| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Endocrine Research Unit (W.C., C.T., T.-H.C., S.A.P., D.S.), Department of Medicine, Department of Dermatology (L.K., Y.O.), Veterans Affairs Medical Center, University of California, San Francisco, California 94121; and Department of Radiobiology (S.M.), School of Medicine, University of Utah, Salt Lake City, Utah 84112
Address all correspondence and requests for reprints to: Dolores Shoback, 111N, Endocrine Research Unit, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: dolores{at}itsa.ucsf.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
140–190 kDa) in membrane fractions isolated from growth
plate cartilage, primary cultures of rat chondrocytes, and several
osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP
responses to high [Ca2+]o were evident in
C5.18 cells and all osteogenic cell lines tested except for SaOS-2
cells. In the latter, high [Ca2+]o reduced
PTH-induced cAMP formation. Raising [Ca2+]o
also increased intracellular free [Ca2+] in SaOS-2
and C5.18 cells. These studies confirm expression of CaRs in cartilage
and bone and support the concept that changes in
[Ca2+]o may couple to signaling pathways
important in skeletal metabolism. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In vitro studies have demonstrated that extracellular [Ca2+] ([Ca2+]o) influences chondrogenic and osteogenic function. Studies by Bonen and Schmid (6) showed that high [Ca2+]o increased type II and X collagen synthesis in tibio-tarsus chondrocytes from chick embryos. In the eggshell-less chicken model, Ca2+ deprivation induced formation of cartilage nodules in the calvaria, a site at which intramembranous bone typically develops (7). Other studies with this model identified a population of calvarial cells that preferentially differentiated into cartilage when maintained at low [Ca2+]o (8). Our previous studies showed that changes in [Ca2+]o modulate matrix protein expression and several functions of the nontransformed chondrogenic C5.18 cells (9). These observations suggest that chondrocytes have the ability to detect and respond to different [Ca2+]o.
Ca2+ is known to affect several aspects of bone cell function. Osteoclasts in vitro respond to increases in [Ca2+]o with an elevation of intracellular free [Ca2+] ([Ca2+]i), and high [Ca2+]o suppresses bone resorption (10, 11). Changes in [Ca2+]o and CaR agonists modulate DNA synthesis and chemotactic responses in osteoblast-like MC3T3-E1 (12, 13, 14) and bone marrow cells (15). The mechanisms underlying this sensitivity to [Ca2+]o are, however, still unclear.
[Ca2+]o regulates diverse cellular functions by activating membrane Ca2+-sensing receptors (CaRs) (16, 17). By multiple approaches, we previously found parathyroid-like CaRs in C5.18 cells (9). Other laboratories have reported that CaRs are expressed in bone marrow cells (15), osteoblastic MC3T3-E1 cells (14), the osteosarcoma cell lines UMR-106 and SaOS-2 (18), and cultured rabbit osteoclasts (11).
To date, no information is available regarding which cells express CaRs in cartilage and bone. To address this issue, we performed in situ hybridization, immunocytochemistry, and immunoblotting. We also assessed whether CaR agonists could couple to signal transduction pathways in bone and cartilage model systems. CaRs are expressed in specific cells in bone and cartilage, and their expression pattern may provide clues critical to understanding how Ca2+ alters skeletal homeostasis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Tissue preparation
The femurs and tibias from 21-day-old rats were obtained after
euthanasia, according to protocols approved by the Animal Welfare
Subcommittee of the San Francisco Department of Veteran Affairs Medical
Center. The tibia growth plate and adjacent articular cartilage from
newborn calves were dissected shortly after death. All tissues were
immediately fixed in paraformaldehyde (5%, vol/vol) plus
glutaraldehyde (0.2%, vol/vol) at 4 C for 24–48 h and decalcified
with EDTA (10%, wt/vol) in PBS at 4 C for 2–3 weeks. After paraffin
embedding, 5-µm sections were cut and mounted on glass slides.
Cell culture
C5.18 cells, derived from fetal rat calvaria (19), were provided
by Dr. Jane E. Aubin (University of Toronto, Canada) and were
maintained as previously described (9). The osteogenic cell lines
MC3T3-E1, ROS 17/2.8, UMR-106, and SaOS-2 cells were cultured in
-modified MEM, Ham’s F-12 media, DMEM, and McCoy’s media,
respectively, supplemented with FCS (10%, vol/vol), penicillin (100
U/ml), and streptomycin (100 µg/ml). Measurements of inositol
phosphates (InsPs) and cAMP were performed on 1- to 2-day
postconfluent cells in 35-mm wells approximately 48–72 h after plating
(5 x 104 cells/cm2) (20, 21, 22).
