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Division of Bone Diseases, Department of Medicine, University of Geneva, Geneva, Switzerland
Address all correspondence and requests for reprints to: Dr. Joseph Caverzasio, Division of Bone Diseases, Department of Medicine, University of Geneva, CH-1211 Geneva 14, Switzerland. E-mail: caverzas{at}cmu.unige.ch
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Abstract |
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Northern blotting analysis revealed the presence of a 4-kilobase Glvr-1
transcript. The expression of Glvr-1 messenger RNA (mRNA) was increased
in response to insulin-like growth factor I (IGF-I). Associated with
this effect, a selective, dose- and time-dependent stimulation of NaPi
transport was observed. Actinomycin D and cycloheximide abolished the
increase in NaPi transport, which thus appeared to be dependent on RNA
and protein synthesis. The increase in Glvr-1 mRNA induced by IGF-I was
dose dependent and transient, peaking after 4 h (
4-fold
increase in response to 10-7 M IGF-I). It
preceded the maximal expression of NaPi transport stimulation
(173–235% of control), which was observed after 18–24 h. Induction
of Glvr-1 mRNA expression by IGF-I was inhibited by actinomycin D,
suggesting that this effect was related to an increase in gene
transcription. The stability of Glvr-1 mRNA was not altered by IGF-I,
and Glvr-1 mRNA induction did not require the synthesis of new
proteins.
These data demonstrate for the first time regulated expression of mRNA encoding the type III NaPi transporter Glvr-1 in osteoblast-like cells. They also suggest that this new transporter family may be involved in Pi handling in osteogenic cells and in its regulation by osteotropic factors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pi uptake in osteoblast-like cells is a saturable, carrier-mediated process that can be regulated by many osteotropic factors, including PTH, platelet-derived growth factor, and fluoride (1, 2). Pi enters the cells through a sodium-dependent Pi (NaPi) transport system present in the plasma membrane (1, 2, 3). A Pi carrier with similar functional properties is also found in the matrix vesicles released by osteogenic cells into the bone extracellular matrix, where accumulation of Pi is thought to play an essential role in the initiation of matrix calcification (1, 2). Although substantial information is available about the functional characteristics of this transport system, its molecular identity is unknown.
During the last years, several mammalian NaPi transporter families have been described (4, 5, 6, 7). Two nonhomologous sodium-phosphate cotransport systems, called type I and II NaPi, were identified in the renal cortex of various species (4). Both types of transporters are almost exclusively expressed in the kidney, where, in particular, type II NaPi plays a major role in the regulation of proximal tubular phosphate transport (4). More recently, the cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphotropic murine retrovirus (Ram-1) have been demonstrated to serve normal cellular functions as sodium-dependent phosphate transporters (5, 6, 7). These proteins belong to a new type III phosphate transporter/retrovirus receptor gene family and are expressed in a wide variety of tissues (5, 7).
The three types of NaPi differ markedly in tissue distribution, structure, and function (4, 7). Characteristics of Pi transport in bone-forming cells suggest that involved carriers are distinct from the type I and II renal transporters (3, 4). Recent studies showed that injection of messenger RNA (mRNA) from rat osteoblast-like cells could induce NaPi transport in Xenopus laevis oocytes. This mRNA did not hybridize to rat renal NaPi-2, suggesting the presence in osteoblast-like cells of a transport system different from that of type II NaPi (8). A distinctive feature of the different types of NaPi is the influence of pH on their activity. Unlike the activity of renal NaPi transporters, which is increased at alkaline pH, Pi uptake mediated by type III NaPi is decreased when pH increases (4, 5, 6, 7). The osteoblastic Pi transport system exhibits a pH dependence similar to that of the type III NaPi (3), and this similarity prompted us to investigate whether type III family members were expressed in osteoblast-like cells.
Insulin-like growth factor I (IGF-I), which is an important regulator of Pi handling in the kidney, in particular during growth, is also one of the most abundant growth factors present in bone (9, 10, 11). As IGF-I has been demonstrated in various in vitro models to stimulate proliferation, differentiation, and Pi transport in osteogenic cells, we were further interested in the potential effect of IGF-I on our experimental system (10, 11, 12, 13).
