Full text of DOI:10.1359/jbmr.2002.17.6.1015

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 6, 2002
©
2002 American Society for Bone and Mineral Research
Role of Inducible Nitric Oxide Synthase in Skeletal
Adaptation to Acute Increases in Mechanical Loading*
1
1
1
1
MAKOTO WATANUKI, AKINORI SAKAI, TAKESHI SAKATA, HIROSHI TSURUKAMI,
2
2
3
3
MASAO MIWA, YASUO UCHIDA, KEN WATANABE, KYOJI IKEDA, and
1
TOSHITAKA NAKAMURA
ABSTRACT
To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we
examined inducible NO synthase (iNOS) gene knockout mice in the tail-suspension model. Histomorphometric
analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and
bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes.
؉
/؉
؉/؊
Both iNOS
and iNOS
mice responded to subsequent 14-day reloading, with increases in BV/TV and
؊/؊
BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS
mice. The osteoblasts
flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS
was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after
؊
/؊
reloading were rescued in iNOS
mice by treatment with an NO donor nitroglycerine (NG). Conversely, the
؉/؉
responses in iNOS mice were inhibited by treatment with an NOS inhibitor aminoguanidine (AG). In bone
marrow cell cultures, mineralized nodules derived from iNOS
؊/؊
mice after reloading were significantly
reduced. Taken together, our results suggest that NO generated by iNOS in osteoblasts plays a critical role in
adjusting bone turnover and increasing osteogenic activity in response to the acute increase in mechanical
loading after tail suspension. (J Bone Miner Res 2002;17:1015–1025)
Key words: inducible nitric oxide synthase knockout mice, tail suspension, osteoblast, mineralized nodule
formation, nitroglycerine
INTRODUCTION
mechanisms mediating the effects of loading and unloading
on bone are unknown.
(8–10)
(11,12)
ECHANICAL STRESS is an important determinant of the
Tail-suspended rats
and mice,
simulating skel-
M
structural and functional integrity of the skeletal etal unloading for hindlimbs, provide a useful model to
(
1–4)
system.
ing space flight or bed rest causes osteopenia in weight- stress. Unloading by tail suspension results in diminished
Skeletal unloading such as weightlessness dur- investigate the regulation of bone turnover by mechanical
(
5–7)
bearing bones in humans.
However, the molecular bone formation and accelerated bone resorption in the ani-
mals, consequently leading to bone loss in the lower
(8–12)
extremities.
Furthermore, proliferation and differenti-
*
Presented in part at the American Society for Bone and Mineral
Research 22nd annual meeting, Toronto, Ontario, Canada, Septem-
ber 22–26, 2000.
ation of osteoprogenitor cells are reduced in the bone mar-
(13,14)
row,
and expression levels of messenger RNAs
All authors have no conflict of interest.
(mRNAs) for insulin-like growth factors (IGFs), transform-
1
Department of Orthopedic Surgery, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, Japan.
Product Research Laboratory, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan.
2
3
Department of Geriatric Research, National Institute for Longevity Sciences, Aichi, Japan.
1
015

1
016
WATANUKI ET AL.
ing growth factor (TGF) ␤2, osteocalcin, and osteopontin and water, and the average amounts of food taken by
(15–17)
are reduced in these animals.
Conversely, subsequent tail-suspended mice were fed also to the ground control
normal weight bearing after a period of unloading results in mice with normal loading. The experimental protocol was
increased bone formation to the level observed at base- approved by the Ethics Review Committee for Animal
(
13,18)
line.
Previous studies using the rat model showed a Experimentation of the University of Occupational and En-
rise in c-fos mRNA expression in periosteal cells within 20 vironmental Health.
minutes after normal reloading and a subsequent increase in
the expression of cyclo-oxygenase 2 mRNA after 2 h in
intracortical bone cells.
Nitric oxide (NO) is a short-lived free radical synthesized
Experimental design and group assignments
(19)
The effects of the iNOS gene on bone during unloading
from L-arginine by NO synthases (NOSs), which has three and normal reloading were examined using histomorphom-
isoforms including constitutively expressed forms of neural etry. Mice were divided into four groups based on the
NOS (nNOS) and endothelial NOS (eNOS) and an induc- experimental protocol: group 1, unloading group, subjected
ible form of NOS (iNOS). In vitro, NO is produced by to hindlimb unloading by tail suspension for 7 days and then
(
20,21)
(22)
osteoblasts
and stimulates their proliferation.
NO killed; group 2, normal-loading control group (without sus-
production in bone cells is enhanced in response to mechan- pension), killed at 7 days; group 3, unloading-reloading
(23,24)
ical stimulation such as fluid shear stress and bending.
group, subjected to unloading for 7 days followed by normal
eNOS gene knockout mice exhibit retarded bone formation reloading for 14 days and then killed on day 21; and group
(25)
during the period of rapid growth and a blunted response 4, normal-loading control group (without suspension),
(
26)
to high-dose estrogen after ovariectomy.
vitro studies provided clear evidence for NO production and genotype groups of the iNOS , iNOS , and iNOS
iNOS expression by bone cells in response to cytokine mice. Six mice from each genotype group were included in
Previous in killed on day 21. Groups 1–4, respectively, comprised three
ϩ/ϩ
ϩ/Ϫ
Ϫ/Ϫ
(
20–22)
stimulation.
in vivo has not yet been well established.
