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Division of Nephrology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
Address all correspondence and requests for reprints to: L. Darryl Quarles, M.D., Box 3036, Duke University Medical Center, Durham, North Carolina 27710. E-mail: quarl001{at}mc.duke.edu
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Introduction |
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-aminoisobutyric
acid, and pheromone receptors (1, 2). Extracellular
calcium is the major ligand for CasR function, and the
predominant physiological function of CasR is to regulate
PTH secretion and parathyroid hyperplasia in response to changes in
extracellular calcium. The central role of CasR in systemic
calcium homeostasis is supported by the presence of inactivating
mutations of CasR in familial hypocalciuric hypercalcemia
and neonatal severe hyperparathyroidism (3), and
activating mutations resulting in hypoparathyroidism (4).
In addition, targeted ablation of CasR results in
hypercalcemia and elevated PTH levels in mice (5). CasR also has been isolated and cloned from kidney (6) and brain (7) cDNA libraries and is purported to be present in many other tissues, including the skeleton, where its physiological role remains to be identified (1). There is a growing body of evidence that extracellular calcium concentrations in the bone microenvironment could play a physiological role in modulating bone remodeling through activation of CasR or a functionally similar, but molecularly distinct, receptor in the skeleton (1, 8, 9, 10). In this model, Ca2+ released from the process of bone resorption is postulated to participate in feedback inhibition of osteoclast-mediated bone resorption and stimulation of osteoblast recruitment and osteoblast-mediated bone formation to replace the bone lost from the previous resorption cycle. A role for calcium sensing in the skeleton is largely supported by indirect evidence. For example, CasR has been variably reported to be expressed in osteoblasts (1, 11, 12, 13), bone marrow stromal cells (11), osteoclasts (12), and monocyte-macrophages (13). In addition, the presence of high ambient Ca2+ concentrations (8–40 mM) at sites of bone resorption is consistent with this hypothesis (14). Moreover, CasR has been reported to stimulate preosteoblastic and/or stromal cell proliferation (11, 15, 16) and to inhibit osteoclastogenesis (12). Others have detected CasR in hypertrophic chondrocytes by in situ hybridization and have demonstrated inhibition of functional responses of cultured chondrocytes to extracellular cations by transfecting antisense CasR constructs (17, 18). Thus, CasR or a related receptor may provide a means by which alterations in mineral homeostasis in the skeleton may directly regulate bone as well as cartilage function.
Definitive in vivo evidence, however, is lacking for a direct physiological role of calcium in the skeleton that is mediated by an extracellular calcium-sensing G protein-coupled receptor. Despite the fact that inactivating CasR mutations in neonatal severe hyperparathyroidism are associated with skeletal manifestations characterized by fractures, these changes have been attributed to hyperparathyroidism, which is indirectly caused by loss of CasR in parathyroid glands (3). Similarly, administration of CasR antagonist to rats causes an increase in circulating PTH levels and bone turnover, but no net change in bone mineral density or disruption in osteoblasts coupling to osteoclasts, as might be expected if CasR is playing a direct role in regulating bone remodeling (19). Targeted deletion of CasR in mice results in growth retardation (5), but to date no studies in these mice have characterized the abnormalities of bone and cartilage in sufficient detail to uncover potential direct consequences of CasR deficiency on the skeleton.
In an effort to identify a function role for calcium-sensing receptors in bone and cartilage in vivo, we evaluated the skeletal phenotype of CasR knockout mice. We observed the unexpected development of severe rickets in mice lacking the CasR rather than the anticipated PTH-mediated skeletal changes. Thus, analogous to the central function of the parathyroid CasR in controlling systemic calcium homeostasis, our studies support the concept that skeletal calcium-sensing receptors may provide a mechanism by which extracellular calcium in the bone microenvironment can directly regulate the mineralization process. Proof of this possibility requires additional studies to remove the confounding effects of excess PTH and hypercalcemia.
