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Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Toru Yamaguchi, M.D., Ph.D., Endocrine-Hypertension Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: toru.yamaguchi{at}nifty.ne.jp
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Introduction |
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The resorption of bone can produce local increases in the extracellular calcium concentration (Ca2+o) beneath a resorbing osteoclast as high as 40 mM (4). Osteoclast formation can be activated by stromal cells through a mechanism involving cell to cell contact (1, 5). It is possible, therefore, that stromal cells that have participated in the process of osteoclast formation remain in close proximity to sites where active bone resorption is taking place and are capable of sensing the locally high levels of Ca2+o that result from osteoclast-mediated breakdown of bone. This high Ca2+o could, therefore, provide the stromal cells with a signal that modulates their subsequent physiological responses.
One possible mechanism by which stromal cells could respond to changes in Ca2+o would be via the Ca2+o-sensing receptor (CaR) that has been cloned from bovine and human parathyroid gland (6, 7), rat kidney (8) and thyroid C cells (9). The physiological relevance of the CaR has been documented in humans by showing that inactivating or activating mutations of the CaR gene cause inherited hyper- or hypocalcemic disorders (10, 11), respectively, rendering affected family members inappropriately resistant or sensitive, respectively, to the usual effects of Ca2+o on parathyroid and renal functions.
In a previous study using immunohistochemistry with CaR-specific antisera, we showed expression of this receptor in diverse cell types in human bone marrow, including alkaline phosphatase-positive, putative osteoblast precursors, nonspecific esterase-positive mononuclear cells, erythroid precursors, and megakaryocytes (12). These findings suggested that the CaR might be involved in the Ca2+o-sensing mechanism of these bone marrow-derived cells. In this study, we used ST2 cells as a model of stromal cells in bone marrow and examined them for the presence and possible role(s) of the CaR in regulating their function(s). We demonstrate herein clear expression of the CaR in ST2 cells, as assessed by immunocytochemical staining and Western blot analysis using a specific anti-CaR antiserum as well as by Northern analysis with a CaR-specific probe and RT-PCR with CaR-specific primers. We also confirm that CaR agonists stimulate both chemotaxis and DNA synthesis in the CaR-expressing ST2 cells examined in this study. These results show that both CaR protein and messenger RNA (mRNA) are expressed in the ST2 cell line; in addition, they suggest that the receptor could potentially play a pivotal role in regulating the function of stromal cells present within the marrow by sensing local changes in Ca2+o related to bone remodeling.
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Materials and Methods |
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Cell culture
ST2 cells were purchased from the RIKEN Cell Bank (Tsukuba,
Japan). ST2 cells were grown in 
MEM (Ca2+, 1.8
mM; Mg2+, 0.81 mM;
H2PO4-, 1.0 mM);
supplemented with 10% FBS and 1% penicillin/streptomycin in 5%
CO2 at 37 C. The medium was changed twice weekly, and the
cells were subcultured into 25-cm2 flasks by detaching them
gently with a cell scraper after reaching subconfluency. For
morphological evaluation, ST2 cells were plated onto 12-mm circular
glass coverslips in 24-well (2.0-cm2) plates. After 24
h of culture, the medium was discarded, and each coverslip with
adherent cells was washed once with PBS, fixed with 4% formaldehyde in
PBS for 5 min, and washed with PBS once again. Each coverslip was
stored at 4 C until assessment of the presence of the CaR as described
below.
Immunocytochemistry for CaR in ST2 cells
A CaR-specific polyclonal antiserum (4637) was provided by Drs.
Forrest Fuller and Karen Krapcho of NPS Pharmaceuticals (Salt Lake
City, UT). This antiserum was raised against a peptide (FF-7;
HNGFAKEFWEETFNC) corresponding to amino acids 345–359 of the bovine
CaR (6) and 344–358 of the human and rat CaRs (7, 8), which resides
within the predicted amino-terminal extracellular domain of the CaR.
