Full text of DOI:10.1359/jbmr.2002.17.4.622

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 4, 2002
© 2002 American Society for Bone and Mineral Research
Vitamin D Hormone Inhibits Osteoclastogenesis In Vivo by
Decreasing the Pool of Osteoclast Precursors in Bone Marrow
TAKESHI SHIBATA,1,2 AYAKO SHIRA-ISHI,2,3 TAKUYA SATO,1 TOSHIMI MASAKI,3 AYA SASAKI,1
YOSHIKO MASUDA,1 AKINORI HISHIYA,1 NOBUYUKI ISHIKURA,1 SAYUMI HIGASHI,3
YASUHIRO UCHIDA,3 MOTO-O SAITO,3 MASAKO ITO,4 ETSURO OGATA,5 KEN WATANABE,1 and
KYOJI IKEDA1
ABSTRACT
Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor ␬B
(NF-␬B) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that
1␣,25-dihydroxyvitamin D3 [1␣,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a
prodrug metabolized to 1␣,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normo-
calcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic
doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly
reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient
amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than
in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less
osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of
osteoclast progenitors in the marrow of vitamin D–treated mice as well as estrogen-treated mice was decreased.
Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and
decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active
vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption.
Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to
estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the
management of osteoporosis. (J Bone Miner Res 2002;17:622–629)
Key words: vitamin D, alfacalcidol, osteoporosis, bone resorption, osteoclasts, receptor activator of nuclear
factor ␬B ligand, ovariectomized mice
calcium and bone metabolism through its action in the
intestine, bone, kidney, and parathyroid gland.(1,2) The
importance of the VDR, especially during the maturation
stage of bone, can be inferred from the consequences of
functional defects in it; these include hypocalcemia, sec-
ondary hyperparathyroidism, and impaired mineraliza-
tion, which are the phenotypes shown in the human
vitamin D–resistant rickets syndrome due to point muta-
tions in the VDR gene(3,4) and also in VDR knockout
mice.(5,6) In contrast, the physiological importance of
INTRODUCTION
ITAMIN D receptor (VDR) liganded with 1␣,25-
dihydroxyvitamin D3 [1␣,25(OH)2D3], the active
and hormonal form of vitamin D, plays a critical role in
V
Dr. Ogata serves as a consultant and receives financial assis-
tance. Dr. Saito, Dr. Uchida, Dr. Masaki, Dr. Higashi, and Dr.
Shiraishi have financial interests in the forms of corporate appoint-
ments. All other authors have no conflict of interest.
1Department of Geriatric Research, National Institute for Longevity Sciences, Aichi, Japan.
2These authors contributed equally to this work.
3Product Research Laboratory, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan.
4Department of Radiology, Nagasaki University School of Medicine, Nagasaki, Japan.
5Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan.
622

VITAMIN D INHIBITS OSTEOCLASTOGENESIS
623
VDR in normal bone remodeling after skeletal matura-
tion remains unclear.
Animals
Seven-week-old female ddy mice were purchased from
Nippon SLC. (Shizuoka, Japan) and acclimated for 1 week
under standard laboratory conditions at 24 Ϯ 2°C and
50–60% humidity. The mice were allowed free access to
tap water and commercial standard rodent chow (CE-2)
containing 1.13% calcium, 1.00% phosphate, and 2.0 IU/g
of vitamin D3 (Clea Japan, Inc., Shizuoka, Japan).
Age-related decline in VDR function is thought to in-
crease the risk of developing osteoporosis, a systemic dis-
ease characterized by excessive bone resorption with rela-
tive deficiency of bone formation.(7,8) Thus, 1␣,25(OH)2D3
and its prodrug 1␣-hydroxyvitamin D3 [1␣(OH)D3; or al-
facalcidol] have been used in some countries for the treat-
ment of osteoporosis.(9) However, how the hormonal form
of vitamin D acts in bone to counter the abnormal remod-
eling process in osteoporosis remains to be clarified.
