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PTH-CALCITONIN-VITAMIN D-BONE |
Double Null Mutant Mice
Institute of Molecular and Cellular Biosciences, University of Tokyo (N.Y., Y.Y., T.Y., K.S., Y.U., S.K.), Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; Biomedical Research Laboratories, Kureha Chemical Industry Co. Ltd. (H.M., Y.N.), Hyakunin-cho, Shinjuku-ku, Tokyo 169-8503, Japan; Institut de Genetique et de Biologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique/INSERM/Université Louis Pasteur/College de France (W.K., P.C.), 67404 Illkirch, Strasbourg, France; First Department of Internal Medicine, University of Tokushima (T.M.), Tokushima 770-8503; and CREST, Japan Science and Technology (S.K.), Kawaguchi, Saitama 332-0012, Japan
Address all correspondence and requests for reprints to: Dr. S. Kato, Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: uskato{at}mail.ecc
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, RXRß, and
RXR
, and it is thought that RXR ligands can also modulate the
trans-activation function of VDR/RXR heterodimers. In
the present study we generated VDR/RXR double null mutant mice to
examine the convergent actions of vitamin D and vitamin A signaling and
to explore the possibility of a functionally redundant VDR. Although
RXR-/- mice exhibited no overt
abnormalities,
VDR-/-/RXR-/-
mice appeared similar to VDR-/-
mice, showing features typical of vitamin D-dependent rickets type II,
including growth retardation, impaired bone formation, hypocalcemia,
and alopecia. However, compared to
VDR-/- mice, growth plate
development in
VDR-/-/RXR-/-
mutant mice was more severely impaired. Normalizing mineral ion
homeostasis through dietary supplementation with high calcium and
phosphorous effectively prevented rachitic abnormalities, except for
disarranged growth plates in
VDR-/-/RXR-/-
mutant mice, and alopecia in both
VDR-/- and
VDR-/-/RXR-/-
mutant mice. Histological analysis of
VDR-/-/RXR-/-
growth plates revealed that development of the hypertrophic
chondrocytes was selectively impaired. Thus, our findings indicated
that the combined actions of VDR- and RXR-mediated signals are
essential for the normal development of growth plate chondrocytes, and
raised the possibility that a functionally redundant VDR is present on
chondrocytes as a heterodimer with RXR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
[1,25-(OH)2D3]
(3, 4). However, its derivatives, although less potent,
also possess biological activity. Most vitamin D effects are exerted
through transcriptional control mediated via VDR binding of the
1,25-(OH)2D3 ligand
(5, 6). VDR is a member of the steroid/thyroid hormone and
vitamin A/D nuclear receptor superfamily and functions as a
ligand-inducible transcription factor with ligand-dependent recruitment
of coactivator complexes (7, 8). VDR binds as a
heterodimer with one of three RXR (RXR, -ß, and -) to vitamin
D-responsive elements in the promotors of vitamin D target genes
(6). Although the trans-activation function of
VDR/RXR heterodimers is induced upon
1,25-(OH)2D3 binding to
VDR, in vitro functional studies suggest ligand binding to
RXR also modulates VDR/RXR heterodimer function (9, 10),
indicating possible convergent activity of vitamin D and A in target
tissues for vitamin D. Like VDR, the RAR (RAR, -ß, and -), TR
(TR and -ß), and other nuclear receptors heterodimerize with RXR
to bind cognate binding sites in target gene promoters
(7). However, unlike other nuclear receptors that
heterodimerize with RXR, only one VDR isotype has been identified in
mammals.
The physiological role of VDR in vitamin D signaling has been
investigated using mutant mice null for VDR (11, 12).
These VDR-/- mice exhibited
phenotypic abnormalities typical of vitamin D-dependent type II
rickets, including growth retardation, impaired bone formation with
disorganized growth plates, and alopecia (13). The
rachitic abnormalities developed only after weaning and were
accompanied by hypocalcemia and hypophosphatemia. However, as lowered
serum mineral levels alone were sufficient to cause such abnormalities
in bone with secondary hyperparathyroidism (14), it
remains unclear whether the rickets-like phenotype was a direct effect
of reduced vitamin D signaling in bone and other target tissues or was
mediated via serum mineral levels. Moreover, VDR inactivation caused
aberrant expression of genes for
25-(OH)2D3
1-hydroxylase, and
25-(OH)2D3 24-hydroxylase
in kidney that are critical for the biosynthesis and catabolism of
1,25-(OH)2D3,
respectively (15, 16), resulting in markedly higher levels
of 1,25-(OH)2D3 with no
detectable levels of
24,25-(OH)2D3. Therefore,
it has been postulated that at least some of the abnormalities in
VDR-/- mice are caused by
the lack of activity of
1,25-(OH)2D3
derivatives, such as
24,25-(OH)2D3. However,
little is known of the receptors for vitamin D metabolites.