The antiserum has recently been fully characterized and used for
studies of the endogenous CaRs expressed in various human and rat
tissues in our laboratory (13, 14, 15, 16). The antiserum was subjected to
further purification using an affinity column conjugated with the FF-7
peptide, and the affinity-purified antiserum was used for
immunocytochemistry and Western blot analysis as described below. Fixed
ST2 cells were treated with Dako Protein Block Serum-Free Solution
(Dako Corp., Carpinteria, CA) for 1 h and then incubated overnight
at 4 C with primary antiserum (anti-CaR antiserum 4637) at a
concentration of 5 µg/ml in blocking solution (Dako Corp.). Negative
controls were carried out by performing the same procedure after
preabsorption of the anti-CaR antiserum with 10 µg/ml of the
synthetic CaR peptide against which it was raised. After washing the
cells three times with 0.5% BSA in PBS for 10 min, alkaline
phosphatase-coupled, goat antirabbit IgG (1:200; Life Technologies) was
added and incubated for 1 h at room temperature. The cells were
then washed three times with PBS for 10 min each time, and the color
reaction was developed for 10–20 min using a solution consisting of 44
µl nitro blue tetrazolium chloride (75 mg/ml) and 33 µl
5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (50
mg/ml) in 10 ml 0.1 M Tris-HCl (pH 9.5), 0.1 M
NaCl, 50 mM MgCl2, and 1 mg/ml levamisole,
which was included for inhibition of endogenous cellular alkaline
phosphatase activity. The color reaction was stopped by washing twice
in the above solution without nitro blue tetrazolium chloride or
5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and
then twice in water.
Western analysis of CaR in ST2 cells
Monolayers of ST2 cells in 75-cm2 flasks were rinsed
twice with 1 mM EDTA in PBS and lysed with 1.0 ml of a
lysis solution (1% SDS and 10 mM Tris-HCl, pH 7.4) heated
to 65 C. The cells were scraped from the flasks, transferred to
microcentrifuge tubes, and heated for an additional 5 min at 65 C. The
viscosity of the sample was reduced by brief sonication, and insoluble
material was removed by centrifugation for 5 min. The resultant whole
cell lysate in the supernatant was stored at -20 C until Western blot
analysis was performed.
Aliquots of 150 µg protein were dissolved in SDS-Laemmli gel loading buffer containing 100 mM dithiothreitol, incubated at 37 C for 15 min, and resolved electrophoretically on 6.5% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose at 240 mA for 40 min in transfer buffer containing 19 mM Tris-HCl, 150 mM glycine, 0.015% SDS, and 20% methanol. The blots were blocked for 2 h with 1% BSA in PBS containing 0.25% Triton X-100 (blocking solution) and then incubated overnight at 4 C with the affinity-purified antiserum (4637) or with peptide-blocked antiserum [the same amount of antiserum preincubated at room temperature for 60 min with twice the amount (wt/wt) of FF-7 peptide] at a concentration of 1 µg/ml in the blocking solution. The blots were washed three times with PBS containing 0.25% Triton X-100 (washing solution) at room temperature for 10 min each time. The blots were further incubated with a 1:2000 dilution of horseradish peroxidase-coupled, goat antirabbit IgG (Sigma Chemical Co.) in the blocking solution for 1 h at room temperature. The blots were then washed three times with the washing solution at room temperature for 40 min each time, and specific protein bands were detected using an enhanced chemiluminescence system (ECL, Amersham, Arlington Heights, IL).
Detection of CaR transcripts by Northern blot analysis
For the purpose of determining the sizes of the CaR transcripts
in ST2 cells, Northern blot analysis was employed on aliquots of 5 µg
polyadenylated [poly(A)+] RNA obtained using
oligo(deoxythymidine)-cellulose chromatography of total RNA.