Generally, it is recognized that the effect of
1␣,25(OH)2D3 on bone is an indirect one, mediated by
stimulation of calcium absorption from the intestine and
resultant suppression of parathyroid hormone (PTH),(7) and
how 1␣,25(OH)2D3 modulates bone cell functions under
vitamin D–replete conditions remains relatively unclear. At
the cellular level, it has been established that 1␣,25(OH)2D3
induces the expression of the receptor activator of nuclear
factor ␬B (NF-␬B) ligand (RANKL) in bone marrow (BM)
stromal cells,(10) which is essential for the differentiation of
bone-resorbing osteoclasts.(11,12) This in vitro property of
1␣,25(OH)2D3 is consistent with the well-known activity of
1␣,25(OH)2D3 in stimulating osteoclastic bone resorption in
organ cultures(13) but is inconsistent with its therapeutic use
in osteoporosis.
Experimental design
Sham-operated and OVX mice were used. The OVX
mice were divided randomly into several groups after sur-
gery. In the sham and OVX control groups, mice received
the vehicle (MCT) orally at a dose of 1 ml/kg body weight
(BW) five times a week. Alfacalcidol was given orally at
0.05 ␮g/kg and 0.2 ␮g/kg BW five times a week, and E2
was injected subcutaneously at 20 ␮g/kg BW five times a
week. These animal studies were carried out in accordance
with the ethical guidelines for animal care at Chugai Phar-
maceutical Co., Ltd., and the National Institute for Longev-
ity Sciences, and the experimental protocols were approved
by the animal care committees of both institutions.
Serum and urine biochemistry
Based on a series of in vivo experiments using an ovari-
ectomized (OVX) rat model of estrogen-deficient osteopo-
rosis, we have shown that contrary to the in vitro activity of
1␣,25(OH)2D3, active vitamin D inhibits osteoclastic bone
resorption as potently as estrogen(14) and that this anticata-
bolic effect is at least in part mediated through a direct
effect on the skeletal tissue independently of the calcium
flux from the intestine and the PTH levels.(15) To gain
further insight into the mechanism by which active vitamin
D inhibits bone resorption, we used murine marrow cultures
for osteoclastogenesis using BM cells from OVX mice
treated in vivo with the drugs. The results indicated that the
hormonal form of vitamin D, at pharmacologic doses that
maintain normocalcemia, does not induce RANKL in bone
but inhibits bone resorption by decreasing the pool size of
osteoclast precursors in BM.
Urine was collected during 24 h of fasting, and blood was
drawn from the abdominal aorta under ether anesthesia.
Blood and urine samples were centrifuged to obtain the
supernatants, which were stored at Ϫ20°C until the assay.
Serum calcium (Ca) and inorganic phosphorus (Pi) concen-
trations were measured with an autoanalyzer (7070; Hitachi,
Tokyo, Japan). The serum osteocalcin concentration was
measured with the Mouse Osteocalcin IRMA Kit (Immu-
topics, Inc., San Clemente, CA, USA). Urinary Ca, Pi, and
creatinine (Cr) were measured with an autoanalyzer. Uri-
nary deoxypyridinoline (D-Pyr) was measured using a
PYRILINKS-D assay kit (Metra Biosystems, Inc., Moun-
tain View, CA, USA), and the data were corrected for
urinary Cr concentrations.
Bone histomorphometry
The distal femora were fixed in ice-cold 70% ethanol.
After dehydration with 2-propanol, the samples were em-
bedded in glycol methacrylate (Wako, Osaka, Japan). Po-
lymerization was performed at 4°C. Specimens were sec-
MATERIALS AND METHODS
Reagents
Alfacalcidol [1␣(OH)D3] was synthesized at Chugai tioned in the center to yield 3-␮m undecalcified sections,
Pharmaceutical Co., Ltd. (Shizuoka, Japan), dissolved in a which were then stained for tartrate-resistant acid phospha-
vehicle (medium-chain triglyceride [MCT]), and diluted to tase (TRAP). The image of the specimen, observed under a
a given concentration. 17␤-Estradiol (E2), purchased from microscope and recorded with a video camera (DK-3000;
Sigma (St. Louis, USA), was dissolved in corn oil (Sigma) Hitachi), was processed using a plotter (Cosmozone 1SA;
and diluted to a given concentration. The stock solutions Nikon, Tokyo, Japan) to measure the primary parameters,
were protected from light and stored at 4°C. Macrophage bone surface (BS, ␮m) and osteoclast number (N.Oc). From
colony-stimulating factor (M-CSF) was purchased from R these primary parameters, the N.Oc per millimeter of tra-
& D Systems, Inc. (Minneapolis, USA), and soluble becular surface (N.Oc/B.S./mm) was calculated. Osteoclasts
RANKL was a gift from Snow-Brand Milk Product Co. were identified as TRAPϩ multinucleated cells that form
(Tochigi, Japan)
resorption lacunae at BS. Nomenclature, symbols, and units

624
SHIBATA ET AL.