One approach to search for nuclear receptors for vitamin D derivatives
is through gene disruption studies (17). Mice deficient
for RXRs are very useful for this approach, as the putative nuclear
receptor, as a nonsteroidal receptor, would probably heterodimerize an
RXR. RXR-deficient mice exhibit no discernible abnormalities
(18), whereas RXRß inactivation leads to impaired
spermatogenesis, and RXR inactivation leads to embryonic lethality
(19). Therefore, in the present study we generated
VDR/RXR double null mutant mice to examine the convergent actions of
vitamin D and A signaling and to explore the possibility of a
functionally redundant VDR-like receptor. The abnormalities observed in
double null mutant mice were basically similar to those in
VDR-/- mice (11, 12), except that growth plate development was severely impaired.
Recent reports have shown that dietary supplementation with high
calcium and phosphorous rescued skeletal rachitic abnormalities, but
not alopecia, in VDR-/- mice
through normalized mineral homeostasis (20, 21). In
contrast, disorganized growth plates in
VDR-/-/RXR-/-
mice were not prevented by dietary supplementation. Histological
analysis of
VDR-/-/RXR-/-
growth plates revealed selective impairment of hypertrophic
chondrocyte development without evidence of altered apoptosis or
osteoclast dysfunction despite normalized mineral homeostasis produced
by the high mineral diet. Thus, our findings indicate that the combined
actions of VDR- and RXR-mediated signals are essential for normal
development of growth plate cartilage and suggest that a functional,
but redundant, VDR-like receptor exists on growth plate
chondrocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-(OH)2D, RRA kit using purified calf
thymus VDR (1,25VD Kit-Med, Japan Mediphysics Co., Nishinomiya,
Japan).
RT-PCR
Total RNA was isolated by the improved
acid-guanidine-phenol-chloroform method (15). A
semiquantitative RT-PCR was performed on total RNA (1 µg) from total
tibia under appropriate conditions to measure each of the transcripts
(22). First strand cDNA was synthesized and amplified with
specific oligonucleotide primers for PTHrP (5'-ATGAATTC
TGCTCAGCTACTCCGTG-3' and 5'-TACCTAGGGAGGTCCTGGA GGTGTG-3'), PTHrPR
(5'-GAAGTTCTGCACACAGC-3' and 5'-TGAAGCCAGAGTAGAGC-3'), osteopontin
(5'-CATTGCCTCCTCCCTCCCGGTG-3' and 5'-GCTATCACCTCGGCCGTTGGGG-3'),
glyceraldehyde-3-phosphate dehydrogenase (5'-ACCACAGTCCATGCCATCAC-3'
and 5'-TCCACCACCCTGTTGCTGTA-3'), VDR (5'-CCT- CGTGG ACATTGGCATG-3' and
5'-TCAGGAGATCTCATTGCCAAA-3') and RXR (5'-CAGGTCTGCCTGGGATTGGA-3' and
5'-GTTGAGTTCTCC ACGTTCATG-3').
Histological analysis and in situ hybridization
Skin sections were fixed in 4% paraformaldehyde and dehydrated
with increasing concentrations of ethanol before paraffin embedding.
Sections were stained with hematoxylin and eosin according to standard
procedures. Undecalcified tibia were fixed in 99.5% ethanol and
embedded in methyl methacrylate. Sections were stained using the
modified Villanueva-Goldner’s Trichrome method (12). For
in situ hybridization (23) metatarsal bones
were fixed in 4% paraformaldehyde and decalcified in 0.46
M EDTA (pH 7.4) for 5–7 d at 4 C before
embedding in paraffin. In situ hybridization using
digoxygenin-labeled riboprobes was performed according to the
manufacturer’s instructions (Nippon Gene, Tokyo, Japan).