Poly(A+)-enriched RNA samples were denatured and
electrophoresed in 2.2 M formaldehyde-1% agarose gels
along with an 0.24–9.5 kb RNA ladder (Life Technologies) and
transferred overnight to nylon membranes (Duralon, Stratagene, La
Jolla, CA). A 577-bp XhoI-SacI fragment
corresponding to nucleotides 721-1298 of the rat CaR complementary DNA
(cDNA) was subcloned into the pBluescript SK+ vector. The
plasmid was then linearized with BglII, and a
32P-labeled riboprobe was synthesized with the MAXIscript
T3 kit (Pharmacia Biotech, Piscataway, NJ) using T3
polymerase and [32P]UTP. Nylon membranes were
prehybridized for 2 h at 52 C in a solution consisting of 50%
formamide, 4 x Denhardt’s solution (50 x Denhardt’s
= 5 g Ficoll, 5 g polyvinylpyrrolidone, and 5 g BSA),
5 x SSPE (20 x SSPE = 2.98 M NaCl and 0.02
M EDTA in 0.2 M phosphate buffer, pH 7.0),
0.5% SDS, 10% dextran sulfate, 250 µg/ml yeast transfer RNA, and
200 µg/ml calf thymus DNA. Labeled complementary RNA probe (2 x
106 cpm/ml) was then added, and the membranes were
hybridized overnight at the same temperature. Washing was carried out
at moderate stringency (0.3 x SSC [20 x SSC = 3
M NaCl and 0.3 M
Na3-citrate·2H2O) and 0.5% SDS at 55 C] for
20 min. Membranes were then exposed to x-ray film (XAR-5, Eastman
Kodak, Rochester, NY) for 4 days at -70 C.
PCR amplification of cDNA products from the mouse homolog of the
CaR expressed in ST2 cells
Total RNA was prepared from ST2 cells using the TRIzol reagent
(Life Technologies). One microgram of total RNA was used for the
synthesis of single stranded cDNA (cDNA synthesis kit, Life
Technologies). The resultant first strand cDNA was used for the PCR
procedure. PCR was performed at a final concentration of 20
mM Tris-HCl (pH 8.4), 50 mM KCl, 1.8
mM MgCl2, 0.2 mM deoxy-NTP, 0.4
µM forward primer, 0.4 µM reverse primer,
and 1 µl ELONGASE enzyme mix (a Taq/Pyrococcus
species GB-D DNA polymerase mixture; Life Technologies). The
primer sequences were: sense, 5'-AGAAGTTCCGAGAGGAAGCC-3'; and
antisense, 5'-ACCTGTTGCCA-CCTTCTTCG-3', designed from the extracellular
domain of the rat CaR. To perform hot start PCR, the enzyme was added
during the initial 3-min denaturation and was followed by 35 cycles of
amplification (30-sec denaturation at 94 C, 30-sec annealing at 47 C,
and 1-min extension at 72 C). The reaction was completed by an
additional 10-min incubation at 72 C to allow completion of extension.
PCR products were fractionated on 1.2% agarose gels. The presence of a
480-nucleotide-long amplified product was indicative of a positive PCR
reaction.
Cloning and sequencing of RT-PCR products derived from the mouse
homolog of the CaR
RT-PCR products were ligated into the pCR 2.1 vector of the TA
cloning kit by incubation overnight at 14 C. Competent cells were
transformed according to the manufacturer’s instructions and placed on
ampicillin-containing agar in the presence of X-galactosidase.
Transformed cells were identified after overnight growth at 37 C as
white colonies. The white colonies were used to inoculate Luria Bertoni
medium, and plasmid DNA was extracted, digested with EcoRI,
and fractionated by agarose gel electrophoresis. DNA from positive
clones was further purified using the Qiagen Plasmid kit (Qiagen,
Chatsworth, CA) and sequenced bidirectionally using M13 forward and M13
reverse primers with an automated sequencer (AB377, Applied Biosystems,
Foster City, CA) in the DNA Sequence Facility of the University of
Maine (Orono, ME), using dideoxy terminator Taq
technology.
Chemotaxis assay of ST2 cells
Chemotaxis was evaluated using a Neuroprobe BW200S blindwell
chamber (Neuro Probe, Gaithersburg, MD) as previously described (17, 18). CaCl2 (1.8 or 4.8 mM), 300
µM neomycin sulfate, or 100 µM
GdCl3·6H2O in serum-free DMEM were loaded
into the lower chamber, which was separated from the upper well by a
5-mm membrane with 5-µm pores. ST2 cells (1 x 105
cells/ml) were dissociated briefly with trypsin-EDTA solution (Life
Technologies), washed twice, suspended in serum-free MEM, and added
to the upper chamber. After a 5-h incubation at 37 C, cells on the
upper surface of the membrane that had not migrated were scraped from
the membrane, and cells that had migrated to the opposite side of the
membrane were fixed with methanol and stained with Giemsa. The cells
that had migrated were then counted in six high power fields (x400)
using a light microscope. For the purpose of comparison between
multiple assays, the data were normalized as the fold increase in
cellular chemotaxis relative to that in the control.