TABLE 1. EFFECTS OF ACTIVE VITAMIN D AND ESTROGEN ON BIOCHEMICAL PARAMETERS IN OVX MICE
Serum
Urine
Dose
(␮g/kg)
Calcium
(mg/dl)
Phosphate
(mg/dl)
Osteocalcin
(ng/ml)
D-Pyr/Cr
(nM/mM)
Operation
Treatment
Calcium/Cr
Sham
OVX
OVX
OVX
OVX
Vehicle
Vehicle
1␣(OH)D3
1␣(OH)D3
E2
9.2 Ϯ 0.6
9.5 Ϯ 0.2
9.2 Ϯ 0.3
9.6 Ϯ 0.5
9.7 Ϯ 0.6
7.7 Ϯ 0.7 152.3 Ϯ 5.6
7.8 Ϯ 0.9 179.6 Ϯ 27.6
8.1 Ϯ 0.9 158.0 Ϯ 16.5
8.8 Ϯ 1.4 174.5 Ϯ 28.2
8.2 Ϯ 1.0 132.1 Ϯ 16.7†
0.103 Ϯ 0.040*
0.186 Ϯ 0.071
0.177 Ϯ 0.064
0.629 Ϯ 0.574
0.179 Ϯ 0.042
31.77 Ϯ 2.58†
43.62 Ϯ 7.92
32.56 Ϯ 6.45*
31.56 Ϯ 5.36†
34.43 Ϯ 4.99*
0.05
0.2
20
Data represent mean Ϯ SEM (n ϭ 6 ϳ 8).
* p Ͻ 0.05; † p Ͻ 0.01 versus OVX group.
used in this study are those described in the Report of the
American Society for Bone and Mineral Research Nomen-
clature Committee.
Three-dimensional analysis of bone by microcomputed
tomography
The microcomputed tomography (micro-CT) apparatus
(␮CT20) and the analysis software used in this study were
provided by Scanco Medical AG (Bassersdorf, Switzer-
land).(16) The whole L5 bodies of the OVX mice and
alfacalcidol- or estrogen-treated OVX mice were scanned
dorsoventrally into 200 slices, each of which was 8 ␮m
thick. On the original three-dimensional (3D) image, mor-
phometric indices were determined directly from the bina-
rized volume of interest (VOI). The total tissue volume
(TV) was the volume of the whole sample examined. Then,
the values of the trabecular bone volume fraction (BV/TV)
and mean trabecular thickness (Tb.Th) were determined
from the local thickness at each voxel representing bone.(17)
Using this technique, thickness can be estimated without a
model assumption. Trabecular separation (Tb.Sp) was cal-
culated by applying the same technique as used for the
direct thickness calculation to the nonbone parts of the 3D
image.
FIG. 1. Active vitamin D decreases N.Oc in OVX mice. OVX ddy
mice (8 weeks old) were given alfacalcidol (ALF) orally (0.2 ␮g/kg
BW) or E2 subcutaneously (20 ␮g/kg BW) five times a week for 2
weeks, and N.Oc/BS at the distal femur was determined as described in
Materials and Methods section. Data are expressed as mean Ϯ SEM
(n ϭ 7). **p Ͻ 0.01, *p Ͻ 0.05 versus the OVX-vehicle group, and
ϩp Ͻ 0.05 versus sham group, using Dunnett’s multiple test.
