the calcium-sensing receptor and 25-hydroxyvitamin d–1α-hydroxylase interact to modulate skeletal...

ORIGINAL ARTICLE JJBMR
The Calcium-Sensing Receptor and 25-HydroxyvitaminD–1a-Hydroxylase Interact to Modulate Skeletal Growthand Bone Turnover
Christian Richard,1 Rujuan Huo,1 Rana Samadfam,1 Isabel Bolivar,1 Dengshun Miao,2 Edward M Brown,3

Geoffrey N Hendy ,1 and David Goltzman1

1Calcium Research Laboratory, Department of Medicine, McGill University, Montreal, Quebec, Canada2Department of Human Anatomy, Nanjing Medical University, Nanjing, People’s Republic of China3Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital,Harvard Medical School, Boston, MA, USA

ABSTRACTWe examined parathyroid and skeletal function in 3-month-old mice expressing the null mutation for 25-hydroxyvitamin

D–1a-hydroxylase [1a(OH)ase�/�] and in mice expressing the null mutation for both the 1a(OH)ase and the calcium-sensing receptor

[Casr�/�1a(OH)ase�/�] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH),

increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous

growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the

Casr�/�1a(OH)ase�/� mice. On a high-lactose, high-calcium, high-phosphorus ‘‘rescue’’ diet, serum calcium and PTH were normal in the

1a(OH)ase�/� mice but increased in the Casr�/�1a(OH)ase�/� mice with reduced serum phosphorus. Growth plate architecture and

mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone

improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr�/�1a(OH)ase�/� mice. These

studies support a role for calcium-stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and

bone and calcium-stimulated CaSR-mediated effects on bone resorption. PTH-mediated bone resorptionmay require calcium-stimulated

CaSR-mediated enhancement of osteoclastic activity. � 2010 American Society for Bone and Mineral Research.

KEY WORDS: CALCIUM-SENSING RECEPTOR; VITAMIN D; PARATHYROID HORMONE ACTION; OSTEOMALACIA; BONE RESORPTION

Introduction

Extracellular calcium ([Ca2þ]e) and the activated form of

vitamin D [1,25(OH)2D] play important roles in modulating

systemic calcium homeostasis. [Ca2þ]e activates a cation-sensing

G protein–coupled receptor (CaSR) to modulate the concentra-

tions of circulating parathyroid hormone (PTH) and to regulate

renal calcium reabsorption.(1) Gain-of-function mutations in the

CASR gene in humans cause autosomal dominant hypocalcemia,

a condition of mild to moderate hypocalcemia associated with

suppression of PTH secretion that can be accompanied by

hypercalciuria.(2) Loss-of-function mutations in the CASR gene

cause familial benign hypocalciuric hypercalcemia in hetero-

zygotes and neonatal severe hyperparathyroidism in homo-

zygotes, both associated with increased PTH secretion and

diminished renal calcium excretion but of differing severities.(3)

Received in original form March 11, 2009; revised form January 18, 2010; accepted

Address correspondence to: David Goltzman, MD, Calcium Research Laboratory, De

West, Montreal, Quebec, Canada H3A1A1. E-mail: [email protected]

Journal of Bone and Mineral Research, Vol. 25, No. 7, July 2010, pp 1627–1636

DOI: 10.1002/jbmr.58

� 2010 American Society for Bone and Mineral Research

Targeted deletion of the Casr gene in mice produced a

phenocopy of the human condition resulting from inactivating

CASR mutations.(4)

1,25(OH)2D is synthesized via the action of the enzyme 25-

hydroxyvitamin D–1a-hydroxylase [1(OH)ase or cyp27B1] to

convert 25(OH)D to 1,25(OH)2D(5) and acts predominantly on a

nuclear vitamin D receptor (VDR).(6) Loss-of-function mutations

in 1(OH)ase in humans produce pseudodeficiency rickets or

vitamin D–deficiency rickets type 1,(7) and loss-of-function

mutations in the VDR gene in humans produce vitamin D–

deficiency rickets type 2.(8) Both disorders manifest rickets and

osteomalacia as a predominant phenotype. Targeted inactiva-

tion of the 1(OH)ase gene in mice reproduces the human

syndrome of vitamin D–deficiency rickets type 1 with hypo-

calcemia, secondary hyperparathyroidism, rickets, and osteo-

malacia.(9) Consequently, the absence of 1,25(OH)2D may have

February 2, 2010. Published online February 8, 2010.

partment of Medicine, Royal Victoria Hospital, Room H4.67, 687 Pine Avenue

1627

profound effects on extracellular calcium ([Ca2þ]e) levels and PTH

secretion, as does [Ca2þ]e per se, but in addition can markedly

alter skeletal homeostasis. More recently, the CaSR has been

reported to function in vitro in a variety of skeletal cells,

including osteoblasts, bone marrow stromal cells, monocyte-

macrophages, osteoclasts, and chondrocytes.(10) By in situ

hybridization, Casr transcripts have been found mainly in

hypertrophic chondrocytes of the epiphyseal growth plate, in

osteoblasts, in osteocytes, and in bone marrow cells but rarely

in mature osteoclasts. Recent in vivo studies have shown that

transgenic mice with a constitutively active mutant CaSR

targeted to mature osteoblasts demonstrated enhanced bone

resorption,(11) whereas mice with osteoblast-specific deletion

exhibited severely undermineralized skeletons.(12) Mice with

chondrocyte-specific deletion of Casr displayed delayed growth

plate development.(12)