Normalization of serum mineral homeostasis
Weanling mice (3 wk old) were supplied minerals by feeding them
for 4 wk a high calcium diet (containing 2.0% calcium, 1.25%
phosphorus, and 20% lactose). Control mice were fed a normal diet
containing 1.2% calcium and 1.0% phosphorus (Japan Clea, Chiba,
Japan). Normalization of serum mineral levels was verified by the
measurement of serum calcium concentrations from 7-wk-old mice. The
mice were killed under the principles and procedures outlined in the
NIH Guide for the Care and Use of Laboratory Animals.
Identification of apoptotic cells
Apoptotic cells were identified by immunostaining with mouse
monoclonal antibody M30 CytoDEATH (Roche Molecular Biochemicals) as the first antibody (24). The
antibody M30 CytoDEATH recognizes a specific caspase
(cysteinyl-aspartic acid protease) cleavage site within cytokeratin 18
that is not detectable in native cytokeratin 18 of nonapoptotic cells
(24). Bone sections prepared for immunostaining were
incubated with M30 CytoDEATH (1:10), and the signal was detected using
the TSA-Direct kit (NEN Life Science Products, Boston, MA)
according to the manufacturer’s instructions.
Osteoclast formation
For in vivo identification of osteoclasts,
undecalcified tibia were fixed with 4% paraformaldehyde and embedded
in methyl methacrylate. Sections were stained for tartrate-resistant
acid phosphatase (TRAP) with a commercially available kit
(Sigma, St. Louis, MO) (12, 25). Osteoclast
formation in vitro was evaluated by cocultures of calvarial
osteoblasts with spleen cells from newborn mice as previously described
(25, 26).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- double
null mutant mice-/-
double null mutant mice were generated through intercrossing
VDR-/+ and
RXR-/+ mice.
RXR-/- mutant mice
exhibit no known growth, behavior, or viability abnormalities
(18). Mutant
VDR-/+/RXR-/+,
and
VDR-/+/RXR-/-
mice appeared completely normal and were not noticeably different from
wild-type littermates.
VDR-/-/RXR-/-
mice showed features typical of vitamin
D-dependent type II rickets, including
growth retardation, hypocalcemia, and hypophosphatemia (Fig. 1
, A and
B), but only after weaning at 3 wk (12, 11, 16).
Altered levels of serum 24,25-(OH)2D and
1,25-(OH)2D were observed due to the aberrant
expression of the two hydroxylases in kidney. VDR inactivation
abolished the positive regulation of 24-hydroxylase in converting
25-(OH)2D3 into
24,25-(OH)2D3 and the
negative regulation of 1-hydroxylase in producing
1,25-(OH)2D3 from
25-(OH)2D3 (Fig. 1C).
Obvious alopecia developed in both
VDR-/-/RXR-/-
and VDR-/- mutant mice after
weaning (Fig. 1, D–G), similar to the skin phenotype seen in
VDR-/- mice at 7 wk
(11, 20, 27) (Fig. 1, H–K).
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in VDR-/- mice causes a more
severe bone phenotype-/-
than in VDR-/- mutant mice,
including wider epiphysis and severe fraying, cupping, and widening of
the metaphysis (3, 4, 5, 6) (Fig. 2A). Comparison of bones from 7-wk-old
mutant mice revealed that bichondylar distances were greater in
VDR-/-/RXR-/-
mice than in VDR-/- mice,
although there was no statistically significant difference in lengths
of the tibia (Fig. 3B). Histological
analysis detected increased osteoid seams in the calvaria (Fig. 2B) and
primary spongiosa of the tibiae due to marked impairment of bone
mineralization in
VDR-/-/RXR-/-
mice (Fig. 3H) compared with
VDR-/- mice (Fig. 3F). Also,
epiphyseal growth plates in the tibia of
VDR-/-/RXR-/-
mice were widened, consistent with the presence of less orderly columns
of hypertrophic chondrocytes. Although compared with
VDR-/- mice (Fig. 3G),
hypertrophic chondrocyte layers in
VDR-/-/RXR-/-
mice appeared expanded (Fig. 3I), osteoclast numbers, present mainly in
the secondary spongiosa, were similar to those in wild-type littermates
(25) (Fig. 3H and Fig. 7, A–D).