Primary chondrocyte cultures for Western blotting were prepared (23)
from articular cartilage from 3-month-old rats, which was aseptically
dissected in ice-cold Medium A [D-MEM plus HEPES (20 mM),
NaHCO3 (45 mM, pH 7.4), penicillin (100 U/ml),
and streptomycin (100 µg/ml)]. After mincing, cartilage pieces were
treated with trypsin (0.25%) in Medium A for 30 min at 37 C. After
inactivating trypsin with Medium A containing heat-inactivated FCS
(10%, vol/vol), tissue was digested in Medium A (5 ml/g tissue) with
collagenase IA (0.18 g/100 ml), hyaluronidase (0.1 g/100 ml), and
deoxyribonuclease I (0.01 g/100 ml), at 37 C, with gentle agitation.
Cells were separated from undigested cartilage with a nylon mesh,
collected by centrifugation at 400 x g for 10 min, and
washed twice with Medium A. Cells released from cartilage were plated
in -modified MEM, supplemented with nucleosides (0.004%, wt/vol),
FCS (15%, vol/vol), penicillin (100 U/ml), and streptomycin (100
µg/ml), for 2 days, to allow for attachment. Chondrocytes were then
replated at a density of 5 x 104
cells/cm2 and allowed to reach confluence before membrane
protein isolation.
In situ hybridization
In situ hybridization of bone sections was adapted
from previously described methods using digoxigenin-labeled sense or
antisense RNA probes (9) prepared from the human keratinocyte CaR
complementary (cDNA) template (nucleotides 2442–2746) (24).
Signals were visualized with diaminobenzidine (DAB) chromogen
substrate. Sections were counterstained with aqueous hematoxylin.
Experiments were repeated at least twice on tissue sections from two or
more animals.
Immunocytochemistry
Immunocytochemistry of rat articular chondrocytes and bone
sections was performed, as previously described (9), with 2 antisera
[21825A (500 nM) and no. 421113A (100 nM)]
(25). To assess specificity, sections were treated with either
antiserum preincubated with excess peptide (100-fold for no. 421113A or
1000-fold for no. 21825A) or nonimmune rabbit IgG. After DAB-staining,
cells or sections were counterstained, either with aqueous hematoxylin
or with alcian green, for the detection of matrix proteoglycans (9).
Experiments were repeated at least four times on tissue sections from
two or more animals.
Immunoblotting
Crude membrane protein fractions were prepared (9, 25) from 1-
to 2-day postconfluent cultures of rat articular chondrocytes; C5.18,
SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1 cells; and bovine growth
plate cartilage. Immunoblotting was performed after electrophoresis of
membrane proteins (50 µg) on 6% SDS-PAGE gels and transfer to
nitrocellulose membranes (9, 25). After blocking with Blotto [150
mM NaCl, 10 mM Tris-HCl (pH 7.4), nonfat dry
milk (5%, vol/vol), Tween-20 (0.05%, vol/vol)], membranes were
incubated with either anti-CaR antiserum (21825A, 50 nM),
the same antiserum preincubated with 1000-fold excess peptide, or
nonimmune rabbit IgG. After three washes and incubation with
peroxidase-conjugated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA; 1:4000), standard ECL assay kits were used
for signal detection (Amersham Pharmacia Biotech).
CaR cDNA amplification and subcloning
RT-PCR was performed, as previously described (26), on RNA
extracted from epiphyseal growth plate cartilage of 2-day-old mice.
Briefly, first-strand cDNA was reverse-transcribed from total RNA using
Superscript II reverse transcriptase and oligo-dT primers (26) and
subsequently used in PCR with Pwo DNA polymerase. Primers
were derived from the nucleotide sequence corresponding to the
extracellular domain of the mouse kidney CaR
cDNA2 (sense primer:
5'-CAAGGTCATTGTCGTTT TCTCCAGC; antisense primer:
5'-GCAATGCAGGAAGTGTAGTTCTCAT). Amplified cDNA was subcloned into pCR
2.1 plasmid by TOPO-TA cloning kit (Invitrogen, Carlsbad,
CA) and sequenced by the Biomolecular Resource Center, University of
California, San Francisco.
Measurement of InsPs and cAMP
InsPs were determined in extracts from 1- to 2-day postconfluent
C5.18, SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1 cells after labeling
membrane polyphosphoinositides with 3H-myoinositol (2
µCi/ml) for 48 h, as previously described (25, 27). In each
experiment, prelabeled cell cultures were washed three times with MEM
and incubated with the same media containing LiCl (10 mM),
CaCl2 (0.5 mM), and MgSO4 (0.5
mM) for the pretreatment period.