The early lethality in the neonatal period of mice with

homozygous Casr deletion (Casr�/�)(4) precludes assessment of

the progression of skeletal abnormalities in this model. This early

lethality is, however, corrected by crossing these mice with mice

that manifest hypoparathyroidism, suggesting that the early

lethality could be corrected by eliminating the hypercalcemia

and hyperparathyroidism.(13,14) We wished to test the hypothesis

that CaSR has an important role to play in skeletal function in

older growing animals and therefore crossed the Casr�/� animals

with 1a(OH)ase�/�mice to ascertain the postnatal consequences

of the absence of the CaSR on skeletal homeostasis and to

examine the interaction of [Ca2þ]e and 1,25(OH)2D in the double

mutants on modulating skeletal function. We exposed the

different genetic models to two different environments: a normal

diet, on which 1a(OH)ase�/�mice are known to be hypocalcemic

and hypophosphatemic, and a high-calcium, high-phosphorus

‘‘rescue’’ diet(15) containing 20% lactose, which is known to

normalize serum calcium and phosphate in the absence of either

active vitamin D or the vitamin D receptor.

Materials and Methods

Derivation of Casr and 1(OH)ase double-null mice

The derivation of the two parental strains of Casr�/� mice and

1a(OH)ase�/� mice by homologous recombination in embryonic

stem cells was described previously by Ho and colleagues(4) and

Panda and colleagues,(9) respectively. Briefly, a neomycin

resistance gene was inserted into exon 5 of the mouse Casr

gene. Western blot analysis of kidney membrane protein

extract from homozygous Casr�/� mice confirmed that no

detectable protein is expressed from this allele.(4) A neomycin

resistance gene replaced exons VI, VII, and VIII of the mouse

1a(OH)ase gene (Cyp27b1), removing both the ligand- and heme-

binding domains. Lack of 1a(OH)ase mRNA expression in kidney

and of circulating concentrations of 1,25(OH)2D has been

demonstrated previously.(9) Mice heterozygous for the null Casr

allele were described previously as being fertile,(4) as were mice

heterozygous for the null 1a(OH)ase allele.(9) Offspring hetero-

zygous at both loci thenweremated with one another in order to

generate pups homozygous for both the Casr and 1a(OH)ase null

alleles [CasR�/�1a(OH)ase�/�]. These mice were maintained on a

1628 Journal of Bone and Mineral Research

mixed genetic background with contributions from BALB/c and

129/SvJ strains, and wild-type littermates were used as controls.

In vivo experiments

Animal protocols were approved by the Institutional Animal Care

and Use Committee at McGill University and were in accordance

with the Canadian Council on Animal Care. Mutant mice and

control littermates were maintained in a virus- and parasite-free

barrier facility and exposed to a 12-/12-hour light/dark cycle. At

approximately 21 days of age, mice were weaned and

maintained on drinking water containing 1.5% calcium gluco-

nate and either a normal diet of autoclaved chow containing 1%

calcium, 0.85% phosphorus, 0% lactose, and 2.2 U/g of vitamin D

(Ralston Purina Co., St. Louis, MO, USA) or a ‘‘rescue’’ diet(15) of

gamma-irradiated chow containing 20% lactose, 2% calcium,

1.25% phosphorus, and 2.2 U/g of vitamin D (TD96348, Harlan

Teklad, Madison, WI, USA). No significant differences in any

parameter determined were observed in wild-type mice on a

normal or a rescue diet.(15) Consequently, control data are shown

for the wild-type mice on the normal diet. Animals were

euthanized at about 3 months of age.

Genotyping of mice

Genomic DNA was isolated from tail fragments by standard

phenol-chloroform extraction and isopropanol precipitation. To

determine the genotypes at both the 1a(OH)ase and Casr loci,

two PCR amplification reactions were required, one for Casr and

one for the 1a(OH)ase locus, respectively. To assay for the

presence of the wild-type Casr allele, samples were amplified

with CaSR forward primer CaR6h50 (50-TCTGTTCTCTTTAGGTCCT-

GAAACA-30) and CaSR reverse primer CaR6h30 (50-TCATTGAT-

GAACAGTCTTTCTCCCT-30). To detect the presence of the null

Casr allele, Neo forward primer r-Neo-2 (50-TCTTGATTCC-

CACTTTGTGGTTCTA-30) was used with the CaSR reverse primer

CaR6h30. PCR conditions were performed using Hot Start Taq

polymerase (Qiagen, Valencia, CA, USA) with 35 cycles of 958C for

17 minutes, 948C for 30 seconds, 558C for 30 seconds, and 728Cfor 45 seconds and then a 7-minute final extension at 728C. Thewild-type and mutant 1(OH)ase alleles were detected using a

multiplex PCR with 1(OH)ase forward primer 1aOHf (50 -

AGACTGCACTCCACTCTGAG- 30) and reverse primer 1aOHr (50-

GTTTCCTACACGGATGTCTC-30) and forward NeoF (50-ACAACA-

GACAATCGGCTGCTC-30) and reverse primer NeoR (50-

CCATGGGTCACGACGAGATC-30) to amplify the inserted neomy-

cin resistance gene. The PCR reaction for 1(OH)ase was 30 cycles

of 948C for 1 minutes, 588C for 60 seconds, and a final elongation

step at 728C for 10 seconds.