DNA synthesis in ST2 cells
We assessed DNA synthesis in ST2 cells using
[3H]thymidine incorporation. ST2 cells were dissociated
with trypsin-EDTA solution (Life Technologies) and seeded in 24-well
(2.0-cm2) plates at a density of 1000 cells/well in 500
µl MEM containing 10% FBS as well as 1.8, 2.8, or 4.8
mM CaCl2; 300 µM neomycin
sulfate; or 100 µM GdCl3·6H2O.
After a 48-h incubation at 37 C, cells were pulsed with
[3H]thymidine (1 µCi/well). Incubations were terminated
after overnight incubation by removal of the medium and addition of 5%
trichloroacetic acid. Cells were then scraped and transferred to
microcentrifuge tubes. After centrifugation at 15,000 x
g and removal of the supernatant, the precipitate was washed
with 75% ethanol and desiccated at room temperature. The residual
pellet was dissolved in 20 mM NaOH and 1% SDS, and a
scintillation cocktail was added. Samples were counted in a liquid
scintillation counter.
Statistics
Experiments on chemotaxis and DNA synthesis were performed three
times on separate occasions. Results are shown as the mean ±
SEM for six determinations from a single representative
experiment. Statistical evaluation for differences between groups was
performed using one-way ANOVA followed by Fisher’s protected least
significant difference. For all statistical tests, P <
0.05 was considered significant.
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Results |
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), which was eliminated by
preincubating the primary antiserum with the peptide against which it
was raised (Fig. 1B).
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). A single major band was stained
in the presence of specific antiserum (lane 1). The molecular mass
corresponding to the position of the stained band (
150–160 kDa) was
of a size consistent with that of the intact, glycosylated CaR (19).
The specificity of the anti-CaR antiserum used in this study was
confirmed by abolition of the band after preabsorption of the anti-CaR
antiserum with the peptide against which it was raised (lane 2).
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shows Northern blot
analysis on poly(A)+ RNA isolated from ST2 cells, which
revealed four transcripts with sizes of 9.5, 8.5, 6.0, and 4.5 kb.
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). No products were observed when the
reverse transcriptase was omitted during the synthesis of cDNA (data
not shown). DNA sequence analysis of the PCR product revealed 95%
sequence identity with the rat CaR cDNA (Fig. 5), showing a difference in its predicted
protein sequence of one amino acid relative to that of the rat CaR at
position 132. These results show that the PCR product corresponded to a
CaR sequence, indicating the presence of bona fide CaR
transcripts in these cells.
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, 4
.8 mM
Ca2+o induced a chemotactic response of ST2
cells over control values at 1.8 mM
Ca2+o (P < 0.05). Both
neomycin sulfate (300 µM) and extracellular gadolinium
ion (Gd3+o; 100 µM) also induced
significant chemotactic responses over the control level
(P < 0.05).
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). Neomycin
sulfate (300 µM) and Gd3+o (100
µM) also significantly stimulated DNA synthesis over its
control level (P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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150–160 kDa) (19). In addition, both Northern
analysis performed on poly(A)+ RNA from ST2 cells and
RT-PCR performed on total RNA from these cells followed by sequence
analysis of the PCR products indicated the presence of bona
fide CaR transcripts. Thus, the present study shows that this
stromal cell line expresses both CaR protein and mRNA. The 9.5- and 4.5-kb transcripts in ST2 cells shown by Northern analysis are of sizes similar to those of transcripts expressed in both mouse kidney and murine AtT-20 cells (20). However, ST2 cells expressed additional 8.5- and 6.0-kb transcripts instead of the 7.5-kb transcript observed in AtT-20 cells or mouse kidney (20). There are previous instances where the relative ratios of the abundance of CaR transcripts vary from organ to organ (reviewed in Ref. 21). Because the 4.5-kb transcript encodes the entire functional CaR protein, the significance of the larger transcripts remains uncertain. However, the possibility of organ/cell type-specific, posttranscriptional regulation of CaR expression as a result of variations in the stabilities of the various CaR transcripts cannot be ruled out.
