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ABSTRACT: Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in
BMP-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 important
role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its
transport 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 by
Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin
red 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 by
increasing the Vmax of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1
but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by
either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization 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 several
markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a
selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by
the 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
NORGANIC PHOSPHATE (Pi) transport represents a particular function of bone-forming cells for extracellular matrix
mineralization.(1,2) As in all eukaryotic cells, osteogenic
cells (i.e., osteoblasts and chondrocytes) are endowed with
Na-dependent Pi transport systems driven by an inwardly
directed Na gradient.(3) The type III Pi transport systems,
namely Pit-1 and Pit-2, are involved in Pi handling by osteogenic cells.(4,5) In situ hybridization analysis revealed expression of the type III transporter Pit-1 (Glvr-1) in a subpopulation of hypertrophic chondrocytes during
endochondral bone formation, suggesting a potential role
1
Division of Endocrinology, Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Japan; 2Service
of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, Geneva, Switzerland; 3INSERM EM
99-03, School of Dental Surgery, Nantes, France; 4Department of Metabolic Diseases, Nagoya University Graduate School of Medicine,
Nagoya, Japan.
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sion no. L20852).(22) The differences in sample loading
were corrected by the expression of a housekeeping gene,
either cyclophilin or GAPDH. Probes were labeled with 50
Ci [-32P] dCTP by random priming. Pre-hybridization
(30 minutes) and hybridization (2 h) were performed in
Quickhyb buffer (Stratagene, La Jolla, CA, USA), which
was supplemented for the hybridization with 100 g/ml
salmon sperm DNA. Membranes were washed twice for 15
minutes in 2 SSC, 0.1% SDS at 25C and for 1030 minutes at 60C in 0.1 SSC, 0.1% SDS before exposition to
Kodak X-OMAT AR films for 1872 h at 80C.
ALP activity
ALP activity was determined as previously described.(24)
Dose-dependent effect of BMP-2 on ALP activity was determined in culture medium containing 1% FCS after 48-h
incubation. Cells were harvested in 0.2% Nonidet P-40 and
SUZUKI ET AL.
disrupted by sonication. The homogenate was centrifuged
at 1500g for 5 minutes, and ALP activity was determined in
the supernatant by the method of Lowry et al.(25)
Statistical analysis
Results are expressed as mean SE. A two-sided unpaired Students t-test or ANOVA for multiple comparison
was used for statistical analysis. A difference between experimental 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 in
MC3T3-E1 cells that responded quite well to BMP-2 for
inducing expression of ALP (Fig. 1B). In confluent cells, we
found that this BMP dose-dependently and selectively increased Pi transport (Fig. 1A). A stimulatory effect was
detected at the dose of 1 ng/ml of BMP-2 with maximal
stimulation at 30 ng/ml (Fig. 1A). Interestingly, the dose
response effect on Pi uptake stimulation was different from
that of ALP induced by BMP-2 (Figs. 1A and 1B). The
stimulation of Pi uptake induced by BMP-2 was detected
after 3 h, further enhanced after 6 h, and reached a maximal
value after 24-h incubation (Fig. 2). Alanine uptake was not
significantly affected in the same conditions (Table 1). Because alanine is transported into mammalian cells mainly
through an Na co-transport system, this observation indicates that the effect of BMP-2 on Pi uptake is mediated by
a selective cellular mechanism. Furthermore, the stimulatory effect of BMP-2 on Pi uptake seemed to be dependent
on RNA and protein synthesis, because it was completely
blocked in cells pretreated with either 2.5 g/ml actinomycin D or 5 M cycloheximide respectively (Table 2). Finally, kinetic analysis indicated that the stimulatory effect
of BMP-2 on Pi uptake is related to an increase in the
maximal rate (Vmax) of the transport system, without a
change in its apparent affinity constant (Km) for phosphate
(Table 1).
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Vmax
(nmol/6 minutes/well)
Km (mM)
Veh
BMP-2
Selectivity
0.398 0.02
0.705 0.03*
0.491 0.04
0.508 0.04
Veh
BMP-2
Alanine transport
(pmol/2 minutes/well)
91.1 6.3
76.7 5.3
gesting that this effect of BMP-2 is, at least in part, independent of new protein synthesis (Fig. 3B).
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SUZUKI ET AL.
ON
89.0 5.6
148.1 7.6*
47.9 3.9
58.5 4.8
108.2 6.3
167.6 6.1*
57.7 5.2
59.0 2.7
Confluent MC3T3-E1 cells were pretreated with either 2.5 g/ml of actinomycin 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.
mineralization. Enhanced accumulation of mineral was detected at the concentration of 3 ng/ml with a gradual increase 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 calcium-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 microscopy (TEM), electron diffraction (EM), and resolution
enhanced Fourier transformed infrared (FTIR) spectroscopy. 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 mineralization induced by BMP-2 was completely blocked by 0.1
mM phosphonoformic acid (Fig. 5A), a competitive inhibitor 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 vehicletreated cells (Fig. 5B), suggesting that part of the blunting
effect of BMP-2 on matrix mineralization observed in PFAtreated 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 reduced matrix mineralization is through its reduction of the
rate of matrix vesicleinduced mineral formation, which
plays an important role in initiating the process of mineralization.(28) Thus, to validate above data suggesting an important role of Pit-1 in matrix mineralization induced by
BMP-2, we constructed MC3T3-E1 cells expressing an antisense Pit-1 oligonucleotide (AS-Pit-1). Of several clones
stably transfected with AS-Pit-1, two expressed lower levels
of Pit-1 mRNA compared with other Pit-1 (Fig. 6, clones 2
and 3) and empty vector transfected cells (data not shown).
Clone 2 was used for further analysis because, in addition to
expressing a lower baseline and a blunted response to
BMP-2 on Pi uptake (Fig. 6B and similar effect obtained
with clone 3, data not shown), this clone had a near normal
stimulation of ALP by BMP-2 (data not shown). The matrix
mineralization induced by BMP-2 in this clone was markedly decreased compared with cells stably transfected with
the empty vector (Fig. 6C), strongly suggesting that Pit-1
plays a critical role in bone matrix mineralization induced
by BMP-2.
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tial components of the TGF- superfamily signaling machinery.(24,2932) Inhibition of the p38 and ERK pathways
by 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 associated 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 alteration in ALP or a cytotoxic effect.
DISCUSSION
Results presented in this study indicate that BMP-2 induces 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
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SUZUKI ET AL.
FIG. 6. Effect of a Pit-1 antisense oligonucleotide on Pit-1 expression, Pi transport, and matrix calcification. (A) MC3T3-E1
cells were stably transfected with either the empty vector
pcDNA3 or a plasmid expressing a Pit-1 antisense mRNA (ASPit-1). Pit-1 mRNA expression was determined by Northern blotting in various clones. (B and C) Confluent pcDNA3 or AS-Pit1
(clone 2) transfected cells were switched to culture medium containing 2% FCS for 24 h. Then they were incubated with either
BMP-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 calcification
were determined as described in Figs. 1 and 4. Each value represents mean SE of four to five determinations from a representative experiment. *p < 0.01 compared with vehicle.
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14.
15.
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ACKNOWLEDGMENTS
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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: Joseph.Caverzasio@medecine.unige.ch