Skeletal radiography

Femurs were removed, dissected free of soft tissue, and fixed in

70% ethanol. Contact radiographs were taken using a Faxitron

Model 805 radiographic inspection system (Faxitron Contact,

Faxitron, Germany) with 22 kV and 4-minute exposure time.

X-Omat TL film (Eastman Kodak, Rochester, NY, USA) was used

and processed routinely.

RICHARD ET AL.

Bone mineral density (BMD) analysis

Densitometry was performed by PIXImus densitometer (Soft-

ware Version 1.46.007, Lunar Corp, Madison, WI, USA) on the

right femur, as described previously.(16) Percent coefficient of

variance (CV%) of BMD for repeated scans was 1% to 3%.

Histology

Femurs, tibias, and thyroparathyroidal tissue were removed and

fixed in PLP fixative (2% paraformaldehyde containing 0.075 M

lysine and 0.01 M sodium periodate) overnight at 48C and

processed histologically as described previously.(17) Distal femurs

and proximal tibias were decalcified in EDTA glycerol solution for

14 days at 48C. Decalcified bones were dehydrated and

embedded in paraffin, after which 5-mm sections were cut on

a rotary microtome. The sections were stained with hematoxylin

and eosin (H&E) or histochemically for tartrate-resistant acid

phosphatase (TRACP),(17) alkaline phosphatase (ALP) activity, and

total collagen, as described below. Alternatively, undecalcified

bones were embedded in methyl methacrylate (MMA), and

1-mm sections were cut on an ultramicrotome. These sections

were stained for mineral with the von Kossa staining procedure

and counterstained with toluidine blue or were stained with the

Goldner trichrome method.

Histochemical staining for collagen, ALP, and TRACP

Total collagen was detected in paraffin sections as described

previously.(18) ALP activity was determined at 378C on

deplasticized, hydrated plastic sections using an ALP substrate

staining kit (SK-5100, Vector Labs, Burlingame, CA, USA). Enzyme

histochemistry for TRACP was performed as described pre-

viously.(17)

Immunohistochemical staining

Decalcified paraffin sections were treated with goat anti-human

type 1 collagen antibody (Southern Biotechnology Associates,

Birmingham, AL, USA). As a negative control, pre–immune serum

was substituted for the primary antibody. Sections of decalcified

bone and of kidney and parathyroid glands were immunostained

with the avidin-biotin-peroxidase complex (ABC) technique as

described previously.(17) Sections were counterstained with

methyl green and mounted with Permount (Fisher Scientific,

Montreal, Canada).

Double calcein labeling

Double calcein labeling was performed by i.p. injection of mice

with 10mg of calcein per gram of body weight (C-0875, Sigma, St.

Louis, MO, USA) at 10 and 3 days before the mice were

euthanized. Bones were harvested and embedded inMMA. Serial

sections then were deplasticized, washed successively in ethanol

and xylene, and mounted with Permount (Fisher Scientific) for

subsequent fluorescence microscopy. The double-calcein-

labeled width of cortex and trabeculae was measured using

Bioquant image analysis software (Version 6, Nashville, TN, USA),

and the mineral apposition rate (MAR) was calculated as the

interlabel width/labeling period.

THE CALCIUM-SENSING RECEPTOR, VITAMIN D, AND BONE

Histomorphometry

Histomorphometric indices were determined as suggested by

the ASBMR Histomorphometry Nomenclature Committee.(19)

Measurements were performed in the secondary spongiosa in

the metaphyseal area (0.5 mm below the growth plate) at the

distal end of the femur. The parameter measured for bone

volume was the total bone volume per tissue volume (BV/TV, %).

The parameters obtained for bone formation were the osteoblast

surface per bone surface (Ob.S/BS, %) and the MAR (mm/day).

The parameter measured for bone resorption was the osteoclast

surface per bone surface (Oc.S/BS, %). After H&E or histochemical

staining of sections from replicate mice of each group, images of

fields were photographed with a digital camera. Images from

sections were processed and analyzed using Bioquant image

analysis software.