For frequency analysis of osteoclast precursors, 1 ϫ 104
ST2 cells were seeded into each well on 96-well plates and
cultured overnight in ␣-MEM/10% FCS. One, 15, and 50
cells (as calculated by limiting dilution) of BM cells diluted
with culture medium were then seeded onto the ST2 cell
layer in each well. Cultures were maintained in ␣-MEM/
10% FCS with 10Ϫ8 M of 1␣,25(OH)2D3 and 10Ϫ7 M of
dexamethasone for 7 days followed by fixation and staining
for TRAP activity using a leukocyte acid phosphatase kit
(Sigma). A well containing TRAPϩ multinucleated cells
was judged to be osteoclast-positive.
In vitro osteoclastogenesis assay
OVX and drug-treated mice were killed by cervical dis-
location, and the tibia and femur were removed and sepa-
rated from the adhering soft tissues. The bone ends were cut
off with a scalpel, and BM cells were collected by flushing
with ␣-modified essential medium (␣-MEM) containing
10% fetal calf serum (FCS). Sorted BM cells were cultured
in ␣-MEM/10% FCS in the presence of M-CSF (5 ng/ml)
and soluble RANKL (50 ng/ml). After 4 days, TRAP solu-
tion assay was performed. In the TRAP solution assay,
enzyme activity was examined by conversion of ␣-naphthyl
phosphate to ␣-naphthol in the presence of 20 mM of
tartrate solution in each well. Absorbance was measured at
405 nm using a microplate reader (Model 550; Bio-Rad
Laboratories, Hercules, CA, USA).
Northern blot analysis
Total RNA was isolated from mice tibias using Trizol
reagent (Life Technologies, Inc., Rockville, MD, USA).

VITAMIN D INHIBITS OSTEOCLASTOGENESIS
625
FIG. 2. Active vitamin D increases bone mass in OVX mice. (A) 3D trabecular bone architecture of the fifth lumbar vertebra in OVX and in
alfacalcidol- or estrogen-treated OVX mice. OVX ddy mice (8 weeks old) were given alfacalcidol (ALF) orally (0.2 ␮g/kg BW) or E2
subcutaneously (20 ␮g/kg BW) five times a week for 1 month, and the fifth lumbar vertebra was analyzed 3D using a micro-CT system, as
described in the Materials and Methods section. Note that treatment with ALF or estrogen markedly increased trabecular bone in OVX mice. (B)
Bone morphometric parameters using micro-CT shown in panel A. BV/TV, Tb.Th, and Tb.Sp were determined as described in the Materials and
Methods section. Data are expressed as mean Ϯ SEM (n ϭ 6–8). **p Ͻ 0.01, *p Ͻ 0.05 versus the OVX-vehicle group, and ϩp Ͻ 0.05 versus
sham group, using Dunnett’s multiple test.
Ten micrograms of RNA was glyoxalated, run on agarose
gel, and transferred to GeneScreen Plus nylon membrane
(NEN Life Science Product, Boston, MA, USA). Mouse
RANKL, OPG, and mouse elongation factor 1 (EF1) com-
plementary DNA (cDNA) were amplified by polymerase
chain reaction (PCR) with oligonucleotide primers. EF1
messenger RNA (mRNA) served as an internal control in
Northern blot analysis. PCR products were subcloned into
pGEM-T (Promega, Madison, USA), and the cDNA probe
was labeled with [␣-32P]deoxycytosine triphosphate (dCTP;
NEN Life Science Product) by the random primer method
(Amersham Pharmacia Biotech, Buckinghamshire, UK).
Prehybridization and hybridization were carried out in
Express-Hyb (Clontech, Palo Alto, CA, USA), and the
filters were washed after hybridization, as recommended by
the suppliers. Then, the filters were exposed to X-ray film or
to the imaging plate of the Fujix Bio-Imaging Analyzer
BAS2500 (Fuji Photo Film Co., Tokyo, Japan).
Statistical analysis
Data were expressed as the mean Ϯ SEM. Statistical
analysis was carried out by analysis of variance (ANOVA)
using the Statistic Analysis System software (SAS Institute,
Inc., Cary, NC, USA). The significances of differences were
determined using Dunnett’s multiple test (for comparison
with OVX-vehicle group) or the Tukey-Kramer test. A
value of p Ͻ 0.05 was considered significant.