Bone marrow stromal cells are known to originate from mesenchymal stem
cells, which are also capable of differentiating into osteoblasts,
chondrocytes, adipocytes, myoblasts, and fibroblasts (22). Stromal
cells provide specific factors for hematopoietic stem cell survival and
differentiation by cell to cell contact through cell surface molecules
and by secreting various humoral factors, such as macrophage
colony-stimulating factor, granulocyte-macrophage colony-stimulating
factor, and IL-7 (1). The ST2 cell line used in this study also retains
such activities and supports both myelopoiesis and B cell lymphopoiesis
under specific culture conditions (2). In addition, this cell line can
support monocyte differentiation into osteoclasts in coculture with
murine spleen cells or mononuclear cells of murine bone marrow in the
presence of 1,25-dihydroxyvitamin D3 and dexamethasone
(3). Recently, ST2 cells themselves have been used as a model for
osteoblast precursors, because they have the capacity to differentiate
into bone-forming osteoblasts in response to BMP-2 (23). Taken
together, these data suggest that the ST2 cell line provides a useful
in vitro model system capable of carrying out functions
intimately related to the processes of both osteoclastic bone
resorption and osteoblastic bone formation. Substantial release of
calcium ions into the extracellular fluid of the bone/bone marrow
compartment takes place during bone turnover (4). Therefore, our
finding of the expression of the CaR in ST2 cells suggests that bone
marrow stromal cells within the immediate vicinity of sites of
resorptive activity could sense such changes in
Ca2+o and modulate the processes of bone
formation and resorption in vivo by cell to cell contact or
secretion of humoral factors. Recently, Ca2+o
was shown to induce the secretion of cytokines from human peripheral
blood mononuclear cells (24, 25), suggesting that a similar process is
possible in ST2 cells.
Our study also shows that high levels of Ca2+o and CaR agonists stimulate both chemotaxis and proliferation of ST2 cells. Although these actions could be mediated by the CaR, additional studies using approaches (e.g. antisense methodologies) that down-regulate the CaR and/or stromal cells derived from mice with targeted disruption of the CaR gene are needed to establish the role of CaR with certainty. Considering that ST2 cells have the potential to differentiate into bone-forming osteoblasts (23), our findings suggest a role for the CaR expressed in stromal cells in the reversal phase of bone remodeling. Bone formation is initiated by the migration of osteoblast precursors into resorption pits after the cessation of osteoclastic bone resorption (26). The CaR in stromal cells might sense the locally high levels of Ca2+o released from mineralized bone matrix during osteoclastic bone resorption (4). As a result, they might then proliferate, migrate into sites where new bone formation is required, and eventually initiate bone formation after their differentiation into mature osteoblasts.
Another possible implication of the CaR agonist-induced chemotaxis and proliferation of ST2 cells is that these responses might reflect the capacity of stromal cells to promote the differentiation of mononuclear precursors for formation of a subsequent generation(s) of new osteoclasts after the disappearance of earlier osteoclasts at a resorptive site. The fluid resorbed from bone by an active osteoclast that contains high levels of Ca2+o (4) is compartmentalized from the surrounding extracellular fluid during active bone resorption by the tight seal of the osteoclast to the underlying bone surface (4). Thus, the resorbed calcium ions are only released into the surrounding microenvironment, where they can potentially serve as a signal to cells within the surrounding bone marrow, if the resorbing osteoclast undergoes apoptotic degeneration or migrates elsewhere, and the concentrated Ca2+o within the solution beneath it is liberated. A previous study showed that Ca2+o could also stimulate the chemotaxis of monocytes (18) as well as the fusion of alveolar macrophages to form multinucleated giant cells (27). Hence, it is possible that stromal cells, by migrating into sites of active bone resorption along with monocytes in response to signals including high local levels of Ca2+o generated by the resorptive process, can first support the differentiation of mononuclear precursors into a new generation of osteoclasts and then later provide a source of osteoblast precursors for eventual initiation of bone formation (26). Clearly, the temporal duration and extent of resorption and the timely initiation of bone formation and full replacement of missing bone are crucial for skeletal integrity. Understanding the mechanisms underlying this highly orchestrated series of events is a challenge for ongoing research in this area.