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Materials and Methods |
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Genotyping
We used a PCR approach to genotype the mice as previously
described (19), using the reverse primer CasR
2144.R (5'-TGAAGCACCTACGGCACCTG-3') specific for the native mouse
CasR gene sequence in combination with a primer designed to
upstream elements in exon 5, CasR 1956.F
(5'-TGATGAAGAGTCTTTCTCGG-3'), or a primer KCM-F
(5'-TCTTGATTCCCACTTTGTGGTTCTA-3') to the inserted neomycin gene
sequence used for targeted disruption of exon 5 (19).
RT-PCR analysis of CasR transcripts in mouse tissues
To detect CasR expression, RT-PCR was performed using
the Titan One Tube RT-PCR Kit (Roche, Indianapolis, IN).
Total RNAs were prepared from mouse kidney, cartilage, bone marrow, and
bone tissue using the TRIzol reagent (Life Technologies, Inc.) as described previously (20). Cartilage was
obtained by dissecting away the growth plate from metaphyseal bone in
4-d-old wild-type mice. To separate bone and bone marrow, the epiphyses
from adult wild-type bone were removed with a scalpel, and the bone
marrow was collected by repeated flushing with HBSS. The remaining bone
shaft was pulverized in liquid nitrogen before RNA extraction.
The RT reaction using 2.0 µg total RNA treated with deoxyribonuclease I (Stratagene, La Jolla, CA) was incubated at 45 C for 60 min, and the template was denatured at 94 C for 2 min. PCR was performed with thermal cycling parameters of 94 C for 30 s, 55 C for 30 s, and 68 C for 45 s for 10 cycles. This was followed by an additional 25 cycles with thermal cycling parameters of 94 C for 30 s, 55 C for 30 s, and 68 C for 45 s plus an additional 5 s with each cycle. The reaction was completed with a final extension at 68 C for 7 min. The mouse specific intron-spanning primer sets used to amplify the regions of the 5'-end of mouse CasR included the mouse-specific forward primer mCasR 640.For (5'-CAGCGAGCCCAAAAGAAAGG-3') and mCasR 1553.Rev (5'-CTTCAGACCGAACCCAATGG-3'). To amplify a region containing the 3'-end of CasR, we used the forward primer mCasR 2150.F (5'-GGAAGATCTTGTGGAGTGGG-3' and reverse primer mCasR 2945.R (5'-GCAAAGAAGAAGCAGATGGC-3'). In addition, using a similar protocol (19), the mouse G3PDH transcript was RT-PCR-amplified as a control using the forward primer G3PDH.F (5'-GACCCCTTCATTGACCTCAACTACA-3') and the reverse primer G3PDH.R (5'-GGTCTTACTCCTTGGAGGCCATGT-3'). The mouse osteocalcin transcript was RT-PCR-amplified using the forward primer mOG+8.F (5'-CAAGTCCCACACAGCAGCTT-3') and the reverse primer mOG+378.R (5'-AAAGCCGAGCTGCCAGAGTT-3'). Amplification products were resolved by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.
Serum biochemistries
Blood was obtained from 4- to 6-d-old mice. Urine was collected
from sodium pentobarbital-anesthetized 4- to 6-d-old mice by aspiration
from the bladder with a 27-gauge needle and tuberculin syringe. Calcium
was measured by the colorimetric cresolphthalein-binding method, and
phosphorus was measured by the phosphomolybdate-ascorbic acid method
(21). Creatinine was measured by the colorimetric alkaline
picrate method (Sigma kit 555, Sigma, St. Louis, MO). PTH
was measured using the rat PTH immunoradiometric assay from Immutopics,
Inc. (San Clemente, CA), which has been previously validated for mouse
PTH (22).
Dry and ash weights of femurs
Femurs collected from 5-d-old
CasR+/+ (n = 6),
CasR+/- (n = 8), and
CasR-/- (n = 6) mice were cleaned of muscle and dried
to constant weight at 110 C. The dried femurs were ashed overnight at
600 C and weighed. The results are expressed as a ratio of ashed to dry
weight. This value represents the total bone mineral content
(23).