We hypothesized that iNOS in bone cells contributes to after suspension in iNOS
However, the role of iNOS in bone cells the study.
The effects of AG administration on normal reloading
ϩ/ϩ
mice and that of NG in
mice were examined also. Groups 5 and 6 each
Ϫ/Ϫ
increased bone formation in response to increased mechan- iNOS
ical loading. In this study, iNOS gene knockout mice un- consisted of five iNOS
ϩ/ϩ
mice. Group 5 mice underwent
derwent tail suspension, and bone and marrow cells in the tail suspension for 7 days followed by normal reloading for
hindlimbs were analyzed after unloading and subsequent 14 days and then were killed on day 21 of the experiment.
normal reloading. We also examined the effects of amino- During the period of normal reloading, mice were treated
ϩ/ϩ
guanidine (AG), an iNOS inhibitor, in iNOϪS/Ϫ mice and with AG (Nacalai, Kyoto, Japan) dissolved in drinking
(28)
nitroglycerine (NG), an NO donor, in iNOS
the period of normal reloading after tail suspension.
mice, during water at a concentration of 4 mg/ml.
Group 6 served as
the nonsuspended normal-loading control group, and mice
from this group were treated in a manner similar to group 5.
The average amount of AG taken by mice was 20 mg/day
MATERIALS AND METHODS
and did not differ between groups 5 and 6. Groups 7 and 8
Ϫ/Ϫ
each consisted of five iNOS
mice. Group 7 mice under-
Animals and tail suspension
went tail suspension for 7 days followed by normal reload-
tm1lau
Mice lacking the iNOS gene (B6.129P2-Nos2
), ob- ing for 14 days and then were killed. During the period of
tained from The Jackson Laboratory (Bar Harbor, ME, normal reloading, mice were treated with NG twice daily by
(
27)
USA), were bred and housed in the facility in the Center dermal application of 0.1 mg of NG with 2% NG tape
(29)
for Laboratory Animal Care at the Product Research Lab- (Nippon Kayaku, Tokyo, Japan).
Group 8 served as the
oratory (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan). normal-loading ground control group and was treated sim-
ϩ/ϩ
Mice of three genotypes, including wild-type iNOS
,
ilarly to group 7.
The impact of the iNOS gene on unloading and reloading
ϩ/Ϫ
heterozygous type iNOS
,
and homozygous type
Ϫ/Ϫ
iNOS
were delivered to the Center for Laboratory An- on bone marrow capacity for bone formation was examined
imal Care unit at the University of Occupational and Envi- by the nodule formation assay. Sixteen mice including eight
ϩ/ϩ
Ϫ/Ϫ
ronmental Health at the age of 7 weeks. Mice were allowed iNOS
mice and eight iNOS
mice underwent tail
to acclimatize for 1 week before the experiments. All mice suspension for 7 days, and then four mice of each genotype
were housed individually in cages in an air-conditioned were killed. The remaining mice were allowed normal re-
environment (temperature, 24 Ϯ 1°C; humidity, 55 Ϯ 5%) loading for 14 days and then were killed on day 21 of the
with illumination from 07:00 to 19:00. Mice were allowed experiment. For the normal-loading ground controls, eight
ϩ/ϩ
Ϫ/Ϫ
free access to water and food, a standard rodent chow mice including four iNOS
mice and four iNOS
mice
containing 1.25% calcium, 1.06% phosphorus, and 2.0 IU/g were killed at baseline, and on days 7 and 21 of the exper-
vitamin D (CE-2; Clea Japan, Inc., Tokyo, Japan) during iment, respectively. For immunohistochemistry of iNOS
3
the acclimatization period. Mice body weight ranged from and enzyme histochemistry of alkaline phosphatase (ALP),
2
0 to 24 g. The mice were divided randomly into body bone specimens were obtained from groups of mice com-
ϩ/ϩ
weight–matched groups. Tail suspension of mice was per- prising five iNOS
formed as described previously by our laboratory.
mice each killed 7 days after tail
(13)
Tail- suspension and at periods of 12 h and 1, 2, 3, 5, 7, and 14
suspended mice were allowed free access to food (CE-2) days after subsequent normal reloading. Two normal-

NOS IN SKELETAL ADAPTATION TO ACUTE INCREASES IN MECHANICAL LOADING
1017
Ϫ/Ϫ
loading iNOS
experiment.