RESULTS
Active vitamin D increases bone mass through
inhibition of osteoclastic bone resorption in OVX mice
Previously, we have shown that active vitamin D is ef-
fective in preventing and treating osteoporosis caused by
estrogen deficiency by suppressing osteoclastic bone resorp-

626
SHIBATA ET AL.
tion in an OVX rat model.(14) First, we examined whether
alfacalcidol also suppresses osteoclastic bone resorption in
OVX mice. As shown in Table 1, oral treatment with
alfacalcidol at 0.05 ␮g/kg and 0.2 ␮g/kg BW decreased
urinary D-Pyr excretion, which was elevated after OVX,
and the magnitude of the effect was comparable with that of
E2. The results of histomorphometry indicate that treatment
of OVX mice with alfacalcidol or E2 decreased the number
of osteoclasts at the distal femur (Fig. 1). Serum osteocalcin
concentration, a marker of bone formation, increased after
OVX and remained elevated after treatment with alfacal-
cidol, and estrogen replacement lowered serum osteocalcin
levels significantly to within the sham range (Table 1).
These results are consistent with our concept that estrogens
depress bone turnover by inhibiting both bone resorption
and formation, whereas active vitamin D suppresses bone
resorption while maintaining bone formation.(14) At the
small doses used in this study alfacalcidol did not raise the
serum calcium concentration (Table 1).
Three-dimensional trabecular structure images obtained
by micro-CT showed that bone mass, which substantially
decreased after OVX, was markedly increased by treatment
with alfacalcidol or estrogen (Fig. 2A). Alfacalcidol or
estrogen increased BV/TV and Tb.Th to the sham levels
(Fig. 2B). Tb.Sp, markedly increased by OVX, was signif-
icantly decreased by alfacalcidol treatment (Fig. 2B). Col-
lectively, these results indicate that active vitamin D, at
pharmacologic doses that do not cause hypercalcemia, in-
hibits osteoclastic bone resorption in vivo and thereby ex-
erts therapeutic effects in estrogen-deficient osteoporosis.
FIG. 3. Treatment in vivo with active vitamin D decreases the
osteoclastogenic potential of in vitro marrow cultures. BM cells, ob-
tained from mice treated in vivo with alfacalcidol (ALF; at 0.05 ␮g/kg
and 0.2 ␮g/kg BW) or E2 (at 20 ␮g/kg BW) after OVX, were cultured
in the presence of M-CSF (5 ng/ml) and soluble RANKL (50 ng/ml),
and their osteoclastogenic potential was assessed by TRAP assay as
described in the Materials and Methods section. *p Ͻ 0.001 (compared
with OVX-vehicle group using Dunnett’s multiple test). Values repre-
sent mean Ϯ SEM (n ϭ 6–8).
active vitamin D decreases the pool of osteoclast progeni-
tors in BM in vivo.
Active vitamin D suppresses the expression of RANKL
mRNA in vivo at suprapharmacologic doses
Active vitamin D inhibits osteoclastogenesis in vivo by
depleting osteoclast precursors
The findings that vitamin D hormone suppresses oste-
oclastic bone resorption in vivo in OVX rats(14) and mice
(indicated in this study) contradict with the long-held
belief that 1␣,25(OH)2D3 induces the expression of
osteoclastogenesis-supporting RANKL(12) and is a bone-
resorbing hormone.(13) To address this discrepancy, we ex-
amined the effects of in vivo treatment with alfacalcidol on
RANKL expression in bone. As shown in Fig. 5A, Northern
blot analysis revealed that RANKL mRNA expression in
the whole femur did not change substantially after OVX or
treatment of OVX animals with pharmacologic doses of
alfacalcidol or E2. When RNA from the diaphysis and
metaphysis was analyzed separately, we did not observe any
significant alteration in RANKL mRNA level among exper-
imental groups at either site (data not shown). OPG mRNA
was undetectable in the diaphysis and abundantly expressed
in the metaphysis, again with no change between sham and
OVX or OVX and drug-treated groups (data not shown).
Interestingly, superpharmacologic or toxic doses of alfa-
calcidol that caused hypercalcemia substantially reduced the
expression of RANKL mRNA at the femur (Fig. 5B).