The aims of this study were thus to investigate the expression of the type III NaPi Glvr-1 in human SaOS-2 cells, an osteosarcoma-derived cell line with many osteoblast-like characteristics, and to test whether expression of Glvr-1 and Pi uptake could be influenced by IGF-I in these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy-CTP, [
-32P]ATP, and
[3H]alanine were obtained from Amersham International
(Little Chalfont, UK). Immobilon S membranes were purchased from
Millipore Corp. (Bedford, MA), and Zeta-Probe GT membranes were
obtained from Bio-Rad Laboratories (Richmond, CA). Quickhyb
hybridization buffer was obtained from Stratagene (La Jolla, CA). All
other reagents were of the highest purity available from standard
laboratory suppliers. Autoradiographies were quantitated using the 300A Computing densitometer from Molecular Dynamics (Sunnyvale, CA).
Cell culture
Human osteoblast-like SaOS-2 cells (14) were cultured in
DMEM-Ham’s F-12 (1:1) containing 10% FCS, 100 IU/ml penicillin, and
100 µg/ml streptomycin. All cultures were maintained at 37 C in a
humidified atmosphere of 5% CO2-95% air. Subcultures were
obtained once a week by removing the cells from the dishes using 0.25%
trypsin in a Ca- and Mg-free Earle’s salt solution containing 2
mmol/liter EDTA. The cells were plated in 16-mm 24-well tissue culture
clusters for Pi and alanine uptake measurements and DNA content
determinations or in 162-cm2 cell culture flasks for RNA
extraction. They reached confluence within 3 days, and the assays were
performed on days 5–6. For all experiments, confluent SaOS-2 cells
were preincubated for 18 h in DMEM-Ham’s F-12 medium containing
0.25% FCS before stimulation with IGF-I or its vehicle for the
indicated times. In some experiments cells were exposed to 1 or 2
µg/ml actinomycin D before or after stimulation with IGF-I.
Actinomycin D (1 µg/ml) has been previously shown to inhibit
transcription in SaOS-2 cells (15). To inhibit translation, cells were
preincubated for 90 min with 5 µM cycloheximide, a
concentration sufficient to inhibit protein synthesis in SaOS-2 cells
(16).
Uptake of Pi or alanine
Transport studies were performed in Earle’s Buffered Salt
Solution (EBSS) as previously described (17). Pi transport was
initiated by adding 0.3 ml EBSS containing 0.1 g/100 ml BSA, 0.1 g/100
ml D-glucose, and 0.1 mM
H3[32P]O4 (1 µCi/ml), except
for kinetic analyses, where
H3[32P]O4 concentrations ranged
from 0.05–1.5 mM. For alanine transport, the EBSS solution
contained 0.1 mM L-[3H]alanine (1
µCi/ml) and 1 mM Na2HPO4. IGF-I,
actinomycin D, cycloheximide, or their respective vehicles were not
added to the uptake solution during the transport determination. After
6 min for Pi or 2 min for alanine, solute uptake was stopped by washing
the cell layer three times with ice-cold (4 C) substrate-free EBSS. The
cells were then solubilized with 300 µl 0.2 N sodium
hydroxide, and the radioactivity was counted by a standard liquid
scintillation technique. The sodium-independent components of Pi and
alanine uptake were determined by replacing NaCl in the uptake solution
with choline chloride.
DNA content
DNA content was measured in parallel to Pi and alanine
uptake in cells that were stimulated with IGF-I or its vehicle for the
indicated times. The cells were detached from the wells by a 10-min
incubation at 37 C with 0.1% collagenase and 0.25% trypsin in Ca- and
Mg-free Earle’s salt solution containing 2 mmol/liter EDTA, and DNA
content was measured as described by Burton (18), using calf thymus DNA
as a standard.
Northern blot analysis
After stimulation of SaOS-2 cells with IGF-I or its vehicle for
the indicated times, the culture medium was removed, and the cells were
stored at -80 C until total RNA was isolated using the RNeasy kit from
Qiagen according to the manufacturer’s instructions. Ten micrograms of
total RNA were denatured in glyoxal and separated by electrophoresis on
a 1.2% agarose gel. RNA was transferred to Hybond N, GeneScreen Plus,
Zeta-Probe GT, or Immobilon S membranes by capillary transfer.
Membranes were UV cross-linked and stained with methylene blue to
control equal sample loading.
The membranes were hybridized to a PCR-amplified fragment (from
bp 1208 to bp 1920) of the human Glvr-1 complementary DNA (cDNA;
GenBank accession no. L20859) (19), which was labeled with 50 µCi
[-32P]deoxy-CTP by random priming. This central region
of the Glvr-1 cDNA exhibits low homology to the related Glvr-2 (GenBank
accession no. L20852) (20). Prehybridization (30 min) and hybridization
(2 h) were performed in Quickhyb buffer (Stratagene), which was
supplemented for the hybridization with 100 µg/ml salmon sperm DNA.
