIG. 2. Time-dependent effect of BMP-2 on Pi transport inMC3T3-E1 cells. Cells cultured for 4 days were switched to-MEM containing 2% FCS for 24 h. Then, they were incubatedwith either 10 ng/ml of BMP-2 () or its vehicle () for varioustime periods. Pi uptake was determined in EBSS containing 0.1mM 32PO4. Each value represents mean SE of five to six deter-minations from a representative experiment. *p< 0.01, comparedwith vehicle.

TABLE1. KINETICANALYSIS OFBMP-2-INDUCED PITRANSPORT

STIMULATION AND

ANALYSIS OF THE

SELECTIVITY OF

THIS

EFFECT BY MEASURINGALANINETRANSPORT

Kineticanalysis

Pi transport

Vmax

(nmol/6 minutes/well) K m

(mM)

Veh 0.398 0.02 0.491 0.04

BMP-2 0.705 0.03* 0.508 0.04Selectivity

Alanine transport

(pmol/2 minutes/well)

Veh 91.1 6.3

BMP-2 76.7 5.3

Confluent MC3T3-E1 cells were stimulated by either BMP-2 (10 ng/ml)

or its vehicle for 6 h. Then Pi and alanine uptake were determined. Valuesare the mean SE of five to six determinations.

*p < 0.001 compared with vehicle.

ROLE OF PI TRANSPORT IN BMP-2INDUCED MINERALIZATION 677


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image analysis after 6-week culture in the presence of 10mM -glycerophosphate. Under this experimental condi-tion, BMP-2 induced a dose-dependent increase in matrix

mineralization. Enhanced accumulation of mineral was de-

tected at the concentration of 3 ng/ml with a gradual in-

crease and near maximal effect between 30 and 100 ng/ml

BMP-2 (Fig. 4A), a dose relationship very close to that of

changes in Pi uptake induced by BMP-2 (Fig. 1A) and quite

different from changes in ALP activity (Fig. 1B). The cal-

cium-phosphate crystals formed in the extracellular organic

matrix of MC3T3-E1 cells cultured for 6 weeks with 30

ng/ml BMP-2 were examined by transmission electron mi-

croscopy (TEM), electron diffraction (EM), and resolution

enhanced Fourier transformed infrared (FTIR) spectros-

copy. As depicted in Fig. 4B, TEM analysis revealed that

thin and relatively long crystals are deposited in association

with collagen fibrils, an observation that is consistent with

physiological mineralization.(26) EM analysis indicated that

the pattern of ring diffraction, with a D-spacing of the first

and second ring of 0.35 and 0.28 nm, respectively (Fig. 4C),

corresponds to an apatite crystal. Resolution enhanced

FTIR analysis showed a typical IR spectrum in the region

of 8001800 cm1, similar to spectra obtained from mouse

trabecular bone (data not shown). Increased matrix miner-

alization induced by BMP-2 was completely blocked by 0.1

mM phosphonoformic acid (Fig. 5A), a competitive inhibi-

tor of Na-Pi cotransport.(27) This effect was observed in

presence of a slightly reduced BMP-2induced stimulation

of ALP in cells exposed to PFA compared with vehicle-

treated cells (Fig. 5B), suggesting that part of the blunting

effect of BMP-2 on matrix mineralization observed in PFA-

treated cells was probably caused by other factors than

merely an inhibition of Pi transport. Another mechanism by

which this analog of inorganic phosphate might have re-

duced matrix mineralization is through its reduction of the

rate of matrix vesicleinduced mineral formation, which

plays an important role in initiating the process of miner-alization.(28) Thus, to validate above data suggesting an im-

portant role of Pit-1 in matrix mineralization induced by

BMP-2, we constructed MC3T3-E1 cells expressing an an-tisense Pit-1 oligonucleotide (AS-Pit-1). Of several clonesstably transfected with AS-Pit-1, two expressed lower levelsof Pit-1 mRNA compared with other Pit-1 (Fig. 6, clones 2and 3) and empty vector transfected cells (data not shown).Clone 2 was used for further analysis because, in addition toexpressing a lower baseline and a blunted response toBMP-2 on Pi uptake (Fig. 6B and similar effect obtainedwith clone 3, data not shown), this clone had a near normalstimulation of ALP by BMP-2 (data not shown). The matrix

mineralization induced by BMP-2 in this clone was mark-edly decreased compared with cells stably transfected withthe empty vector (Fig. 6C), strongly suggesting that Pit-1plays a critical role in bone matrix mineralization inducedby BMP-2.