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-/- mice-/-
mutant mice from impaired bone formation. Both
VDR-/-/RXR-/-
and VDR-/- mutant mice
showed considerable recovery from growth retardation after being fed
the mineral-enhanced diet, although recovery was less pronounced in
VDR-/-/RXR-/-
mice (Fig. 3C
). The diet also restored serum calcium and phosphorous
levels in both single and double null mutants to near wild-type levels
(black columns in Fig. 3A). The development of alopecia was
not prevented in
VDR-/-/RXR-/-
mice (data not shown), as previously reported for
VDR-/- mice
(20). Bone mineralization in both mutant mouse strains was
restored, as indicated by the disappearance of widely spread osteoid
seams (Fig. 3, L and N), with significant, but not complete,
improvement in tibial lengths of both
VDR-/-/RXR-/-
and VDR-/- mice (Fig. 3B).
However, despite the recovery from impaired mineralization, the
expanded chondrocyte layers, as estimated by bichondylar distance (Fig. 3B) and histological analysis (Fig. 3, I and O), failed to show full
recovery in
VDR-/-/RXR-/-
mice in contrast to VDR-/-
mice.
We then examined the expression of the VDR and RXR genes in growth
plates. As previously reported (28), the VDR gene was
expressed abundantly in the growth plates of 7-wk-old wild-type
littermates, whereas RXR expression levels were low (
10-fold less
than VDR levels by semiquantitative RT-PCR; Fig. 4A), with only very weak signals detected
in chondrocytes by in situ hybridization (data not shown).
The observation of reduced RXR gene expression in growth plate
chondrocytes is supported by the pattern of RXR gene expression in
the ATDC5 cell line when differentiated into hypertrophic
chondrocyte-like cells (29) (Fig. 4A).
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-/- mice) among the
different genotypes, even in mutant mice not rescued by dietary
supplementation. However, reduced OPN gene expression was
observed (Fig. 4B). OPN is a marker of hypertrophic chondrocytes,
whereas the other genes tested are markers for proliferative and
prehypertrophic chondrocyte layers (34). We then performed
a more detailed analysis of metatarsal growth plates of 5-wk-old
VDR-/-/RXR-/-
mice by in situ hybridization (Fig. 5) (23). In agreement with
the RT-PCR findings,
VDR-/-/RXR-/-
and VDR-/- null mutant mice
fed a normal diet showed an altered pattern of
OPN gene expression (Fig. 5, E–L), whereas the expression
patterns of the other genes, including ColXA1 (Fig. 5, A–D), were normal. Consistent with the histological analysis (Fig. 3, D–O) and measurements of bichondylar distance (Fig. 3B), the
supplemental diet corrected the scattered OPN gene
expression pattern in VDR-/-
mice, but failed to restore the normal pattern in
VDR-/-/RXR-/-
mice (Fig. 5, M and N).
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-/- mouse growth
plates-/-
mice, we examined the possibility that receptor inactivation suppressed
apoptosis, resulting in layer expansion due to the prolonged life span
of the hypertrophic chondrocytes (35). However,
immunohistological analysis using an anti-DEATH protein antibody
(24) showed no apparent change in apoptosis in the growth
plates or the adjacent metaphyseal bone in 5-wk-old
VDR-/-/RXR-/-
mice (Fig. 6, A–D). Furthermore,
mRNA levels of apoptosis-related genes Fas,
c-myc, bax, bad,
bcl-xL, and bcl-w in the
growth plates of
VDR-/-/RXR-/-
mice (36), as estimated by semiquantitative RT-PCR, also
appeared normal (Fig. 6E).
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-/-
mice fed a normal diet and that osteoclast cell numbers were normal
(Fig. 7D). Also, a coculture system of
splenic osteoclast precursor cells and calvarial osteoblasts (Fig. 7E)
used to induce mature TRAP-expressing osteoclasts (Fig. 7F) (25, 26) suggested that RXR inactivation did not modulate
responsiveness to osteoclast differentiation factors, including vitamin
D, PTH (Fig. 7G), and IL-1 (data not shown). Thus, these results
indicate that only specific functions of hypertrophic chondrocytes were
impaired by combined VDR and RXR inactivation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice exhibit no
overt phenotype, it is possible RXR inactivation would have a
greater effect on target tissues for this putative receptor in
VDR-/- mice. Therefore, to
test this hypothesis we generated double null mutant mice for VDR and
RXR (18).