Biochemical and hormonal analyses

Serum and urine calcium and serum phosphate and creatinine

were determined by autoanalyzer (Beckman Synchron 67,

Beckman Instruments, Mississauga, Ontario, Canada). Serum

samples were obtained at the time of sacrifice, and urine samples

were obtained by bladder aspirate just prior to sacrifice. Serum

intact PTHwasmeasured by a two-site immunoradiometric assay

(Immutopics, San Clemente, CA, USA). A mouse osteocalcin two-

site immunoradiometric assay (IRMA; Immutopics, Inc.) was used

for measurement of serum osteocalcin levels according to the

manufacturer’s specifications. A mouse TRACP 5b assay (IDS, Inc.,

Fountain Hills, AZ, USA) was used according to the manufac-

turer’s specifications for determination of osteoclast-derived

TRAP 5b.

Statistical analysis

Statistical comparisons employing Graph-Pad Prism Version 4.00

analysis software (GraphPad Software, Inc., San Diego, CA, USA)

were made using Student’s t test or ANOVA, followed by a

Bonferroni adjustment or a Neuman-Keuls multiple-comparison

test. p< .05 was considered significant.

Results

Serum biochemistry and parathyroid gland size

On a normal diet, at 3 months of age, both 1a(OH)ase�/� and

Casr�/�1a(OH)ase�/� mice were hypocalcemic (Fig. 1), with

enlarged parathyroid glands and elevated circulating serum PTH

concentrations and hypophosphatemia. On the rescue diet,

serum calcium, phosphate, and PTH concentrations were normal

in the 1a(OH)ase�/� mice, but in the double mutants, despite a

mean elevation in serum calcium, serum PTH and parathyroid

gland size remained increased, and animals displayed marked

hypophosphatemia (Fig. 1). Urine calcium levels were reduced in

the Casr�/�1a(OH)ase�/� mice both on a normal and a rescue

diet (Fig. 1). Serum creatinine levels were not significantly

different in thewild-type, 1a(OH)ase�/�, and Casr�/�1a(OH)ase�/�

mice both on a normal diet and a rescue diet. Thus, on a rescue

diet, serum creatinine levels (normal range 18 to 71mM)

were 32.3� 3.5, 38.5� 6.2, and 45.4� 7.1 mM (mean� SE of five

Journal of Bone and Mineral Research 1629

Fig. 1. Serum calcium (A) and urine calcium (B) concentrations, parathyroid gland histology (C), parathyroid gland histomorphometry (D), and serum PTH

(E) and phosphate concentrations ( F) in 1a(OH)ase�/� (D�/�), Casr�/�1a(OH)ase�/� (DKO), and wild-type (WT) mice on a normal diet or a rescue diet. Each

biochemistry value is themean� SEM of 3 to 10 replicates, and each parathyroid histomorphometry value is themean� SEM of 4 to 10 replicates. �p< .05

compared with WT mice. ��p< .01 compared with WT mice. ��p< .001 compared with WT mice. þp< .05 compared with 1a(OH)ase�/� mice on the

corresponding diet. þþp< .01 compared with 1–(OH)ase�/� mice on the corresponding diet. þþþp< .001 compared with 1a(OH)ase�/� mice on the

corresponding diet. In the representative photomicrographs, arrows denote parathyroid glands. Magnification �100.

determinations) inwild-type, 1a(OH)ase�/�, and Casr�/�1a(OH)ase�/�

mice, respectively.

Effects on growth and the growth plate

At birth, the wild-type, 1a(OH)ase�/�, and Casr�/�1a(OH)ase�/�

mice each had a mean weight of 1.5 g. After weaning, on a

normal diet, 1a(OH)ase�/�mice grewmore slowly than wild-type

mice (Fig. 2A, B), and femur length (Fig. 2C) was reduced; the

growth of Casr�/�1a(OH)ase�/� animals was severely stunted,

and the femur length was even more markedly reduced. On a

rescue diet, the growth of 1a(OH)ase�/�mice (Fig. 2B) and femur

length (Fig. 2C) were increased, but they were still below those of

wild-type animals. Although Casr�/�1a(OH)ase�/� mice

improved their growth markedly on the rescue diet, they

continued to display retarded growth compared with wild-type

mice (Fig. 2B), and femur length was below that of the

1a(OH)ase�/� mice (Fig. 2C).

Examination of the cartilaginous growth plates (Fig. 2D)

revealed an enlarged and distorted growth plate in the

1a(OH)ase�/� mice on the normal diet with little mineralization

compared with wild-type mice. The double mutants showed

similarly widened growth plates with even less evidence of


mineralization. On a rescue diet, the organization of the growth

plate and mineralization were markedly improved in the

1a(OH)ase�/� mice, but the growth plates remained wider than

in wild-type mice. In the Casr�/�1a(OH)ase�/� animals on the

rescue diet, although mineralization was improved, the growth

plate remained enlarged, and the cellular architecture, particu-

larly in the hypertrophic zone, was disorganized (Fig. 2D).

Mineralization of bone

Both von Kossa and trichrome staining (Fig. 3A, B) demonstrated

increased unmineralized bone in both mutant models on the

normal diet compared with wild-type mice, and quantitation of

osteoid volume revealed a marked increase (Fig. 3C). In addition,

on the normal diet, double fluorochrome labels were not

observed in bone after calcein administration in either mutant

model, so the MAR could not be calculated (Fig. 3D). On the

rescue diet, osteoid volume was markedly reduced in both

models and was not significantly different from that of wild-type

mice (Fig. 3A, B). In addition, the MAR of 1a(OH)ase�/� mice was

not significantly different from that of wild-type mice, and the

MAR of Casr�/�1a(OH)ase�/� mice also was markedly improved

(Fig. 3D).