[Ca2+]o was then varied by adding fresh media
containing either 0.5 or 10 mM CaCl2 for 60
min. Total 3H-InsPs were extracted and analyzed as
described (25, 27).
Accumulation of cAMP was determined after incubation of cells with assay medium [MEM + BSA (0.1%, wt/vol), IBMX (0.4 mM), MgSO4 (0.5 mM)] containing 0.5, 2.5, 5.0, or 10.0 mM CaCl2 with or without PTH (10-6 M) for 10 min (25, 27).
Measurement of [Ca2+]i
[Ca2+]i was determined in either
subconfluent (60–80%) or 1- to 2-day postconfluent C5.18 cells and 1-
to 2-day postconfluent SaOS-2 cells using an InCyt Im2
imaging system (Intracellular Imaging, Inc., Cincinnati,
OH) with a 40x Nikon Fluor objective. Briefly, cells were
loaded with Fura-2 AM (3–10 µM) in Buffer B [HEPES (20
mM, pH 7.4), NaCl (120 mM), KCl (5
mM), MgCl2 (1 mM), pyruvate (1
mg/ml), glucose (1 mg/ml), and CaCl2 (1.0 mM)]
at 37 C for 30–40 min. After three washes with Buffer B, cells were
incubated at 37 C for 15–30 min before recording. Fluorescent emission
(510 nm) was detected by a COHU high-performance CCD camera (COHU, Inc,
San Diego), digitized, and stored in a microcomputer. The 340/380
excitation ratio (R340/380) of emitted fluorescence was
calculated and presented.
Statistics
Data, normalized to baseline activity in individual experiments,
were combined and reported as mean ± SE. Statistical
significance was assessed by Student t test or ANOVA with
f-test using Microsoft Corp. Excel (Microsoft Corp., Seattle, WA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, a and
c, arrowheads). Proliferating chondrocytes demonstrated
weaker signals (Fig. 1, a and c, arrows). Staining was
evident in the majority of articular chondrocytes (Fig. 1b) and was
specific, because it was absent when the complementary sense cRNA probe
was used (Fig. 1d, and data not shown).
|
) and rat femur (Fig. 2b) showed
expression of CaR transcripts in most osteoblasts
(arrowheads), osteocytes (arrows), and some cells
in the bone marrow (asterisks). Staining was, however, weak
to absent in osteoclasts (Fig. 2, a and b, double
arrowheads). Staining was judged specific, because it was absent
in the hybridization with sense cRNA probe (Fig. 2c).
|
, there was strong
immunostaining (depicted by brown color) in chondroblasts
within the chondrogenic layer (arrows) but no apparent
staining in chondroblasts within the fibrous perichondrium
(asterisk). Higher magnification confirmed both membrane and
cytoplasmic staining in these chondroblasts (Fig. 3b). CaR
immunostaining was also evident in the epiphyseal growth plate of the
bovine proximal tibia (Fig. 3, c and d). Staining was weak in the
proliferating zone (Fig. 3, c and d, arrows) and increased
as the cells progressed to the maturation (Fig. 3d, arrowhead) and hypertrophic zones (Fig. 3c, arrowhead; Fig. 3d, double arrow). In articular
cartilage, staining was strong in chondroblasts and chondrocytes (Fig. 3e, arrowheads). CaR immunoreactivity was specific, because
staining was not seen when either anti-CaR antiserum preincubated with
excess peptide (data not shown) or nonimmune serum was used (Fig. 3f).
Identical staining patterns were observed with another anti-CaR
antiserum (21825A), raised against an extracellular epitope of the
bovine CaR, confirming the above results (data not shown).
|
, and data
not shown). The staining patterns with these antisera in the adjacent
rat distal femur growth plate were comparable with those noted in Fig. 3
for bovine proximal tibia. Lower-power views showed weak staining in
the columns of proliferating cells (Fig. 4b) that increased in
hypertrophic chondrocytes (Fig. 4b, arrow). This was
confirmed in high-power views (Fig. 4c). The specificity of the
staining pattern was confirmed by its abolition after preincubation of
antiserum with excess peptide (data not shown).
|
). CaR staining was more intense in
cells within the nodules and absent in cultures incubated with
nonimmune IgG (Fig. 5b), underscoring specificity of the staining.
|
shows the primary
spongiosa of bovine tibia in which CaR immunoreactivity is present in
most osteoblasts (arrowheads). Staining was comparable, in
intensity, with adjacent hypertrophic chondrocytes (arrows).