Skeleton staining
Whole mouse carcasses were collected from
CasR+/+ (n = 6),
CasR+/- (n = 12), and
CasR-/- (n = 7) mice
after they were killed by sodium pentobarbital overdose, skinned,
eviscerated, and fixed for more than 3 d in 95% ethanol. They
were then defatted for 2–3 d in acetone and stained sequentially with
Alcian blue and Alizarin red S in 2% KOH (24). The
stained skeleton preparations were cleared with 1% KOH/20% glycerol
and stored in 50% ethanol/50% glycerol (25).
Quantitative histomorphometric analysis of nondecalcified
bone
Skeletons of CasR+/+,
CasR+/-, and
CasR-/- mice were prelabeled
with calcein (Sigma C-0875; 30 µg/g BW, sc injection) 1
and 3 d before collection of tibias. Femurs and tibias were
removed from 5-d-old CasR+/+,
CasR+/-, and
CasR-/- mice, fixed in 70%
ethanol, prestained in Villanueva stain (26), and
processed for methyl methacrylate embedding. We also collected bone
samples from 2-d-old mice that were not prelabeled. Five-micron
sections were stained with either Toluidine blue or Goldner’s stain
and analyzed under transmitted light, and 10-µm Villanueva-prestained
sections were evaluated under fluorescent light as previously reported
by our laboratory (27). The histomorphometric examination
was performed with an Osteomeasure digitizing system (OsteoMetrics,
Inc., Atlanta, GA) using a drawing tube attached to a microscope.
Growth plate measurements were performed at a magnification of x40,
whereas measurement of the bone secondary spongiosa was performed at a
magnification of x400. The terminology and units used are those
recommended by the histomorphometry nomenclature committee of the
American Society of Bone and Mineral Research (28). RZ.Th,
PZ.TH., and HZ.Th are the abbreviations used for the resting,
proliferative, and hypertrophic zones of the growth plate cartilage,
respectively. BV/TV represents the trabecular bone volume of the
secondary spongisa; OV/BV is the relative osteoid volume; OS/BS is the
percentage of bone surfaces covered with osteoid; O.Th. is the osteoid
seam width; Ob/Bs is the osteoblast perimeter; ES/BS is the resorptive
perimeter; N.Oc is the number of osteoclasts per bone perimeter; MAR
represents the adjusted mineral apposition rate; MLT is the
mineralization lag time, MS/OS represents the mineralizing osteoid; BFR
represents the surface referenced bone formation rate.
Skeletal radiography
Five-day-old mice were radiographed using a Hewlett-Packard
Faxitron 43807 (Palo Alto, CA) and X-Omat film (Eastman Kodak Co., Rochester, NY).
Statistics
We evaluated differences between groups by one-way ANOVA. All
values are expressed as the mean ± SEM. All
computations were performed using the Statgraphic statistical graphics
system (STSC, Inc., Rockville, MD).
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) and showed an obvious reduction in the
size of the skeleton (Fig. 1). Comparison
of skeletal radiographs of CasR+/+ and
CasR-/- littermates (Fig. 1, C vs. D) demonstrated a more radiolucent skeleton in
CasR-/- mice. In addition,
the epiphyseal cartilage of the wrist was wider, the mineralization of
the os calcis decreased, and the number of mineralized caudal vertebrae
was less than those in the wild-type
CasR+/+ littermates. The apparent decrease
in mineralization of the skeleton in the
CasR-/- mice was confirmed
by a significantly reduced bone ash (Table 1). In
CasR+/- mice the radiographic appearance
of the skeleton (data not shown), growth, and dry ashed weight of bone
(Table 1) were identical to those in
CasR+/+ mice.