RICHARD ET AL.

Fig. 2. Photomicrographs of 60-day-old WT, 1a(OH)ase�/� (D�/�), and Casr�/�1a(OH)ase�/� (DKO) mice on a normal diet (A). Growth curves of WT mice

and 1a(OH)ase�/� (D�/�) mice on a normal diet and on a rescue diet and Casr�/�1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue diet (B).

Contact radiographs of femurs of 120-day-old WT mice and 1a(OH)ase�/� (D�/�) and Casr�/�1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue

diet. Vertical white lines depict the lengths of the femurs to the left of the lines (C). Histology of growth plates of WT, 1a(OH)ase�/� (D�/�), and Casr�/�

1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue diet (D). In each case, photomicrographs of the growth plate are shown stained by trichrome

stain (left) and by von Kossa stain for mineralization (right). Magnification�200. White vertical arrows denote the width of the cartilaginous growth plate.

Black staining in von Kossa–stained sections denotes mineral. Arrows depict region below the hypertrophic zone of the growth plate wheremineralization

normally occurs.

Bone matrix deposition, bone volume, and BMD

Collagen staining revealed dramatically increased bone matrix

deposition in both 1a(OH)ase�/� mice and Casr�/�1a(OH)ase�/�

mice while on a normal diet (Fig. 4A). This was substantiated

when immunohistochemistry for type 1 collagen was performed

on bone (Fig. 4B). Therefore, when trabecular bone volume was

determined in the secondary spongiosa of collagen-stained

bones, BV/TV was increased in bothmutants on the high-calcium

diet and especially in the Casr�/�1a(OH)ase�/� mice (Fig. 4C).

Despite the increased BV/TV, however, BMD was decreased in

both mutants and notably in the Casr�/�1a(OH)ase�/� mice. On

a rescue diet, bone matrix deposition was considerably reduced

in 1a(OH)ase�/� mice but remained markedly increased in the

Casr�/�1a(OH)ase�/� mice (Fig. 4A, B). BV/TV fell markedly in the

1a(OH)ase�/� mice on the rescue diet (Fig. 4C) along with BMD

(Fig. 4D). However, the BV/TV remained dramatically increased in

the double mutants on the rescue diet (Fig. 4C) but with normal

BMD (Fig. 4D).

Effects on bone turnover

The ALP-positive area lining the bone perimeter was determined

to assess osteoblasts. Osteoblasts were increased significantly in

both mutants on the normal diet but fell markedly in

1a(OH)ase�/� mice on the rescue diet (Fig. 5A). Osteoblasts


remained substantially increased in Casr�/�1a(OH)ase�/� mice

on the rescue diet (Fig. 5A). Serum osteocalcin levels generally

paralleled the changes in osteoblasts observed by bone

histomorphometry (Fig. 5B).

Osteoclast numbers were significantly reduced in the Casr�/�

1a(OH)ase�/� animals on the normal diet and remained reduced

in the double-mutant mice on the rescue diet (Fig. 6A). Serum

TRACP 5b levels, reflecting osteoclast activity, generally

paralleled the changes seen in osteoclast numbers (Fig. 6B).

Discussion

We exposed animals deficient in the active form of vitamin D and

those with, in addition, targeted disruption of the Casr gene to

two environmental manipulations—exposure to a normal diet

and exposure to a rescue diet. On the normal diet, the

1a(OH)ase�/� mice remained hypocalcemic, with severe sec-

ondary hyperparathyroidism, consistent with our previous

findings.(15) After deletion of the full-length CaSR in the double

mutants, serum calcium remained very low in the absence of

active vitamin D, both parathyroid gland size and circulating PTH

concentrations were even greater than in the 1a(OH)ase�/�

mice, and hypophosphatemia was present. On the rescue diet,

mean serum calcium levels were normalized in 1a(OH)ase�/�


Fig. 3. Photomicrographs of von Kossa (A) and trichrome (B) stains of the metaphyses of femurs fromWTmice and from 1a(OH)ase�/� (D�/�) and Casr�/�

1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue diet. The black deposits in sections stained with von Kossa indicate mineralized bone, and light

blue stain abutting trabeculae denotes unmineralized osteoid (yellow arrows). In trichrome stains, green staining denotes mineralized trabeculae, and red

staining denotes unmineralized osteoid (also indicated by yellow arrows). Magnification �100 and �200 for von Kossa– and trichrome-stained sections,

respectively. Quantitation of osteoid volume (OV) relative to bone volume (BV) is plotted in panel C for WT mice and for 1a(OH)ase�/� (D�/�) and Casr�/�

1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue diet. Each value is the mean� SEM of 3 to 6 determinations. ��p< .01 relative to values in WT

mice. Quantitation of MAR in WT mice and in 1a(OH)ase�/� (D�/�) and Casr�/�1a(OH)ase�/� (DKO) mice on a rescue diet. Inadequate calcein labeling of

bone in 1a(OH)ase�/� and Casr�/�1a(OH)ase�/� mice on a normal diet precluded calculation of MAR for these models. Each value is the mean� SEM of 5

to 9 determinations.

mice, as described previously,(15) but rose above normal in the

double mutants, likely because of impaired renal calcium

excretion owing to the absence of the renal action of CaSR in

the setting of increased gastrointestinal absorption of calcium

because of the rescue diet. The lower urine calcium excretion in

Casr�/�1a(OH)ase�/� mice relative to wild-type mice and to

1a(OH)ase�/� mice on the rescue diet therefore confirms the

reduced CaSR activity in the Casr�/� mice we employed.