There was no immunoreactivity in cells with an osteoclastic or
chondroclastic morphology (double arrowheads). Higher-power
micrographs in Fig. 6b confirmed the cytoplasmic and membrane
distribution of the staining in osteoblasts (arrowheads) and
lack of staining in osteoclasts (double arrowheads). In the
same field, some mononuclear bone marrow cells (arrows) were
faintly stained. CaR immunoreactivity was also detected in most
osteocytes (Fig. 6c, arrows) at levels similar to those of
adjacent osteoblasts (Fig. 6c, arrowheads).
|
, a–c, double arrows), some osteoclasts, farther away from
the growth plate, stained positively with anti-CaR antiserum. The
intensity of staining in a large osteoclast in Fig. 6d
(arrow) was comparable with that of adjacent osteoblasts
(arrowheads). Staining was weak to absent, however, in three
smaller osteoclasts in the same field (double arrows). CaR
expression in osteoclasts may vary, therefore, with their location in
bone and perhaps their stage of differentiation, given that the large
osteoclasts tend to be older than small osteoclasts.
In rat tibia shown in Fig. 6e, CaR immunoreactivity was detected in
osteoblasts (arrowheads), cells in the bone marrow, and
osteocytes (arrows) but not in osteoclasts (double
arrowheads), confirming the above observations in bovine bone
sections. Some osteocytes in the same field were not stained
(double arrows). Specificity of staining in both bovine and
rat tissues was confirmed by its abolition after preincubation of
antiserum with excess peptide (data not shown).
Detection of CaR protein by immunoblotting
To assess the size of CaRs, we immunoblotted crude membrane
fractions from cultured rat articular chondrocytes, C5.18 cells, bovine
growth plate cartilage, and four osteoblastic cell lines. Antisera
blotted three major bands in membrane preparations from HEK-293 cells
expressing the bovine CaR cDNA (Fig. 7a, upper panel, lane 1), as previously observed (9, 25). Bands
of approximately 140 and approximately 160 kDa in CaR-expressing
HEK-293 cells are thought to be the variably glycosylated forms of the
CaR, which has a protein core of approximately 120 kDa (16, 25). The
protein band of more than 205 kDa in these membranes is specific and
likely represents receptor aggregates (16, 25).
|
, upper panel, lane 2) (9). Specific
immunoreactivity, larger than our highest protein standard, was also
detected in these cells. In rat articular chondrocytes, the most
prominent band was approximately 155 kDa (Fig. 7a, upper
panel, lane 3), although there were several less discrete, but
specific, bands between approximately 170 and approximately 200 kDa and
more than 205 kDa. In lysates from bovine growth plate cartilage, there
was a predominant band of approximately 140 kDa and 2 fainter bands of
approximately 168 and approximately 175 kDa (Fig. 7a, upper
panel, lane 4). Immunoreactivity in these cells was specific,
because it was absent when preimmune serum was used and was abolished
by preincubation of antiserum with excess peptide (Fig. 7a, lower
panel). These results suggest that CaRs in articular chondrocytes
and growth plate share epitopes with the bovine parathyroid CaR and are
likely to be glycoproteins of similar molecular weight.
In cultured osteoblastic model systems, such as SaOS-2 cells, we
detected a prominent band at approximately 190 kDa, a fainter band of
approximately 158 kDa, and two bands of more than 200 kDa in membrane
fractions (Fig. 7b, upper panel, lane 1). A similar pattern
was observed in membrane fractions prepared from MC3T3-E1 cells, with
one band at approximately 155 kDa, a weaker signal at approximately 190
kDa, and the strongest signal at more than 205 kDa (Fig. 7b, upper panel, lane 3). In contrast, we detected only one band
of approximately 150 kDa in ROS 17/2.8 cells (Fig. 7b, upper
panel, lane 2). Two protein bands of approximately 140 and
approximately 150 kDa were detected in UMR-106 membrane fractions. The
immunoreactivity of the above bands was specific, because it was
abolished by preincubation of antiserum with excess peptide (Fig. 7b, lower panels).