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90-fold increase compared with wild-type
controls) accompanied by a modest, but significant, increase in serum
total Ca and a decreased serum inorganic P. The effects of the elevated
PTH are also evident on urinary excretion of calcium, which was
decreased in CasR-/-
compared with CasR+/+ mice. We were unable
to detect differences in serum PTH, calcium, or phosphorus or urinary
calcium excretion in CasR+/- compared with
CasR+/+ controls (Table 1
).
Endochondral bone formation
We examined the overall development and mineralization of the
skeleton in CasR+/+,
CasR+/-, and
CasR-/- mice by Alizarin red S and
Alcian blue staining of mineralized bone and cartilage (Fig. 1, A and
B, and Fig. 2). We observed no
differences between CasR+/- and
CasR+/+ mice (data not shown). In
contrast, we observed profound rachitic changes (Fig. 2, B, D, and F)
and delayed endochondral bone formation (Fig. 2, H and J) in
CasR-/- mice compared to
wild-type controls (Fig. 2, A, C, E, G, and I). These changes were
observed in 2- and 6-d-old mice. These consisted predominantly of a
widening of the metaphyseal regions of long bone, ribs, caudal
vertebrae, sternum, and other areas of growth plate cartilage.
Ossification of other areas of the skeleton as well as the skeleton’s
overall appearance were normal in
CasR-/- mice (Fig. 1B). In
addition, the craniofacial bone appeared normal in
CasR-/- mice (compare Fig. 1, B and A).
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, B and D, and F) compared with
CasR+/+ controls (Fig. 2, A and C and D).
Consistent with the skeletal radiographs (Fig. 1, C and D), the wrist
of CasR-/- demonstrated
widened growth plate compared with CasR+/+
(compare Fig. 2, B and A). Secondary ossification of the femoral
epiphysis was also delayed in 6-d-old
CasR-/- compared with
CasR+/+ mice (compare Fig. 2, D and C).
Similarly, secondary mineralization centers were observed in ventral
ribs in CasR+/+ mice by 6 d of age,
whereas no mineralization was observed in the ribs in
CasR-/- mice. The ventral
ribs also showed classic rachitic rosary in
CasR-/-, giving a beaded
appearance to the cartilage (Fig. 2F) compared with the normal
appearing rib cartilage of CasR+/+ mice
(Fig. 2E). The sternum was not mineralized in
CasR-/- mice (Fig. 1B).
Skeletal development is recapitulated in the postnatal period in the
mouse tail vertebra and is characterized by an orderly caudal
progression of endochondral bone formation that can be quantified in
Alizarin red- and Alcian blue-stained skeletal preparations. We
characterized the temporal aspects of endochondral bone formation in
tail vertebrae these mice. Representative Alizarin red- and Alcian
blue-stained cleared tail preparations from
CasR+/+ and
CasR-/- mice are shown in
Fig. 2, G–J. In CasR+/+ we observed
a time-dependent progressive sequence of mineralization of tail
vertebrae, such that an average of 24.3 ± 0.5 of the 29 caudal
vertebrae had mineralized by d 6 (Fig. 2, G and I). We observed no
significant delay in the mineralization of caudal vertebrae in
CasR+/- mice, which averaged 22.2 ±
1.3 mineralized vertebra (data not shown). In contrast, we found an
average of 11± 0.6 of 29 mineralized caudal vertebrae in 6-d-old
CasR-/- mice (Fig. 2, H and
J). In the few animals that survived to d 12, these changes were
partially reversed, as evidenced by foci of endochondral ossification
in the knees and progression of caudal vertebrae mineralization in
CasR-/- mice. Nevertheless,
the extent of caudal vertebrae mineralization remained significantly
less in 12-d-old CasR-/-
compared with CasR +/+ mice (24
± 0.25 vs. 28 ± 0.2; P = 0.001).