Although circulating PTH concentrations normalized in the

normocalcemic 1a(OH)ase�/� mice, PTH concentrations

remained markedly increased in the mice deficient in CaSR

despite the hypercalcemia, confirming the deficient transduction

of the [Ca2þ]e signal in the parathyroid gland in the absence of

the CaSR.

Previous studies have implicated the CaSR in modulating

skeletal function. Initial studies demonstrated abnormal miner-

alization of cartilage and bone associated with Casr deficiency,(20)

but correction of the severe hyperparathyroidism in Casr�/�

mice resulted in healing of the rickets and osteomalacia,(13)

suggesting that the demineralization observed was due to the

effect of severe hyperparathyroidism in the neonate. [Ca2þ]e and

the CaSR, however, have been reported to modulate growth

plate chondrocyte differentiation in vitro,(21–23) and targeted


deletion of the CaSR from chondrocytes has been reported to be

lethal in utero before embryonic day 13 but to produce viable

mice with delayed growth plate development if conditional

targeted deletion in these cells is induced between E16 and

E18.(12) In our studies, mutant mice deficient in both the CaSR

and the 1a(OH)ase enzyme grew extremely poorly on the normal

calcium intake and were significantly smaller than those

deficient in only vitamin D. This reduction in growth was

associated with major abnormalities of the growth plate,

including widening and disorganization of the cellular progres-

sion from proliferative to hypertrophic zones observed in wild-

type mice and with less mineral deposition at the chondrooss-

eous junction than was seen even in the single-mutant

1a(OH)ase�/� mice. The reduced mineralization may have been

secondary at least in part to the more severe hypophosphatemia

observed in the double mutants,(24) who had more severe

hyperparathyroidism.

In 1a(OH)ase�/� mutants on a rescue diet, mineralization of

the growth plate was improved, and growth increased, but the

growth plates remained wider than in wild-type mice, and the

rate of growth remained consistently below that of wild-type

mice. Consequently, factors other than the ambient levels of

calcium and phosphorus, both of which were normalized, must

RICHARD ET AL.

Fig. 4. Photomicrographs of collagen stain of proximal ends of tibias (A) and of type 1 collagen immunohistochemical stain of tibial metaphyses (B) from

WTmice and from 1a(OH)ase�/� (D�/�) and Casr�/�1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue diet. Magnification�25 in A and�200 in B.

Trabecular bone volume (BV/TV) (C) and bone mineral density (BMD) of femurs (D) from 1a(OH)ase�/� (D�/�) and Casr�/�1a(OH)ase�/� (DKO) mice on a

normal diet and on a rescue diet. Each value is the mean� SEM of 3 to 13 determinations. �p< .05 relative to WT mice. ��p< .001 relative to WT mice.þp< .05 relative to 1a(OH)ase�/� mice on the corresponding diet. þþþp< .001 relative to 1a(OH)ase�/� mice on the corresponding diet.

have contributed to the persistent growth plate abnormality,

notably the vitamin D deficiency per se. In Casr�/�1a(OH)ase�/�

animals on the rescue diet, mineralization of the growth plate

and long bone growth both improved on the rescue diet in

association with the change from hypo- to hypercalcemia.

Recent studies of Casr�/� mice revealed an alternatively spliced

product lacking exon 5. This was apparently generated from the

construct designed to disrupt Casr expression and which was

the construct used to generate the Casr�/� mice employed in

these and previous studies.(4,26) This alternatively spliced product

was identified in multiple tissues, including the growth plate,

skin, and kidney.(27) Although it has not been possible to express

the alternate spliced product in a functional form in hetero-

logous cell systems, it has been suggested that this alternative

receptor form potentially could provide partial or full functional

compensation in some tissues of Casr�/� mice. Thus, in growth

plate chondrocytes from Casr�/� mice, high [Ca2þ]e increased

inositol phosphate production and promoted the differentiation

of Casr�/� growth plate chondrocytes in vitro.(26) The improve-

ments in the growth plate, notably in mineralization, and in

growth that we observed in the double mutants in the presence

of hypercalcemia are indeed in keeping with activation of a

hypomorphic Casr allele in chondrocytes by [Ca2þ]e. Never-

theless, the growth plate remained somewhat enlarged, and the

cellular architecture, particularly in the hypertrophic zone, was


disorganized. However, in the Casr�/�1a(OH)ase�/� animals on a

rescue diet, vitamin D deficiency still was present, and

hypophosphatemia, which has been reported to impair

caspase-mediated apoptosis of hypertrophic chondrocytes,(25)

also persisted. Therefore, the fact that complete normalization of

the growth plate did not occur may indicate that persistent

hypophosphatemia and absent active vitamin D precluded

complete recovery.