Role of MAP kinase pathways in the stimulation of

Pi transport and matrix calcification induced

by BMP-2

Previous studies have shown that both Smad and mito-gen-activated protein kinase (MAPK) pathways are essen-

TABLE2. EFFECT OFACTINOMYCIND OR CYCLOHEXIMIDE ONBMP-2INDUCED PITRANSPORT

Pi transport (pmol/6 minutes/well)

Vehicle 89.0 5.6BMP-2 148.1 7.6*

Actinomycin D 47.9 3.9

Actinomycin D + BMP-2 58.5 4.8Vehicle 108.2 6.3

BMP-2 167.6 6.1*Cycloheximide 57.7 5.2

Cycloheximide + BMP-2 59.0 2.7

Confluent MC3T3-E1 cells were pretreated with either 2.5 g/ml of ac-

tinomycin D, 5 M of cycloheximide, or their vehicle for 3 h and exposed

to BMP-2 (10 ng/ml) or its vehicle for 6 h. Then, Pi uptake was determined.

Values are the mean SE of six determinations.

*p < 0.001 compared with vehicle.

FIG. 3. Effect of BMP-2 on mRNA expression of type III Na-Pitransporters Pit-1 and Pit-2 in MC3T3-E1 cells. (A) Cells were

prepared as described in Fig 2 for Pi transport analysis. Then, theywere exposed to BMP-2 (10 ng/ml) or its vehicle for various timeperiods before analysis of Pit-1, Pit-2, and cyclophilin (CYCLO)mRNA expression by Northern blotting analysis. (B) In anotherset of experiments, cells were pretreated with either 5 M ofcycloheximide (CYCLOHEX) or 2.5 g/ml actinomycin D(ACTINO) as well as their vehicles for 3 h before exposure toBMP-2 (10 ng/ml) or its vehicle for 8 h and determination of Pit-1and cyclophilin mRNA expression by Northern blot analysis.

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tial components of the TGF- superfamily signaling ma-

chinery.

(24,2932)

Inhibition of the p38 and ERK pathwaysby 10 M of SB203580 and U0126, respectively, had no

effects on Pi transport stimulation induced by BMP-2 (data

not shown). In contrast, the JNK inhibitor, SP600125 (SP),

dose-dependently reduced this effect without influencing

basal Pi transport activity (Fig. 7A). This effect was associ-

ated with a marked decrease in BMP-2induced matrix

mineralization by 10 M SP600125 and complete inhibition

at 20 M (Fig. 7B). As previously observed,(30) SP600125

significantly enhanced basal (1.5 0.04-fold after 11 days,

p < 0.01) and BMP-2stimulated ALP activity (data not

shown). Enhanced basal ALP activity was associated with

increased baseline matrix mineralization (data not shown),

indicating that the blunting effect of SP600125 on matrix

mineralization induced by BMP-2 was not caused by alter-

ation in ALP or a cytotoxic effect.

DISCUSSION

Results presented in this study indicate that BMP-2 in-

duces a selective, dose- and time-dependent increase in Pi

transport in MC3T3-E1 osteoblasts and that this effect is

involved in the mineralization of the bone matrix induced

by this BMP. Pi transport stimulation by a member of the

TGF- family has already been previously reported for

TGF-in chondrocytes,

(20)

indicating that members of thisfamily regulate Pi transport in several type of osteogenic

cells. Pi transport stimulation by BMP-2 is dependent on

RNA and protein synthesis and reflects a change in the

Vmaxof the transport system. These characteristics are simi-

lar to those previously reported for the regulation of Pi

uptake by several hormones or growth factors in different

types of bone-forming cells and suggest that the observed

increase in Pi uptake may require the synthesis of new Pi

carriers and their insertion into the plasma mem-

brane.(10,11,13,20) Consistently, Northern blotting analysis

showed a time-dependent increase in the expression of

transcripts encoding the type III Pi transporter Pit-1 in re-

sponse to BMP-2 in MC3T3-E1 cells. The induction of Pit-1

preceded expression of Pi transport stimulation and this

effect is dependent on gene transcription. Interestingly, al-

though the related type III Pi transporter Pit-2 was also

present in MC3T3-E1 cells, its expression level was not

affected by BMP-2. A similar observation has been re-

ported in ATDC5 chondrocytic cells with TGF-,(20) sug-

gesting distinct cellular mechanisms for the regulation of

these two transport systems in osteogenic cells previously

characterized as housekeeping Pi transport systems.(33)