Cloning of a partial CaR cDNA from epiphyseal growth plate
cartilage
To determine the molecular identity of a putative CaR in
cartilage, we performed RT-PCR on RNA isolated from mouse epiphyseal
growth plate and kidney (the latter as a control tissue). A cDNA with
an expected size of approximately 1 kb was amplified from both
cartilage and kidney (see Fig. 8). The
PCR reaction was specific, because no cDNA was amplified from control
RNA samples incubated without reverse transcriptase. We subcloned this
PCR product and analyzed its sequence. Both the nucleotide and deduced
amino acid sequences have a high degree of similarity (89% and 95%,
respectively) to the extracellular domain (residues no. 265–599) of
the human parathyroid CaR (28), suggesting that this CaR cDNA is the
cartilage homologue of the CaRs identified in other systems.
|
). This increment in
[Ca2+]o, however, had no significant effect
on InsP production in SaOS-2 cells (see Table 1).
|
, table (n
= 12); P > 0.1 for 2.5 and 5 mM Ca2+vs. controls; and P < 0.03 for 10
mM Ca2+ vs. controls]. Incubation
of SaOS-2 cells with PTH (10-6 M) at 0.5
mM Ca2+ profoundly increased cAMP accumulation,
by 16.5 ± 1.1-fold over basal (P < 0.001; see
Fig. 9). PTH increased cAMP levels by 12.4 ± 1.3, 10.7 ±
1.1, and 9.3 ± 0.7-fold at 2.5, 5.0, and 10 mM
Ca2+, respectively (Fig. 9). The ability of PTH to
stimulate cAMP accumulation was modestly blunted by raising
[Ca2+]o. Similar inhibitory effects of high
[Ca2+]o on cAMP production were also observed
in C5.18 cells (data not shown). These findings suggest that the cAMP
responses of osteoblastic and chondrogenic cells to PTH can be
modulated by the [Ca2+]o.
|
, PTH did not affect high
[Ca2+]o-stimulated Ca2+
mobilization (a) or basal [Ca2+]i at 0.5
mM Ca2+ (b). These and the above observations
indicate that signaling pathways coupled to PTH receptors and CaRs
differ.
|
). In approximately
20% of cells, high [Ca2+]o induced a rapid
(within 20 sec) and transient (duration, <180 sec) peak increase in
[Ca2+]i. This was followed by a sustained
elevation in [Ca2+]i lasting more than 10 min
(Fig. 11a). In the majority of cells (80%),
[Ca2+]i rose slowly over approximately 2 min,
until it reached a plateau, which was sustained for at least 10 min
(Fig. 11b). Figure 11c shows the composite kinetics of the changes in
[Ca2+]i recorded from 31 cells in which the
sharp initial phase of the response is not evident (n = 3
experiments). In contrast, in 1- to 2-day postconfluent cells, more
cells (
60%) expressed a fast transient intracellular
Ca2+ response. As a result, the sharp transient phase of
the response was more evident in the ensemble recording of the cell
populations (Fig. 11d). To determine whether changes in
[Ca2+]i are sensitive to
[Ca2+]o within the physiological range, we
performed dose-response studies. Raising
[Ca2+]o from 0.5 to 1.0 mM
substantially increased [Ca2+]i (see Fig. 11e), suggesting that physiologic levels of extracellular
Ca2+ could readily modulate
[Ca2+]i in these cells. Similar results were
obtained in 2 other experiments.
|
, LaCl3 (100 µM) promptly and profoundly
suppressed the sustained increase of [Ca2+]i
caused by high [Ca2+]o. GdCl3
(100 µM) produced effects similar to those of
LaCl3 (data not shown). These results suggest that high
[Ca2+]o elevates
[Ca2+]i in C5.18 cells by increasing
Ca2+ uptake across the membrane. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
These studies are the first to demonstrate CaR expression in growth plate in a distinctive pattern. Expression is strong in chondroblasts and hypertrophic chondrocytes and absent to weak in proliferating and maturing chondrocytes. This may be important because chondroblasts and hypertrophic chondrocytes serve very different functions. Chondroblasts synthesize matrix proteins, such as proteoglycans and type II collagen, which comprise early hyaline cartilage (3). Hypertrophic chondrocytes, in contrast, participate in later steps of chondrogenesis. They synthesize type X collagen, express alkaline phosphatase, produce matrix vesicles, and accumulate Ca2+ in their intracellular compartments (3, 33, 34). These and other steps lead to the mineralization of cartilage and ultimately to new bone formation (1, 3, 33, 35).
Variable expression of CaRs in different populations of chondrocytes may occur because [Ca2+]o is important to some (but not all) chondrocytes. [Ca2+]o is likely to be relevant to hypertrophic chondrocytes, which sequester Ca2+ and secrete matrix proteins that eventually become mineralized. Perhaps [Ca2+]o may be less important to the functions of proliferating and maturing chondrocytes in which we found weaker CaR expression. Alternatively, Ca2+-sensing mechanisms other than the CaR may be present in those cells.