Histological analysis
The earliest time point that we evaluated was 2 d, although
detailed analysis was performed in 5-d-old mice to permit the
administration of fluorescent labels. The overall gross features of
neonatal bone in both
CasR-/- and
CasR+/+ mice included a distinct bone
cortex, a well developed marrow cavity with a primary and secondary
spongiosa, and an adjacent growth plate merging with a cartilaginous
epiphysis. The growth plate in
CasR-/- mice was widened
compared with that in CasR+/+ littermates
(compare Fig. 3, B and A), consistent
with the observed rachitic changes in skeletal radiographs and Alcian
blue-stained skeletal preparations. Growth plate enlargement was mainly
due to an increased height of the hypertrophic zone (Table 2). The width of the hypertrophic zone
was approximately twice as wide in
CasR-/- compared with
CasR+/+ mice (Table 2) due to a greater
number of chondrocytes in this zone rather than an increase in cell
size or extracellular matrix. Indeed, five to seven cell layers
constituted the hypertrophic zone in
CasR+/+ growth plates, whereas the zone of
hypertrophic chondrocytes extended to greater than 15 cell layers in
CasR-/- mice. The widths of
the proliferative and resting zones were not different between
CasR-/- and
CasR+/+ mice (Table 2). In addition,
examination of the zone of calcified cartilage and primary spongiosa
under fluorescent light in mice labeled with calcein (Fig. 4) demonstrated a normal pattern of
mineralization in CasR+/+ (Fig. 4C), but a
significant reduction in mineralized cartilage and bone in
CasR-/- mice (Fig. 4D). The
growth plate and calcified cartilage in
CasR+/- mice were indistinguishable from
those in CasR+/+ mice (data not shown).
The histological pattern of a widened zone of hypertrophic-like
chondrocytes in CasR-/- mice
is consistent with the widening of the growth plates observed by
Alizarin red S/Alcian blue-stained whole skeletons (Fig. 2).
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, C and D).
The undecalcified bone histology of the trabecular and cortical bone
from the tibial metaphysis of CasR+/+ and
CasR-/- mice also is shown
in Figs. 3 and 4. The most striking finding was the excessive amounts
of osteoid in CasR-/- (Fig. 3, D and H) compared with CasR+/+
littermates (Fig. 3, C and G). The relative osteoid volume was 7-fold
greater in CasR-/- compared
with CasR+/+ mice (Table 2). This
increase in osteoid volume in
CasR-/- mice was due both to
a 3-fold increased extent of osteoid covered bone surfaces, or OS/BS
(Table 2 and Fig. 3D), and to a 4-fold increase in the osteoid seam
width, or O.Th (Table 2 and Fig. 3H). Approximately half of the
trabecular bone surface was covered with osteoid in
CasR-/- mice (Fig. 3, F and
H, and Fig. 4B), whereas CasR+/+ mice
displayed a normal osteoid surface (e.g. 17%; Fig. 3, E
and G, and Fig. 4A). This hyperosteoidosis in
CasR-/- mice is consistent
with the reduction in dry ashed weight in these animals compared with
CasR+/+ mice (Table 1). This increase in
osteoid was the result of impaired mineralization, as evidenced by the
marked reduction in surfaces undergoing mineralization (Table 2 and
Fig. 4, D and F) in CasR-/-
mice. Compared with the normal percentage of mineralizing surfaces
(Fig. 4C) and number of double labeled surfaces (Fig. 4E) in
CasR+/+ mice, the osteoid surfaces of
CasR-/- mice either
exhibited no calcein label or a diffuse band of calcein at the
bone-osteoid interface (Fig. 4, D and F). These are classic
histological features of osteomalacia. Indeed, the mineralization lag
time exceeded 100 d in
CasR-/- mice, consistent with the
presence of osteomalacia (Table 2). As a consequence, the bone
formation rate was suppressed in
CasR-/- mice (Table 2) despite the
excess circulating PTH levels that typically stimulate mineralized bone
formation rates.