Recent in vitro and in vivo studies have suggested an

important role of Pi transport for the mineralization of the

FIG. 4. Dose-dependent effect of BMP-2on MC3T3-E1 cell matrix calcification. (A)Cells cultured for 5 days were treated withvarious concentrations of BMP-2 or its ve-hicle every other day for 6 weeks in -MEMmedium containing 10% FCS and 10 mM-glycerophosphate. Matrix mineralizationwas determined by alizarin red staining andby NIH image analysis. (B and C) Cells ex-posed to 30 ng/ml BMP-2 for 6 weeks as de-

scribed in A were fixed in cacodylate/glutaraldehyde buffer and embedded inEpon, and 80-nm-thick sections and crystalswere analyzed by transmission electron mi-croscopy and electron diffraction. (B) Trans-mission electron microscopy: crystal localiza-tion (7500) associated with collagen fibrils(#) is shown by arrows. *Cellular compart-ment. (C) Electron diffraction analysis: ar-rows indicate various diffraction rings withtheir corresponding D-spacing values.



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bone matrix. Essentially, it has been documented that Pitransport in osteogenic cells not only provide sufficient Pifor metabolic processes but also plays a specialized functionin matrix vesicles, microstructures that likely serve as nucle-ation sites for mineralization of the extracellular matrix ofcertain tissues.(9,34) This hypothesis, mainly supported by in

vitro studies, was reinforced by the in vivo observation ofPit-1 expression in a subset of hypertrophic chondrocytesduring endochondral bone formation.(6) In this study, en-hanced Pi uptake by BMP-2 was associated with a corre-sponding increase in physiological matrix mineralization.Interestingly, the dose effect of BMP-2 on Pi uptake corre-

FIG. 5. Effect of PFA on BMP-2induced MC3T3-E1 matrixcalcification and ALP activity. Cells cultured for 5 days weretreated with phosphonoformic acid (PFA, 0.1 mM), an inhibitorof Pi transport, for 1 h before exposure to BMP-2 (30 ng/ml) or itsvehicle every other day for 6 weeks for analysis of matrix miner-

alization (A) and 24 h for ALP activity (B). Matrix mineralizationwas determined by alizarin red staining and by NIH image analy-sis and ALP activity by the method of Lowry et al. (25) Each valuerepresents mean SE of five to six determinations from a repre-sentative experiment. *p< 0.01, compared with vehicle.

FIG. 6. Effect of a Pit-1 antisense oligonucleotide on Pit-1 ex-pression, Pi transport, and matrix calcification. (A) MC3T3-E1cells were stably transfected with either the empty vectorpcDNA3 or a plasmid expressing a Pit-1 antisense mRNA (AS-Pit-1). Pit-1 mRNA expression was determined by Northern blot-ting in various clones. (B and C) Confluent pcDNA3 or AS-Pit1(clone 2) transfected cells were switched to culture medium con-taining 2% FCS for 24 h. Then they were incubated with eitherBMP-2 (30 ng/ml) or its vehicle during 6 h for Pi transport (Pi

TRANSP) analysis (B) and 6 weeks for matrix calcification(CALCIFIED AREA) (C). Pi transport and matrix calcificationwere determined as described in Figs. 1 and 4. Each value repre-sents mean SE of four to five determinations from a represen-tative experiment. *p< 0.01 compared with vehicle.

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lated quite well with changes in matrix mineralization butpoorly with alteration in ALP expression, suggesting thatthe rate-limiting step of this process is probably not theamount of extracellular Pi available in the extracellularcompartment but rather the capacity of the cells to trans-port Pi, caused by an increased number of Pit-1 carriers

inserted in the plasma membrane. Because matrix vesiclesare derived from the plasma membrane of osteogenic cells,it is likely that matrix vesicles derived from BMP-2treatedcells also have a higher capacity to accumulate Pi and min-eralize, which could partly explain the enhanced matrixmineralization observed in MC3T3-E1 cells treated withBMP-2. Increased matrix mineralization by BMP-2 was