Our findings suggest that CaRs may play a role in the differentiation of or matrix production by chondrocytes. We previously found that C5.18 cells [which resemble hypertrophic chondrocytes by their expression of late chondrogenic markers (e.g. type X collagen and alkaline phosphatase)] respond to high [Ca2+]o. High [Ca2+]o suppresses aggrecan, type II and type X collagen, and alkaline phosphatase mRNA levels and inhibits cartilage matrix production (9). We also found that high [Ca2+]o promotes matrix mineralization and the expression of type I collagen, osteonectin, and osteopontin mRNA in C5.18 cells3 [markers typically associated with the terminal differentiation of hypertrophic chondrocytes (36, 37)]. Thus, CaRs may participate in as-yet-undefined ways in differentiation and mineralization of chondrocytes.
Our studies revealed distinct kinetic patterns of intracellular Ca2+ responses in C5.18 cells. There was a fast, transient phase in the intracellular Ca2+ response analogous to CaR receptor-mediated Ca2+ mobilization in other systems. This response is thought to be caused by the release of Ca2+ from intracellular stores (38). This transient Ca2+ mobilization was observed in only a small number of cells in subconfluent cultures but became the prevalent response in confluent and postconfluent cultures. It is still unclear how the stage of growth in culture can affect signal transduction responses.
Previous studies did not demonstrate significant changes in CaR expression in C5.18 cells in subconfluent and up-to-10-days postconfluent cultures (9). It is, therefore, likely that the intracellular Ca2+ responses we observed at different stages of confluence are caused by postreceptor signaling molecules or possibly to other Ca2+-sensing mechanisms.
Increases in [Ca2+]i, with or without a preceding rapid transient, were sustained for more than 10 min. This prolonged mobilization of Ca2+ could be blocked by La3+ and Gd3+ (suggesting that Ca2+ entry via membrane channels is important in sustained Ca2+ mobilization). CaRs couple to nonselective cation channels, permeable to Ca2+, in other cells (39). During cartilage differentiation, chondrocytes actively mobilize and store Ca2+ in their intracellular compartments, which is later released for matrix mineralization (5, 34). Whether increased CaR expression in hypertrophic chondrocytes amplifies the ability of cells to mobilize and store Ca2+ and produce mineralized cartilage is yet to be explored. As in C5.18 cells, high [Ca2+]o also induced a sustained increase in [Ca2+]i in osteoblastic SaOS-2 cells. Because osteoblasts are important in bone matrix synthesis and mineralization, their ability to detect and mobilize Ca2+ could be crucial in maintaining mineral homeostasis.
Our findings in rat and bovine bone sections were predictable, in part, by previous studies. CaRs were expressed in most osteoblasts and bone marrow cells and in some (but not all) osteocytes. Studies with RT-PCR and Western and Northern blotting demonstrated CaR expression in UMR-106, SaOS-2, and MC3T3-E1 cells (14, 18). House et al. (15) also showed CaR expression in cultured human bone marrow mononuclear cells. Whereas the latter cells may be the precursors of osteoblasts and osteoclasts, the remainder of the studies from other groups were done with transformed osteoblastic (osteosarcoma) cell lines maintained in long-term culture. How closely they resemble normal osteoblasts is uncertain. Our studies are the first to identify CaRs by immunocytochemistry and in situ hybridization in normal bone. The fact that high [Ca2+]o can activate signal transduction pathways in osteoblastic and chondrogenic cell lines, similar to those coupled to classical CaRs, suggests that these receptors may serve similar Ca2+-sensing functions in these systems.
In SaOS-2 cells, increases in [Ca2+]o produce
dual effects on cAMP metabolism. In the absence of PTH, high
[Ca2+]o modestly increased cAMP production.
In contrast, high [Ca2+]o suppressed cAMP
production in the presence of PTH. The difference could be caused by
the ability of adenylate cyclases to be activated by high
[Ca2+]i (40, 41) in the absence of PTH and
the ability of PTH to couple to Gs and to
Gi activation. Further studies will be needed to address
these possibilities definitively.
The distribution of CaRs among osteoclasts was unexpected. CaR
immunoreactivity was absent in osteoclasts close to the growth plate
but was apparent in osteoclasts in the secondary spongiosa distal to
the growth plate. In the secondary spongiosa, the intensity of CaR
staining seemed to increase with osteoclast size (see Fig. 4d).
Osteoclasts are preferentially formed in the primary spongiosa during
bone growth (42), and their age is estimated to be greater as a
function of their distance from growth plate (43). Thus, CaR expression
may be restricted to the mature osteoclasts. This finding requires
confirmation, and its significance is unknown. The following is,
however, clear: osteoclasts in vitro respond to changes in
[Ca2+]o with increases in
[Ca2+]i; high
[Ca2+]o suppress bone resorption (10); and
local [Ca2+] in bone during active resorption may reach
concentrations as high as 40 mM (44). Thus,
[Ca2+]o, potentially via CaR activation, may
regulate osteoclast function.