Membranes were washed twice for 15 min each time in 2 x SSC
(standard saline citrate)-0.1% SDS at 25 C and for 10–30 min at 60 C
in 0.1 x SSC-0.1% SDS before exposition to Kodak X-Omat AR films
(Eastman Kodak, Rochester, NY) for 18–72 h at -80 C. To correct for
differences in sample loading by assessing the expression of a
housekeeping gene, membranes were stripped in 0.1 x SSC-0.1% SDS
at 95 C and rehybridized with a BamHI fragment of the human
cyclophilin cDNA (21), using hybridization conditions similar to those
used for Glvr-1. Alternatively, equal sample loading was confirmed by
rehybridizing the membranes to a 5'-end-labeled 32-mer oligonucleotide
complementary to the human 18S rRNA
(5'-CCTGTATTGTTATTTTTCGTCACTACCTCCCC-3'). Hybridization was carried out
at 63 C in Quickhyb buffer supplemented with 100 µg/ml salmon sperm
DNA. Membranes were washed for 5 min in 2 x SSC-0.5% SDS at 25
C, for 15 min in 2 x SSC-0.1% SDS at 25 C, and for 20 min in
0.1 x SSC-0.5% SDS at 37 C. Signals were quantitated by
densitometry.
Statistical analysis
Results are expressed as the mean ± SEM.
Significance of differences was calculated by two-tailed unpaired
Student’s t test. A difference between experimental groups
was considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The Na-independent component of Pi
uptake accounted for less than 20% of total Pi uptake in
vehicle-treated cells and was not affected by IGF-I. This component was
thus neglected in further experiments. Sodium-dependent Pi uptake was
preferentially increased compared with sodium-dependent alanine
transport, which was not changed after 6-h incubation with
10-7 M IGF-I and was only slightly increased
after 24 h (Table 1). This observation suggests that the change in
Pi uptake induced by IGF-I is mediated by a selective cellular
mechanism rather than by a general alteration in the Na gradient
driving force. In the same experimental conditions, DNA content was not
changed in IGF-I-treated cells after 6 h and increased by only
15% after 24 h (Table 1).
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). A significant increase was observed
in response to 10-11 M IGF-I, and maximal
stimulation was achieved at 10-8–10-7
M. The effect of IGF-I was time dependent and progressive;
it was first detectable after 4 h and reached a maximal value
after 18 h (Fig. 1B). Furthermore, the stimulation of Pi transport
by IGF-I appeared to be dependent on RNA and protein synthesis, as it
was completely blocked in cells pretreated with either actinomycin D
(Fig. 2A) or cycloheximide (Fig. 2B).
Kinetic analysis indicated that the stimulatory effect of IGF-I on Pi
uptake was related to an increase in the maximal rate
(Vmax) of the transport system, without a significant
change in the apparent Km (Fig. 3 and Table 2).
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). Glvr-1 mRNA levels then
returned to baseline within 18 h. Expression of the housekeeping
gene cyclophilin was not significantly affected by IGF-I at any time
point (Fig. 4B).
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) and, taking into account the
lower sensitivity of Northern blot analysis compared with Pi uptake
measurements, was approximately parallel to the dose-dependent increase
in Pi transport (Fig. 1A
). An increase in Glvr-1 mRNA was indeed
detectable from 10-10 M IGF-I and progressive
up to 10-7 M IGF-I. Steady state Glvr-1 mRNA
levels in cells exposed to 10-7 M IGF-I for
4 h were consistently more than 4-fold higher than those observed
in vehicle-treated cells (Figs. 4C and 5C).
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, A and B).
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, C and D). SaOS-2 cells were
preincubated with 5 µM cycloheximide for 90 min before
stimulation with 10-7 M IGF-I for 4 h.
The inhibition of protein synthesis did not interfere with the
induction of Glvr-1 mRNA by IGF-I. This observation suggests that the
synthesis of new intermediary gene products is not necessary to enable
SaOS-2 cells to increase Glvr-1 mRNA expression in response to
IGF-I.