completely blocked by PFA, a competitive inhibitor of Na-Pi cotransport,(27) suggesting a role of Pi transport in thisprocess. However, whether this blunting effect of PFA onmatrix mineralization was caused by alteration in Pi trans-port remained unclear because this analog of inorganic Pihas been recently shown to interact with the rate of mineralformation in matrix vesicles.(28) However, analysis of ma-trix mineralization in MC3T3-E1 cells expressing a Pit-1antisense oligonucleotide strongly suggests that Pi transportplays a critical role in matrix mineralization induced byBMP-2. Indeed, in these cells having a lower expression ofPit-1 but a nearly normal response of ALP induced byBMP-2, enhanced matrix mineralization by this BMP wasmarkedly impaired. Data from this experiment also indicatethat Pi transport is not the only factor involved in the cal-cification of the organic matrix (Fig. 6). Indeed, in cellsexpressing the Pit-1 antisense oligonucleotide and having alower capacity to transport Pi, matrix mineralization wasnot lower but slightly increased compared with cells ex-pressing the empty vector. This apparent discrepancy is bestexplained by enrichment of cells having a higher capacity tomineralize during clonal selection. Among factors likely toinfluence this process, one could mention the amount ofmatrix vesicles released or their capacity to transport cal-cium or other molecules involved in accumulation of min-eral inside the vesicles as well as many other parametersinvolved in the control of the deposition, maturation, and

calcification of the bone matrix. Obviously, the formationof a collagenous matrix and its mineralization is a complexprocess that is not yet very well understood.

In MC3T3-E1 cells, we recently documented that, in ad-dition to activation of the Smad-signaling pathway, BMP-2also activates the p38 and JNK pathways with a selectiverole of each pathway in the differentiation of these cells.(30)

In this study, we found that JNK is probably involved inmediating the stimulation of Pi transport by BMP-2 in os-teoblastic cells. Whereas the ERK and p38 inhibitors hadno effect on Pi transport stimulation induced by BMP-2, theJNK inhibitor, SP600125, was quite effective in blunting thisresponse in MC3T3-E1 cells. A dose of 20 M that selec-tively inhibits activation of JNK by BMP-2 in these cells(30)

completely blocked this effect without influencing basal Pitransport activity. Associated with this blunting effect on Pitransport, inhibition of JNK completely prevented BMP-2induced matrix mineralization. Interestingly, this effect wasobserved despite preservation of the increase in ALP ac-tivity induced by BMP-2 in presence of this inhibitor, sug-gesting distinct roles of Pi transport and ALP in initialevents of primary calcification of the bone matrix. To ourknowledge, this is the first observation for an implication ofthis pathway in the regulation of a Pi transport system inmammalian cells. Further studies are required to determinedownstream regulatory molecules activated by this pathway

FIG. 7. Effect of the JNK inhibitor, SP600125, on BMP-2induced changes in Pi transport and matrix calcification. MC3T3-E1 cells were prepared for either (A) Pi transport (Pi TRANS) or(B) matrix calcification (CALCIFIED AREA) analysis as de-scribed in Figs. 1 and 4. Cells were preincubated for 1 h with orwithout various doses of the JNK inhibitor SP600125. Then, theywere exposed to 10 ng/ml BMP-2 in presence of the inhibitor or itsvehicle for 6 h or 4 weeks (every 23 days) before the determi-

nation of Pi transport and matrix calcification, respectively. Pitransport and matrix mineralization were determined as describedin Figs. 1 and 4, respectively. Each value represents mean SE offour to five determinations from a representative experiment.*p< 0.01, compared with vehicle.



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that mediate the stimulation of Pi transport by BMP-2 inosteoblastic cells.

In conclusion, data presented in this study indicate thatBMP-2 stimulates both Pi uptake and Pit-1 expression inMC3T3-E1 cells. This effect is probably mediated by acti-vation of the JNK pathway. They also provide strong evi-dence that this effect is involved in bone matrix mineraliza-

tion induced by BMP-2, further supporting the concept thatPi handling by osteogenic cells is an important parameterfor the primary calcification of the bone matrix.

ACKNOWLEDGMENTS

The authors thank P Apostolides for expert technicalassistance. We also acknowledge the contribution of PaulPilet, Thierry Bouillon, and Pierre Weiss for the biominer-alization analysis by transmission electron microscopy andelectron diffraction. This study was supported by the SwissNational Science Foundation (3100AO-100607) and aGrant-in-Aid for Scientific Research from the Ministry of

Education, Culture, Sports, Science and Technology of Ja-pan (15590987).

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Address reprint requests to:Joseph Caverzasio, PhD

Service of Bone Diseases

Department of Rehabilitation and Geriatrics

University Hospital of Geneva

CH-1211 Geneva 14, Switzerland

E-mail: [email protected]

Received in original form December 6, 2005; revised form January30, 2006; accepted February 13, 2006.


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