Studies by Quarles et al. (13) and Yamaguchi et al. (14) showed that changes in [Ca2+]o and CaR agonists modulate DNA synthesis and chemotaxis in osteoblast-like MC3T3-E1 cells. Membrane CaRs were proposed by both groups to mediate the responses of osteoblasts to [Ca2+]o (13, 14). There is controversy, however, regarding the molecular identity of these receptors. Sequences homologous to known CaRs, were identified by Yamaguchi et al. (14). Quarles et al. (13), however, did not detect CaR sequences by RT-PCR. The reasons for these differences may be methodologic (14). Our studies to assess CaR expression using immunocytochemistry, in situ hybridization, and immunoblotting support the idea that molecules homologous to known parathyroid and kidney CaRs are present in bone (16, 17).
Although full-length cDNAs encoding chondrocyte or osteoblast CaRs have not yet been isolated, our data underscore the biochemical and functional similarities between such putative receptors and known CaRs. In situ hybridization with a CaR probe indicated the presence of transcripts in cartilage and bone with homology to known CaRs. RT-PCR demonstrated a product whose sequence was highly homologous to CaR cDNAs identified in other tissues. Antisera raised against intracellular and extracellular epitopes of the bovine parathyroid CaR revealed similar staining patterns in both rat and bovine cartilage and bone sections. Western blotting suggested interesting biochemical differences between CaRs in cartilage and bone and those in the parathyroid. Variations in the number and sizes of CaR proteins by Western blotting were also reported in UMR-106, SaOS-2, and MC3T3-E1 cells (14, 45). Variable degrees of glycosylation could explain the differences in molecular weights among these putative CaRs. Alternatively spliced variants of the CaR may be expressed in osteogenic and chondrogenic cells similar to those reported in keratinocytes (26). Molecular cloning of full-length CaR cDNAs in cartilage and bone will be required to address these issues. The present studies, taken together, begin to address potentially key aspects of the control of chondrogenic and skeletal function that may be subject to local regulation by extracellular Ca2+.
|
Footnotes |
|---|
2 Y. Oda and co-workers, manuscript in
preparation; deposited sequence in GenBank (Accession no.
AF110178)
3 W. Chang and D. Shoback, unpublished
observations.
Received April 7, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  P. Wellendorph, L. D. Johansen, A. A Jensen, E. Casanova, M. Gassmann, P. Deprez, P. Clement-Lacroix, B. Bettler, and H. Brauner-Osborne No evidence for a bone phenotype in GPRC6A knockout mice under normal physiological conditions J. Mol. Endocrinol., March 1, 2009; 42(3): 215 - 223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Belozeroff, W. G. Goodman, L. Ren, and K. Kalantar-Zadeh Cinacalcet Lowers Serum Alkaline Phosphatase in Maintenance Hemodialysis Patients Clin. J. Am. Soc. Nephrol., March 1, 2009; 4(3): 673 - 679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Chang, C. Tu, T.-H. Chen, D. Bikle, and D. Shoback The Extracellular Calcium-Sensing Receptor (CaSR) Is a Critical Modulator of Skeletal Development Sci. Signal., September 2, 2008; 1(35): ra1 - ra1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Brown and J. B. Lian New Insights in Bone Biology: Unmasking Skeletal Effects of the Extracellular Calcium-Sensing Receptor Sci. Signal., September 2, 2008; 1(35): pe40 - pe40. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nakagawa, E. C. Perez, J. Oh, F. Santos, A. Geldyyev, M.-L. Gross, F. Schaefer, and C. P. Schmitt Cinacalcet does not affect longitudinal growth but increases body weight gain in experimental uraemia Nephrol. Dial. Transplant., September 1, 2008; 23(9): 2761 - 2767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Cheng, C. Tu, L. Rodriguez, T.-H. Chen, M. M. Dvorak, M. Margeta, M. Gassmann, B. Bettler, D. Shoback, and W. Chang Type B {gamma}-Aminobutyric Acid Receptors Modulate the Function of the Extracellular Ca2+-Sensing Receptor and Cell Differentiation in Murine Growth Plate Chondrocytes Endocrinology, October 1, 2007; 148(10): 4984 - 4992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Chang, C. Tu, Z. Cheng, L. Rodriguez, T.-H. Chen, M. Gassmann, B. Bettler, M. Margeta, L. Y. Jan, and D. Shoback Complex Formation with the Type B {gamma}-Aminobutyric Acid Receptor Affects the Expression and Signal Transduction of the Extracellular Calcium-sensing Receptor: STUDIES WITH HEK-293 CELLS AND NEURONS J. Biol. Chem., August 24, 2007; 282(34): 25030 - 25040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Dvorak, T.