To investigate whether IGF-I affected the stability of Glvr-1 mRNA,
SaOS-2 cells were stimulated for 4 h with 10-7
M IGF-I before transcription was inhibited with actinomycin
D (Fig. 7). Glvr-1 mRNA levels decreased
rapidly after the addition of 1 µg/ml actinomycin D (Fig. 7A). The
half-life of the Glvr-1 transcript appeared to be shorter than 1
h, and no difference in Glvr-1 mRNA stability could be detected between
IGF-I- and vehicle-treated cells (Fig. 7, C and D). Together with the
results shown in Fig. 6, A and B, these observations suggest that the
increase in Glvr-1 mRNA steady state levels observed in response to
IGF-I in SaOS-2 cells mainly reflects a stimulatory effect of IGF-I on
the transcription of this gene.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I is one of the most abundant growth factors present in bone and has been shown to stimulate bone growth in vivo as well as proliferation and activity of bone-forming cells in vitro (10, 11, 12). In this study, IGF-I significantly enhanced Pi transport and Glvr-1 mRNA expression in SaOS-2 cells. The stimulation of Pi transport was selective, and dose and time dependent. The increase in Pi uptake was related to a change in the Vmax of the transport system and was blocked by transcriptional and translational inhibitors, suggesting that IGF-I may induce the synthesis of new Pi carriers and their insertion into the plasma membrane. IGF-I was previously reported to stimulate Pi transport in two different types of osteogenic cells: rat UMR-106–01 osteoblast-like cells and chicken growth plate chondrocytes (12, 13). Pi transport stimulation by IGF-I in chicken growth plate chondrocytes was comparable in terms of selectivity, and dose and time dependency to that observed in SaOS-2 cells (13). In contrast, higher concentrations of IGF-I were required in UMR-106–01 cells to observe a significant increase in Pi uptake, and the effect was independent of protein synthesis, suggesting a different mechanism of action for IGF-I in UMR-106–01 compared with SaOS-2 cells (12).
IGF-I dose dependently increased Glvr-1 expression in SaOS-2 cells. It appeared to act mainly on the transcription of the Glvr-1 gene without affecting the stability of the Glvr-1 mRNA, and the induction of Glvr-1 mRNA by IGF-I did not require the synthesis of intermediate gene products. The increase in Glvr-1 mRNA expression was transient, and its peak preceded by several hours the maximal expression of Pi transport stimulation. The parallel between the induction of Glvr-1 mRNA expression and Pi transport stimulation strongly suggests that Glvr-1 mediates at least part of the stimulatory effect of IGF-I on Pi transport in SaOS-2 cells. Consistently with this hypothesis the delay observed between the peak of Glvr-1 mRNA expression and the maximal increase in Pi uptake might reflect the time required for the translation of the newly transcribed mRNA and for the insertion of the protein into the plasma membrane to form new functional transport sites.
The apparent Km observed for Pi uptake in SaOS-2 cells (450–500 µM) is on the same order of magnitude as those reported for other osteoblast-like cell lines (3, 17). This apparent Km is higher than those measured for Glvr-1 expressed in oocytes (24 µM) or overexpressed in 208F rat embryo fibroblasts or Mus dunni tail fibroblasts (70 and 53 µM, respectively) (5, 6). These differences could reflect cell type-specific features, such as cell membrane properties, posttranslational modifications, or expression of distinct regulatory proteins; however, the potential existence in SaOS-2 cells of other Pi transport systems in addition to Glvr-1 has not been investigated to date and thus cannot be excluded.
Several in vitro observations suggested that Pi transport plays an important functional role in osteogenic cells and in their matrix vesicles for the initiation of bone matrix calcification (1, 2, 13). The identification of a NaPi transporter expressed in bone-forming cells now provides new molecular tools to study the role of Pi transport and its regulation during bone formation, in particular in relation to bone matrix mineralization. We are currently investigating the expression of Glvr-1 mRNA during the development of murine metatarsals by in situ hybridization. Preliminary data suggest that in this model of endochondral bone formation, Glvr-1 is selectively expressed in a subpopulation of early hypertrophic chondrocytes in a region close to the onset of matrix mineralization (our unpublished data). These observations, which are consistent with a potential role for the Glvr-1 phosphate transporter in initial events of matrix calcification, further suggest important functions for type III NaPi transporters in the context of bone formation in vivo.
In conclusion, this study shows for the first time the expression of a type III NaPi transporter in osteoblast-like cells. Our data also indicate that both the expression of this transporter and Pi transport are stimulated by IGF-I in SaOS-2 cells and thus suggest that type III NaPi transporters may be involved in regulated Pi handling in osteogenic cells. The identification of a NaPi transporter exhibiting regulated expression in bone-forming cells, besides providing new tools to investigate the molecular mechanisms of Pi transport regulation, should also lead to a better understanding of the role of Pi in the metabolism, growth, and differentiation of osteogenic cells as well as in initial steps of bone matrix calcification.
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Acknowledgments |
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Footnotes |
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Received May 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3-treated rat osteoblast-like
cells. Pflugers Arch Eur J Physiol 430:64–67[CrossRef][Medline]
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