Additional approaches that could potentially be taken to clarify these mechanisms is the investigation of bone turnover and metabolism in patients with autosomal dominant hypocalcemia and neonatal severe hyperparathyroidism (NSHPT), human disorders caused by activating and inactivating CaR mutations (10, 11), respectively, or using a homozygous CaR-deficient mouse, which is an animal model for NSHPT (28). The skeletal abnormalities found in NSHPT and the homozygous CaR-deficient mouse are thought to be caused secondarily by the associated severe hyperparathyroidism (29, 30), which makes it difficult to analyze the consequences of the CaR deficiency per se in these models. However, an in vitro study using primary cultures of bone marrow cells from homozygous CaR-deficient mice could circumvent this difficulty and provide insights into the role of the CaR in stromal cells in the control of bone turnover. Autosomal dominant hypocalcemia, in contrast, causes more modest changes in symptoms, bone metabolism, and circulating levels of calciotropic hormones and mineral ions (31); thus, investigating skeletal tissue and its metabolism in this disorder may also provide some clues to the biological importance of the CaR in bone turnover.
Recently, Quarles et al. reported that a Ca2+o-sensing mechanism in the mouse osteoblast-like MC3T3-E1 clonal cell line is functionally similar to but molecularly distinct from the CaR (32). After failing to detect expression of the CaR by Northern analysis and RT-PCR in MC3T3-E1 cells, they identified nucleotide sequences of putative CaR-related receptors (Casr-rs) in mouse genomic libraries by PCR (33). The deduced protein sequence of one of these putative receptors (Casr-rs1) was 63% similar and 40% identical to the CaR over the available transmembrane region. This putative receptor might also exist in bone marrow stromal cells and could sense the local changes in Ca2+o caused by bone turnover. However, this CaR-related nucleotide sequence, initially identified in MC3T3-E1 cells by RT-PCR and used as a probe to screen mouse genomic libraries to identify other related sequences, could only be identified in subsequent analyses of mouse tissues, including MC3T3-E1 cells, by RT-PCR and not by Northern analysis (33), suggesting very low levels of expression of uncertain physiological significance. Thus, additional studies are necessary to determine whether these CaR-related nucleotide sequences are actually expressed as mature proteins in MC3T3-E1 cells using specific antisera raised to their predicted protein sequences and what, if any, function(s) they serve in these cells.
On the basis of our results in this study using the murine clonal ST2 cell line, we suggest that the CaR is expressed in bone marrow stromal cells in vivo and that in this cell type it could potentially play physiological roles in both bone formation and bone resorption. Recently, we also found that two other murine cell lines, the monocyte-macrophage-like J774 cell line and the osteoblast-like MC3T3-E1 cell line, express the CaR. Furthermore, CaR agonists stimulate chemotaxis and proliferation of both cell lines (34, 35). As stromal cells and osteoblasts both belong to the mesenchymal lineage, and monocytes-macrophages belong to the hematopoietic lineage (22), these findings suggest that the CaR is expressed in diverse bone marrow cells, confirming our previous observation with primary cultures of human and murine bone marrow (12). Both cell types are known to appear at sites of bone resorption at the end of the resorptive phase of bone remodeling and to initiate bone formation (26). Therefore, our findings are consistent with an important role for the CaR in the key reversal phase of bone remodeling through its sensing of calcium ions released by osteoclast-mediated bone resorption and ensuring the chemotaxis and proliferation of cell populations needed for the orderly transition from bone breakdown to its subsequent replacement by newly formed bone.
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Footnotes |
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Received February 5, 1998.
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C. S. Kovacs, L. L. Chafe, M. L. Woodland, K. R. McDonald, N. J. Fudge, and P. J. Wookey Calcitropic gene expression suggests a role for the intraplacental yolk sac in maternal-fetal calcium exchange Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E721 - E732. [Abstract] [Full Text] [PDF]
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