-H. Chen, B. Orwoll, C. Garvey, W. Chang, D. D. Bikle, and D. M. Shoback Constitutive Activity of the Osteoblast Ca2+-Sensing Receptor Promotes Loss of Cancellous Bone Endocrinology, July 1, 2007; 148(7): 3156 - 3163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rodriguez, C. Tu, Z. Cheng, T.-H. Chen, D. Bikle, D. Shoback, and W. Chang Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2+-Sensing Receptor in Growth Plate Chondrocytes Endocrinology, December 1, 2005; 146(12): 5294 - 5303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rodriguez, Z. Cheng, T.-H. Chen, C. Tu, and W. Chang Extracellular Calcium and Parathyroid Hormone-Related Peptide Signaling Modulate the Pace of Growth Plate Chondrocyte Differentiation Endocrinology, November 1, 2005; 146(11): 4597 - 4608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yamauchi, T. Yamaguchi, H. Kaji, T. Sugimoto, and K. Chihara Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E608 - E616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Chattopadhyay, S. Yano, J. Tfelt-Hansen, P. Rooney, D. Kanuparthi, S. Bandyopadhyay, X. Ren, E. Terwilliger, and E. M. Brown Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts Endocrinology, July 1, 2004; 145(7): 3451 - 3462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Dvorak, A. Siddiqua, D. T. Ward, D. H. Carter, S. L. Dallas, E. F. Nemeth, and D. Riccardi Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones PNAS, April 6, 2004; 101(14): 5140 - 5145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Purroy and N. K. Spurr Molecular genetics of calcium sensing in bone cells Hum. Mol. Genet., October 1, 2002; 11(20): 2377 - 2384. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Chang, C. Tu, S. Pratt, T.-H. Chen, and D. Shoback Extracellular Ca2+-Sensing Receptors Modulate Matrix Production and Mineralization in Chondrogenic RCJ3.1C5.18 Cells Endocrinology, April 1, 2002; 143(4): 1467 - 1474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Chang, S. Pratt, T.-H. Chen, L. Bourguignon, and D. Shoback Amino Acids in the Cytoplasmic C Terminus of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Cell-surface Expression and Phospholipase C Activation J. Biol. Chem., November 16, 2001; 276(47): 44129 - 44136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Shoback and W. Chang Editorial: Starvation Amidst Plenty--Rickets and Hypercalcemia in Calcium Receptor Knockout Mice Endocrinology, September 1, 2001; 142(9): 3733 - 3735. [Full Text] [PDF]
|
|
|
|
|
|
S. C. Garner, M. Pi, Q. Tu, and L. D. Quarles Rickets in Cation-Sensing Receptor-Deficient Mice: An Unexpected Skeletal Phenotype Endocrinology, September 1, 2001; 142(9): 3996 - 4005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. D. Bennett, U. Alvarez, and K. A. Hruska Receptor-Operated Osteoclast Calcium Sensing Endocrinology, May 1, 2001; 142(5): 1968 - 1974. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. Yamaguchi, N. Chattopadhyay, O. Kifor, C. Ye, P. M. Vassilev, J. L. Sanders, and E. M. Brown Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line Am J Physiol Cell Physiol, February 1, 2001; 280(2): C382 - C393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Brown and R. J. MacLeod Extracellular Calcium Sensing and Extracellular Calcium Signaling Physiol Rev, January 1, 2001; 81(1): 239 - 297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Sanders, N. Chattopadhyay, O. Kifor, T. Yamaguchi, R. R. Butters, and E. M. Brown Extracellular Calcium-Sensing Receptor Expression and Its Potential Role in Regulating Parathyroid Hormone-Related Peptide Secretion in Human Breast Cancer Cell Lines Endocrinology, December 1, 2000; 141(12): 4357 - 4364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Huang, S.-L. Cheng, and E. Slatopolsky Sustained Activation of the Extracellular Signal-regulated Kinase Pathway Is Required for Extracellular Calcium Stimulation of Human Osteoblast Proliferation J. Biol. Chem., June 8, 2001; 276(24): 21351 - 21358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-L. Tu, W. Chang, and D. D. Bikle The Extracellular Calcium-sensing Receptor Is Required for Calcium-induced Differentiation in Human Keratinocytes J. Biol. Chem., October 26, 2001; 276(44): 41079 - 41085. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