We then used murine marrow cultures for osteoclastogen-
esis to explore the mechanism by which vitamin D hormone
suppresses osteoclastic bone resorption. BM cells, obtained
from mice treated in vivo with alfacalcidol or estrogen after
OVX, were cultured in the presence of M-CSF and
RANKL, and their osteoclastogenic potential was assessed
by TRAP staining. As shown in Fig. 3, osteoclastogenic
activity was increased in marrow cultures from OVX mice,
and this was significantly and dose-dependently suppressed
by in vivo treatment with alfacalcidol as well as by estrogen.
Because the osteoclastogenesis-supporting compounds
M-CSF and RANKL were present in excess in these cul-
tures and, therefore, were not rate-limiting, these results
suggest that the pool of osteoclast progenitors in the marrow
of vitamin D–treated mice may have been decreased.
To substantiate this concept, frequency analysis was per-
formed. Theoretically, single marrow cells from OVX and
drug-treated mice were cultured on the stromal layer of ST2
cells in the presence of 1␣,25(OH)2D3, and the number of
osteoclast progenitors originally present in the marrow was
assessed by determining the number of wells with TRAPϩ
osteoclasts (not the number of TRAPϩ osteoclasts formed).
As shown in Fig. 4, the number of osteoclast progenitors
was increased in the BM of OVX mice and was lowered by
DISCUSSION
The previous observations that vitamin D hormone in-
in vivo treatment with alfacalcidol or estrogen, showing that duces the expression of RANKL in stromal cells,(12) thereby

VITAMIN D INHIBITS OSTEOCLASTOGENESIS
627
1␣,25(OH)2D3 is a bone-resorbing hormone.(10) Our results
indicate that active vitamin D, at pharmacologic doses that
maintain normocalcemia, does not increase RANKL ex-
pression in vivo, whereas toxic doses of active vitamin D
that cause hypercalcemia markedly reduce the expression of
RANKL. These results suggest that vitamin D hormone
causes hypercalcemia primarily by increased calcium ab-
sorption from the intestine, and hypercalcemia in turn may
reduce the expression of RANKL as a compensatory mech-
anism.
This study suggests that the main pharmacologic action of
vitamin D hormone in vivo, at least in a high bone turnover
state such as estrogen deficiency, is to suppress osteoclastic
bone resorption. Based on our previous findings that the
therapeutic effects of vitamin D hormone on bone take place
in parathyroidectomized animals,(18,19) it is suggested that at
least part of the skeletal action of active vitamin D can take
place independently of serum calcium and PTH levels,
pointing to the direct action of vitamin D on bone. Further-
more, these data provide an important clue as to how
vitamin D hormone acts in bone, not by suppressing the
osteoclastogenesis-supporting microenvironment (“soil”),
FIG. 4. Treatment with active vitamin D in vivo decreases the
number of osteoclast precursors in BM. Single marrow cells (as cal-
culated by limiting dilution) from OVX and alfacalcidol- (ALF; at 0.05
␮g/kg and 0.2 ␮g/kg BW) or E2 (at 20 ␮g/kg BW)-treated mice were represented by RANKL, but by decreasing the number of
cultured on the stromal layer of ST-2 cells in the presence of
osteoclast progenitors, that is, “seeds” of osteoclasts, in BM.
In this respect, it is interesting to note that vitamin D
1␣,25(OH)2D3 (10Ϫ8 M) and dexamethasone (10Ϫ7 M), and the num-
ber of osteoclast progenitors originally present in the marrow was
deprivation in growing pigs is associated with an enlarged
determined from the number of wells with TRAPϩ osteoclasts. *p Ͻ
pool of osteoclasts,(20) a mirror image of the situation in our
0.01 and **p Ͻ 0.001 (vs. the OVX-vehicle group) by the Tukey-
vitamin D–treated mice. It also is noted that estrogen defi-
Kramer test.