Trabecular bone volume was enhanced both in 1a(OH)ase�/�

mice and in Casr�/�1a(OH)ase�/�mice on a normal calcium diet,

and increased evidence of alkaline phosphatase–positive

osteoblasts was observed. These findings are consistent with

augmented synthesis of bone matrix by the high circulating

concentrations of PTH that were present in each mutant. In

previous studies in mice with targeted deletion of Casr from

osteoblasts, it was reported that the expression of genes

encoding osteoblast markers was reduced, suggesting that the

CaSR is required for osteoblast differentiation. The presence of

augmented osteoblasts and increased bone matrix volume in

our studies, even in the face of CaSR deletion, suggests that any

requirement of the CaSR for osteoblast activation is readily

overcome by the osteoblast-stimulating effects of elevated PTH.

Nevertheless, severe osteomalacia was observed on a normal

diet, as indicated by the increased osteoid volume and absence

of double fluorochrome labeling; similar impairment of miner-


Fig. 5. ALP-positive osteoblast perimeter (Ob.perim) relative to bone

perimeter (B.perim) (A) and serum osteocalcin concentrations (B) in WT

mice and in 1a(OH)ase�/� (D�/�) and Casr�/�1a(OH)ase�/� (DKO) mice

on a normal diet and on a rescue diet. Each value is the mean� SEM of 3

to 5 determinations. �p< .05 relative to WT mice. ��p< .001 relative to

WTmice. þp< .05 relative to 1a(OH)ase�/�mice on a corresponding diet.þþþp< .001 relative to 1a(OH)ase�/� mice on a corresponding diet.

6Normal Diet Rescue Diet

m)

3

4

5

+++B.P

m (N

/mm

0

1

2 *** ***+++ +++

N.O

c/B

D-/- DKO WT D-/- DKO

40 Normal Diet Rescue Diet

30

(U/ µ

l)

10

20

TRA

P 5b

D-/- DKO WT D-/- DKO

0

Fig. 6. Number of TRACPþ osteoclasts (N.Oc) relative to bone perimeter

(B.perim) in number (n) per millimeter (upper panel) and serum TRACP 5b

concentrations (lower panel) in WT mice and in 1a(OH)ase�/� (D�/�) and

Casr�/�1a(OH)ase�/� (DKO) mice on a normal diet and on a rescue diet.

Each value is the mean� SEM of 3 to 8 determinations. ��p< .001

relative to WT mice. þþþp< .001 relative to 1a(OH)ase�/� mice on a

corresponding diet.

alization of bone also was evident in the hypocalcemic and

hypophosphatemic double mutants on the normal diet.

Consequently, the increased bone matrix remained largely

unmineralized, as also indicated by the reduced BMD. On a

rescue diet, mineralization of bone in 1a(OH)ase�/� mice was

normalized and was markedly improved in double mutants. This

improved mineralization of bone in 1a(OH)ase�/� mice on the

rescue diet was no doubt facilitated by the reduced circulating

PTH and consequent reduced production of bone matrix, as well

as by the normalization of calcium and phosphorus. The

contribution of hyperparathyroidism similarly may explain, at

least in part, the mineralization defect previously reported in

mice with parathyroid-specific CaSR deletion.(12)

In Casr�/�1a(OH)ase�/� mice on a rescue diet, however, PTH

levels in the circulation remained high, and osteoblast numbers

and activity remained elevated, resulting in persistently

increased trabecular bone volume. Despite this evidence of

persistently augmented bone matrix synthesis, osteoid volume

was reduced, and the MAR and BMD were improved in the

presence of ambient hypercalcemia, even in the face of

hypophosphatemia. This constellation of hypercalcemia and

elevated PTH, without significant osteomalacia, that was

observed in the double mutants on a rescue diet is also

observed in murine models of primary hyperparathyroidism(28)

and in human hyperparathyroidism(29) despite hypophosphate-

mia. Thus our present studies demonstrating improved miner-


alization in hypercalcemic Casr�/�1a(OH)ase�/� mice also

support a direct role for calcium in bone mineralization. At

least part of the effects on transosteoblastic or paraosteoblastic

[Ca2þ]e transport in bone via the skeletal CaSR therefore may be

analogous to the active transcellular or passive paracellular renal

transport of [Ca2þ]emediated by the renal CaSR.(30) Other studies

have suggested the presence of an alternate calcium receptor in

bone, GPRC6A, that might be involved in skeletal mineraliza-

tion,(31,32) although conflicting reports on the skeletal phenotype

of GPRC6A null mice have been published recently.(32,33) Our

results cannot exclude the possibility that other receptor

mechanisms or nonreceptor mechanisms are involved in

mineralization of bone via increased [Ca2þ]e.