Normalization of impaired mineral homeostasis failed to rescue
aberrant growth plate development in
VDR-/-/RXR-/- mice
After weaning,
VDR-/-/RXR-/-
mice developed typical features of rickets, such as growth retardation,
impaired bone formation, and alopecia that were almost identical to
those in VDR-/- mice.
Detailed phenotypic analyses detected no differences between
VDR-/- mice and
VDR-/-/RXR-/-
mice in tissues such as skin and intestine and no differences in
impaired mineral and vitamin D metabolism, except in bone. Despite the
similar reduction in serum mineral levels between
VDR-/- and
VDR-/-/RXR-/-
mice, growth plates of
VDR-/-/RXR-/-
mice were more affected than those of
VDR-/- mice. Histological
analysis of the bone revealed that growth plate development,
particularly at the terminal layer of hypertrophic chondrocytes, was
impaired in
VDR-/-/RXR-/-
mice even when mineral homeostasis was normalized through the feeding
of a diet supplemented with high calcium and phosphorous. This is in
contrast to the disorganized growth plates of
VDR-/- mice that were
recovered by dietary supplementation as previously reported (20, 21). However, similar to that observed in
VDR-/- mice, alopecia was
not prevented by the supplemented diet in
VDR-/-/RXR-/-
mice. As the supplemented diet maintained almost normal serum mineral
levels in both VDR-/- and
VDR-/-/RXR-/-
mice, it is therefore unlikely that the aberrant development of growth
plate cartilage was simply due to hypocalcemia, hypophosphatemia, or
secondary hyperparathyroidism. This is further supported by the
findings that phenotypic abnormalities of bone and bone formation in
mice with similar pathophysiological states (14) were not
consistent with those observed in
VDR-/-/RXR-/-
mice. Thus, our findings raise the possibility that a functionally
redundant VDR-like receptor heterodimerized with RXR is involved in
chondrocyte differentiation.
VDR and RXR as regulatory factors for chondrocyte
differentiation
Continuous chondrogenesis in growth plates
supports longitudinal bone growth, allowing vertebrate development.
Proliferating chondrocytes at epiphyseal growth plates differentiate
into prehypertrophic chondrocytes, then to hypertrophic chondrocytes
with secretion of cartilage matrix, and finally the hypertrophic
chondrocytes are replaced by bone. Reflecting the complexity of
cartilage differentiation and bone formation, known as endochondrial
ossification, distinct classes of regulatory factors, such as cytokines
and transcription factors, have been identified from a variety of
approaches (39, 40). Although disruption of these
regulatory factor genes perturbs cartilage development, in most cases
the phenotypic abnormality and time of onset are distinct from the
phenotype of
VDR-/-/RXR-/-
mice. Interestingly, similar phenotypes involving expanded layers of
hypertrophic chondrocytes have been described in mice deficient for
matrix metalloproteinase (MMP)-9/gelatinase B (41)
and vascular endothelial growth factor (VEGF) proteins
(42). However, the CoLXA1 gene expression
pattern in early layers of hypertrophic chondrocytes was normal in
VDR-/-/RXR-/-
mice, but not in MMP-9- and VEGF-deficient mice.
These results suggest that the VDR/RXR heterodimer acts later than
MMP-9 and VEGF, and that the malfunction of a specific factor(s) caused
by VDR and RXR inactivation appears to be responsible for the
altered development of hypertrophic chondrocytes. To more precisely
define the function of VDR and RXR in growth plate chondrogenesis,
studies looking at stage- and cell type-specific gene disruption of VDR
and RXR in growth plates are required in addition to the
identification of downstream target genes expressed in hypertrophic
chondrocyte layers.
A functionally redundant nuclear receptor for vitamin D
is expressed as a heterodimer with RXR in growth plate
cartilage?