ciency is not associated with substantial up-regulation of
RANKL in bone, at least at the RNA level (Fig. 5A), but
stimulating osteoclastogenesis in vitro,(10) and that with an enlarged pool of osteoclastogenic precursor cells
1␣,25(OH)2D3 stimulates bone resorption in an organ (Fig. 4). Thus, the mechanisms by which the turnover of
culture system(13) led to the widespread belief that osteoclast progenitors is determined in BM under various
FIG. 5. The effects of active vitamin D in vivo
on RANKL mRNA expression in the bone of
OVX mice. (A) OVX ddy mice (8 weeks old) were
given alfacalcidol (ALF) orally (at 0.05 ␮g/kg and
0.2 ␮g/kg BW) or E2 subcutaneously (at 20 ␮g/kg
BW) five times a week for 2 weeks, and RANKL
mRNA expression in the whole femur was deter-
mined by Northern blot analysis. EF1␣ mRNA
was used as an internal control. The bars represent
mean Ϯ SEM of RANKL mRNA level corrected
for EF1␣ mRNA (n ϭ 3 for each group). There
was no statistical significance between the groups.
(B) OVX mice were treated with the indicated
doses (0.2, 1, 5, 10, and 20 ␮g/kg BW) of ALF for
2 weeks, and RANKL mRNA expression in the
whole femur was determined by Northern blot
analysis. The figures below the RANKL session
indicate the serum calcium concentrations of the
respective animals in milligrams per deciliter.

628
SHIBATA ET AL.
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enriched in osteoclast precursors such as c-Kitϩ, Mac-1low
,
and c-Fmsϩ cells(21,22) in the BM of drug-treated animals.
The molecular mechanism by which vitamin D hormone
decreases the pool size of hematopoietic progenitors under-
going differentiation to osteoclasts remains unknown at
present and requires further investigation. During the prep-
aration of this study, Shevde et al. reported that estrogen
inhibits M-CSF/RANKL-induced osteoclast differentiation,
probably by interfering with c-Jun activity in osteoclast
precursors.(23) In view of the tumor necrosis factor (TNF)
receptor–associated factor (TRAF), NF-␬B and c-Jun
N-terminal kinase (JNK)/activator protein 1 (AP-1) path-
ways having been shown to represent essential signaling
molecules acting downstream of RANKL binding to RANK
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dependent kinase inhibitors such as p21Cip1 and p27Kip1 (24)
,
and because p27Kip1 has been implicated as a factor deter-
mining the pool size of hematopoietic progenitors,(25) it is
possible that the vitamin D hormone regulates the pool of
osteoclast progenitors by modulating the activity of these
cell cycle regulators.
In conclusion, we have established, using an OVX mouse
model of osteoporosis and in vitro murine marrow cultures
for osteoclastogenesis, that the pharmacologic action of
active vitamin D in vivo is to decrease the pool of osteoclast
progenitors in BM, thereby inhibiting bone resorption. In
view of the unusual activity of the vitamin D hormone (it
maintains bone formation while suppressing bone resorp-
tion,(18,19) unlike estrogens, which depress both processes),
active vitamin D may provide a tool for designing useful
VDR-based drugs for the management of osteoporosis.
13. Raisz LG, Trummel CL, Holick MF, DeLuca HF 1972 1,25-
Dihydroxycholecalciferol: A potent stimulator of bone resorp-
tion in tissue culture. Science 175:768–769.
14. Shira-ishi A, Takeda S, Masaki T, Higucvhi Y, Uchiyama Y,
Kubodera N, Sato K, Ikeda K, Nakamura T, Matsumoto T,
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ulates formation in an ovariectomized rat model of osteopo-
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770–779.
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Ezawa I, Ikeda K, Ogata E 1999 The advantage of alfacalcidol
over native vitamin D in the treatment of osteoporosis. Calcif
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ACKNOWLEDGMENTS
We thank Dr. Richard Thornhill (Chugai Pharmaceutical
Co., Ltd.) for editing this article. This work was supported
in part by a grant for Comprehensive Research on Aging
and Health from the Ministry of Health and Welfare of
Japan (to K.I.).
16. Ruegsegger P, Koller B, Muller R 1996 A microtomographic
system for the nondestructive evaluation of bone architecture.
Calcif Tissue Int 58:24–29.
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Address reprint requests to:
Kyoji Ikeda, M.D.
Department of Geriatric Research
National Institute for Longevity Sciences (NILS)
36–3 Gengo, Morioka
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