As noted previously and seen again in these studies,

hypocalcemic 1a(OH)ase�/� mice on a normal diet(15,34) and

mice with targeted deletion of the VDR gene on a normal diet(35)

show the absence of significantly augmented osteoclast activity

relative to wild-type controls despite the presence of markedly

elevated circulating concentrations of PTH. In our current studies,

inactivation of the Casr gene in the 1a(OH)ase�/� mice further

reduced osteoclast numbers relative to those seen in the

1a(OH)ase�/�mice. Moreover, in the double mutants on a rescue

diet, despite persistent hyperparathyroidism, osteoclast num-

bers and activity again were not elevated. It has been reported

previously, from studies in vitro, that macrophages, which are

osteoclast precursors, bind poorly to demineralized bone.(36)

RICHARD ET AL.

However, other studies have reported that osteoclasts bind, in

vitro and in vivo, to both demineralized andmineralized bone,(37)

and although they will not form ruffled borders(38) and secrete

acid, they can continue to release degradative enzymes and

resorb bonematrix; that is, they adhere and remain at least partly

functional.(37) In our studies, the reductions in osteoclasts

appeared to be similar both on the nonmineralized and

mineralized bone surfaces of these models when they were

osteomalacic on a normal diet. Furthermore, as shown in Fig. 6,

osteoclast numbers remained markedly reduced in the CaSR�/�

1a(OH)ase�/� mice on a rescue diet, even when the majority of

bone surfaces were mineralized (Fig. 3). Consequently dimin-

ished functional CaSR activity rather than reduced osteoclast

binding to demineralized surfaces appeared to be a common

element determining low osteoclast numbers.

It has been reported previously that there is reduced capacity

of bonemarrow cells isolated from Casr�/�mice, relative to bone

marrow cells isolated from wild-type mice, to differentiate into

TRACPþ multinucleated osteoclasts in vitro.(38) Consequently,

irrespective of the presence or absence of a splice variant in the

osteoclasts of Casr�/� mice (and therefore in the Casr�/�

1a(OH)ase�/� mice), the capacity of osteoclasts to differentiate

appears reduced in vitro. The reduced numbers of TRACPþ

osteoclasts in the Casr�/�1a(OH)ase�/� mice in our study in vivo

therefore most likely were due, at least in part, to decreased

osteoclastogenesis, consistent with these previous in vitro

observations. In addition, in studies in vivo, it has been reported

that transgenic mice expressing a constitutively active mutant

Casr in mature osteoblasts display increased expression of

RANKL, an increased number and activity of osteoclasts, and

reduced bone volume.(11) Reduced RANKL signaling in the Casr�/�

1a(OH)ase�/� mice therefore may represent an additional

mechanism of reduced osteoclastogenesis in the absence of

the CaSR. Consequently, both hypocalcemia in the 1a(OH)ase�/�

mice and CaSR deficiency in the double mutants appear to lead

to inappropriately low or reduced calcium signaling in bone,

resulting in diminished osteoclastogenesis, an impediment that

cannot be overcome by high circulating PTH.

In view of the fact that osteoclast/chondroclast production at

the chondroosseous junction also may be defective, diminished

removal of hypertrophic chondrocytes may occur in this region,

leading to altered cartilage growth plate remodeling. Therefore,

the enlargement of the cartilaginous growth plate, notably the

hypertrophic zone, observed both in the hypocalcemic

1a(OH)ase�/� mice and in the hypocalcemic and hypercalcemic

Casr�/�1a(OH)ase�/� mice also may be due in part to reduced

activation of the CaSR on the chondroclast/osteoclast system.

In summary, our studies support the important roles of [Ca2þ]enot only in parathyroid function but also in growth plate

maturation, in skeletal mineralization, and in osteoclastic bone

resorption. In the parathyroid gland, in the absence of the CaSR, a

signal by [Ca2þ]e could not be transduced, even in the presence

of hypercalcemia, thus resulting in persistent hyperparathyroid-

ism. In contrast, the capacity of hypercalcemia to improve

growth and mineralization in the absence of the CaSR suggests a

different [Ca2þ]e/CaSR relationship, perhaps owing to a hypo-

morphic Casr allele or to another mechanism of [Ca2þ]e action to

enhance mineralization. In view of the fact that hypercalcemia


did not restore bone resorption in the absence of the CaSR, the

mechanism modulating osteoclasts appears to involve a [Ca2þ]e-

stimulated skeletal CaSR that behaves in a fashion analogous to

the parathyroid CaSR.

Disclosures

DG has served as a consultant to Abbott, Genzyme, Amgen, Lilly,

and NPS Allelix. All the other authors state that they have no

conflicts of interest.

Acknowledgments

CR was a Bone Scholar of the Skeletal Health Training Program of

the Canadian Institutes for Health Research (CIHR). This work was

supported by grants to DG and GNH from the CIHR. We thank the

staff of the Centre for Bone and Periodontal Research, McGill

University, Montreal, Quebec, Canada, for excellent technical

assistance with biochemical, histomorphometric, and imaging

analyses.

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RICHARD ET AL.

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