We observed an increase in the number of osteoblasts in
CasR-/- mice due to the
greater percentage of osteoid covered surfaces (Table 2). In addition
the morphology of the osteoblasts in
CasR-/- displayed a
tendency to be transformed from the normal cuboidal form to an
irregular polygonal morphology that is typically seen with PTH excess
(Fig. 3H). We also observed entrapment of individual
osteoblasts/osteocytes in the osteoid of
CasR-/- mice, consistent
with the presence of woven osteoid in the animals (Fig. 3H).
Interestingly, despite the elevated PTH in
CasR-/- mice, their bone
marrow had no peritrabecular fibrosis, a finding that is commonly
observed in PTH excess states. The resorptive surfaces and osteoclastic
number were also similar in the CasR+/+
and CasR-/- mice (Table 2
and Fig. 3, E and F), but the osteoclast morphology in
CasR-/- exhibited giant
osteoclasts with several nuclei and prominent nucleoli that are typical
in hyperparathyroid bone disease.
CasR expression in bone, cartilage, and bone marrow
We used two different primer sets to amplify the 5'-end and 3'-end
of the CasR cDNA sequence to evaluate CasR
expression in bone, bone marrow, and cartilage. As a positive control
we amplified CasR from RNA derived from mouse kidney. The
RT-PCR results were identical with the two primer sets. The results
from the primer set that amplifies the 5'-end of CasR are
shown in Fig. 5. We successfully
amplified the predicted 914-bp product in kidney, bone marrow, and
cartilage (Fig. 5A). In contrast, we failed to amplify CasR
in bone (Fig. 5A). To assure that the bone sample contained
osteoblasts, we tested the bone-derived RNA for osteocalcin expression.
Using osteocalcin-specific primers, we were able to amplify the
predicted size product from bone (Fig. 5B). Osteocalcin also was
present in bone marrow, but to a lesser extent (Fig. 5B). As a control
for RNA integrity, we successfully amplified G3PDH transcripts from all
tissues (Fig. 5, A and B).
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The etiology of the rachitic changes is not clear. The early appearance of rachitic changes in 2-d-old CasR-/- mice is too soon for nutritional and/or vitamin D deficiencies to manifest. Although CasR-/- mice have severe hyperparathyroidism accompanied by hypercalcemia, hypophosphatemia, few of the phenotypic changes of the skeleton in CasR-/- resemble the anticipated skeletal effects of PTH excess. For example, CasR-/- mice appear to have a more selective enlargement of hypertrophic zone of the growth plate, whereas transgenic mice models of PTH/PTHrP overactivity typically have increases in both proliferative and hypertrophic chondrocytes (29, 30, 31, 32, 33, 34). Also, neonatal transgenic mice expressing constitutively active PTH/PTHrP receptors exhibit a profound inhibition of endochondral bone formation, leading to the absence of a normal marrow space and defined metaphyseal bone (31), whereas CasR-/- mice have a grossly normal skeletal architecture with a clearly demarcated primary and secondary spongiosa and a normal appearing bone marrow cavity. More importantly, hyperparathyroidism, in the absence of hypocalcemia and vitamin D deficiency, does not typically cause rickets or osteomalacia in metaphyseal bone (35, 36). Rather, PTH typically stimulates osteoblast and osteoclast activity, leading to accelerated mineralized bone formation rates, bone resorption, and bone marrow fibrosis (37). The severity of the rachitic changes and the absence of the typical skeletal changes induced by PTH excess support a more complex pathophysiology of these skeletal alterations in CasR-/- mice that may involve a skeletal calcium-sensing receptor-like mechanism.