As RXRß- and
RXR-/- single-null mutant
mice exhibit no observable rickets-related abnormalities (17, 18), these two RXRs appear to be functionally redundant for
VDR/RXR heterodimer function in vitamin D target tissues. However, the
enhanced growth plate phenotype of
VDR-/-/RXR-/-
was not seen in
VDR-/-/RXRß-/-
mice, even though
VDR-/-/RXRß-/-
mutants developed
RXRß-/--associated
abnormalities (17, 18) as well as rickets (our unpublished
results). Due to the embryonic lethality of the
RXR-/- mutation
(19), generation of
VDR-/-/RXR-/-
mutant mice is impossible, although a cartilage-specific RXR
inactivation appears possible, as performed in skin (27).
Nonetheless, the present study indicated that a functionally redundant
VDR-like receptor may play a critical role in normal chondrocyte
differentiation and appears to form a heterodimer with at least RXR.
A candidate for this receptor is one of the RAR subtypes, as vitamin A
signaling is known to modulate skeletal development and bone formation
from a series of genetic studies of RAR and RXR (17).
Moreover, a recent report claims that retinoic acid is a potent
negative regulator of growth plate chondrogenesis by reducing
chondrocyte hypertrophy and chondrocyte proliferation
(43), although a hypertrophic chondrocyte-specific
abnormality with normal bone development has not yet been observed in
mice deficient for any of the RAR subtypes.
A more interesting candidate would be an unknown receptor functionally
regulated by the binding of vitamin D derivatives such as
24,25-(OH)2D3
(2).
24,25-(OH)2D3 is one of the
major serum vitamin D metabolites and is reported to stimulate growth
plate chondrogenesis (44). Serum
24,25-(OH)2D3 levels were
substantially lowered in
VDR-/- and
VDR-/-/RXR-/-
mice due to abnormal expression of vitamin D hydroxylases (12, 15, 16), suggesting that disorganized growth plates may be due
to the lack of
24,25-(OH)2D3 actions on
chondrocytes. This implies that a
24,25-(OH)2D3 receptor, if
it exists, may be expressed in chondrocytes and exhibit similar
functions as VDR by heterodimerizing with RXR. To explore this
possibility, the screening of a cDNA library made from growth
plate chondrocytes of
VDR-/-/
RXR-/- mice for a receptor that forms
a heterodimer with RXR is currently under way.
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Acknowledgments |
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Footnotes |
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1 N.Y., Y.Y., and T.Y. contributed equally to this work. 
Abbreviations: ColXA1, Collagen type XA1;
1,25-(OH)2D3, 1,25-dihydroxyvitamin
D3; MMP, matrix metalloproteinase; OPN, osteopontin; TRAP,
tartrate-resistant acid phosphatase; VEGF, vascular endothelial growth
factor.
Received April 11, 2001.
Accepted for publication August 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dependent rickets. Curr Opin Nephrol Hypertens 7:377–383[Medline]
null mice are apparently normal and compound
RXR+/-/RXRß-/-/RXR-/-
mutant mice are viable. Proc Natl Acad Sci USA 93:9010–9014 developmental function: convergence of RXR and RAR signaling
pathways in heart and eye morphogenesis. Cell 78:987–1003[CrossRef][Medline]
mutations in mouse epidermis. Nature 407:633–636[CrossRef][Medline]
,25-dihydroxyvitamin
D3 receptors and their action in embryonic chick
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1 gene for
collagen II develop a skeleton with membranous and periosteal bone but
no endochondral bone. Genes Dev 9:2821–2830This article has been cited by other articles:
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), RXR
),
VDR-/- (
), and
VDR-/-/RXR
) mice. Results are expressed as the mean ± SE
for four mice. B and C, Analyses of endocrine indicators in mice of
each genotype at 7 wk of age. Concentrations of serum calcium,
phosphate (B), and vitamin D metabolites
(C) are shown. Results are expressed as the mean ±
SE for five samples. D–G. The side view of
wild-type (D), RXR
, P < 0.05 between
VDR-/- and
RXR
). After 8 d, the number of TRAP-positive multinucleated cells
(F) per well was counted. The combination of the cells from different
genotypes is shown in the lower panel. Results are
expressed as the mean ± SE for three samples.
When both cells were prepared from wild-type mice,
1,25-(OH)2D3 stimulated TRAP-positive
multinucleated cells formation, whereas when osteoblast cells from
VDR-/- mice were used, no
osteoclasts were formed.