A direct role for calcium in mediating the mineralization process in not a novel concept. Recent observations in VDR null mice demonstrate a direct role of calcium in the pathogenesis of rickets, as evidenced by the ability of calcium to correct the mineralization abnormalities in the absence of a functional VDR (35, 36). Except for the growth retardation, the bone and growth plate abnormalities in CasR-/- mice resemble the calcium-responsive lesion in hypocalcemic VDR null mice (35, 36). As extracellular fluid supersaturates the calcification of crystal hydroxyapatite, the selective mineralization of bone and cartilage must be regulated by additional organic factors that modulate the mineralization process (38). Our studies raise the interesting possibility the extracellular calcium-sensing receptor in cartilage and bone marrow may regulate this process. The possibility that a calcium-sensing receptor expressed in the skeleton has a direct role in regulating skeletal mineralization will require further proof. It would be interesting in future studies to evaluate the effects of extracellular calcium to regulate the osteoblast and/or chondrocyte production of mineralization modulators or to regulate matrix vesicle function.
On the other hand, our findings are inconsistent with some of the prevailing ideas regarding the possible role of the skeletal calcium receptor as predicted by in vitro studies. For example, in cell culture models extracellular calcium inhibits osteoclastic activity and stimulates osteoblastic proliferation and chemotaxis. Consequently, the absence of CasR in osteoclasts is predicted to stimulate osteoclastogenesis, whereas the lack of CasR in preosteoblasts or marrow stromal cells should inhibit osteogenesis (1, 15, 16, 39). In the current studies we failed to find support for CasR regulation of osteoclast activity and osteoblast recruitment, but found multinucleated osteoclasts and increased osteoblast-covered bone-forming surfaces in CasR-/- mice.
Despite our unexpected and compelling findings, we cannot establish a direct role for CasR in the skeleton based on our current investigations. Our results are confounded by the severe hyperparathyroidism and the accompanying hypercalcemia and hypophosphatemia. Also, because of the early mortality of CasR-/- mice, our findings are limited to the neonatal skeleton. Extrapolating a role for CasR to the adult skeleton awaits additional studies in CasR-/- mice lacking excess PTH, which may allow them to survive to adulthood. In addition, implicating a direct role for CasR in the skeleton is potentially complicated by redundant calcium-sensing mechanisms in bone. There is an ongoing controversy as to whether all of the functional responses to cations in osteoblasts and osteoclasts are mediated solely by CasR or whether another CasR-like receptor is present (10, 40, 41, 42) that differs from CasR with regard to cation specificity, response to calcimimetics, and signaling pathways. If so, the seemingly paradoxical idea that some of the osseous changes in CasR-/- mice might be mediated by another calcium sensing receptor has validity. In this regard, aluminum, which stimulates the putative novel osteoblast-sensing receptor, but not CasR (43), causes osteomalacia in both humans and animal models (27, 44). In addition, in the rare CasR-/- mice that survived for 12 d, we observed partial reversal of the mineralization abnormalities in whole skeletal preparations. As these mice were not exposed to different environmental conditions, their longer survival may represent selection of an unspecified genetic trait imparting resistant to the loss of CasR. Consequently, the reversal of their skeletal abnormalities may be related to the osseous effects of these undefined compensatory events and suggest the skeletal abnormalities may be modulated by factors other than CasR itself.
In conclusion, we have observed abnormalities of mineralization of extracellular matrix in CasR-/- mice that are not completely explained by excess PTH. We speculate that the ablation of CasR in cartilage and bone marrow, the presence of a novel skeletal CasR, or some unanticipated effects of PTH excess in the absence of CasR may be responsible for the observed mineralization abnormalities in bone and cartilage. The major importance of our current findings is to direct research toward identifying the function of calcium-sensing receptors in the skeleton. Before any direct consequences of skeletal calcium receptors can be confirmed, additional studies are needed to remove the confounding skeletal effects of PTH in CasR-/- mice and to identify the molecular mechanisms and physiological role of the persistence of calcium sensing in CasR-/- osteoblasts.
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Acknowledgments |
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Footnotes |
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Abbreviations: CasR, Cation-sensing receptor; G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
Received February 2, 2001.
Accepted for publication May 14, 2001.
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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]
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 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]
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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]
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 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]
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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]
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