mice were killed also on day 7 of the Histomorphometry
Right tibias were fixed with 10% buffered formalin and
embedded in methylmethacrylate (MMA) resin after Villa-
nueva’s bone staining. Serial nondecalcified 5-␮m-thick
frontal sections were obtained using a microtome (model
Anesthesia and tissue samples
For histomorphometry specimens, mice underwent bone
labeling using injections of calcein (6 mg/kg body weight)
intraperitoneally at 6 days and 2 days, respectively, before
death. At the time of death, mice were anesthetized with
ether and exsanguinated, and then both tibias were har-
vested. The left femur was used for size measurement and
genotyping. For bone marrow cell culture specimens, both
femurs and tibias were harvested from mice in which their
bones were not labeled with calcein. For immunohisto-
chemistry specimens for iNOS and enzyme histochemistry
2
050 Supercut; Reichert-Jung, Heidelberg, Germany). Left
tibias were fixed with ice-cold 5% PFA in 0.1 M of PB
containing 2% sucrose, pH 7.4. Samples were then embed-
ded in a mixture of MMA, hydroxyglycol methacrylate, and
(32)
2
-hydroxyethylacrylate.
at 4°C. The specimens were sectioned in the center to yield
-␮m frontal nondecalcified sections, which were stained
Polymerization was performed
5
for tartrate-resistant acid phosphatase (TRAP).
We measured the secondary spongiosa in the tibial spec-
(32)
for ALP, mice were anesthetized with an intraperitoneal imens as described previously. MMA sections were mea-
injection of pentobarbital (50 mg/kg body weight) and per- sured at 100-fold magnification to determine the percentage
fused via the ventricle with physiological saline and then by of trabecular bone volume to tissue volume (BV/TV, %) and
a solution of 2% paraformaldehyde (PFA) in 0.1 M of trabecular bone surface (BS, mm). Double-labeled surfaces
phosphate buffer (PB; pH 7.2), followed by isolation of to total labeled surfaces (dLS, mm) in MMA sections were
bilateral tibias.
obtained from measurement of the trabecular perimeter at
00-fold magnification and the interlabel thickness (Ir.L.Th,
m) at 200-fold magnification. Percentages of dLS to bone
1
␮
Body weight and size of femur
surface (dLS/BS, %) were obtained. The mineral apposition
rate (MAR, ␮m/day) was calculated as ␲/4 ϫ Ir.L.Th/3
Body weight of mice was measured weekly from the start
of the experiment. At the end of the experiment, femur sizes
were measured with a digital caliper (Digimatic Caliper;
Mitsutoyo, Kanagawa, Japan). The length of the femur
(␮m/day). The ratio of bone formation to bone surface
(BFR/BS, %-␮m/day) was calculated by the formula
(MAR ϫ dLS/BS). The percentage of trabecular osteoclast
represented the distance between the top of the greater surface to bone surface (Oc.S/BS, %) was obtained from
trochanter to the distal end of the lateral femoral condyle. measurement of TRAP-stained sections at 200-fold magni-
ϩ
The anteroposterior diameter was measured at the midpor- fication. TRAP cells that formed resorption lacunae at the
tion of the femur.
surface of trabeculae and contained one or more nuclei were
designated as osteoclasts.
Genotyping
Bone marrow cell cultures for nodule formation
Genotyping for the iNOS gene was performed by reverse-
transcription polymerase chain reaction (RT-PCR) amplifi-
cation of RNA isolated from the normal-loading control
mice. Total RNA was extracted from the femur using the
RNEasy kit (Qiagen, Hilden, Germany). First-strand com-
plementary DNA (cDNA) was reverse-transcribed from to-
tal RNA (3 ␮g) using Moloney murine leukemia reverse
transcriptase (SuperScript; Life Technologies, Inc., Rock-
ville, MD, USA) and oligo(dT)12–18 primer (Life Technol-
ogies, Inc.). PCR amplification was performed using gene-
specific PCR primers and Taq polymerase (EX Taq; Takara
Biomedicals Shuzo Co., Shiga, Japan). The cycling condi-
tions were set as follows: 28 cycles of denaturation at 95°C
for 1 minute, primer annealing at 55°C for 1 minute, and
extension at 72°C for 2 minutes. For amplification of iNOS,
sense primer 5Ј-ACCTACTTCCTGGACATTACGACCCC-
Bone marrow cells were obtained from both tibias and
femurs after cleaning of soft tissues. Marrow cell cultures
(32)
were prepared using the method reported previously.
Bone marrow cells were disseminated into 6-well plates
Corning, New York, NY, USA) at a concentration of 5 ϫ
(
1
6
0 cells/ml in 2 ml of ␣-modified Eagle’s medium (Flow
Laboratories, Irvine, UK). The medium was supplemented
with 15% fetal calf serum (FCS; Life Technologies,
Inc.), 2.0 g/liter of NaHCO , 100 ␮g/ml of streptomycin,
3
1
00 U/ml of penicillin, 1.25 U/ml of nystatin (Sigma
Chemical Co., St. Louis, MO, USA), 50 ␮g/ml of ascorbic
acid (Wako, Osaka, Japan), 10 mM of sodium
␤
-glycerophosphate (Sigma Chemical Co.), and 10 nM of
dexamethasone (Wako). On day 21 of culture, cells were
fixed for 24 h in a 1:1:1.5 solution of 10% formalin, meth-
anol, and water, stained for 15 minutes with a saturated
3
Ј and antisense primer 5Ј-AAGGGAGCAATGCCCG-
(30)
TACCAGGCCC-3Ј were used.
␤-actin (sense primer,
5
5
Ј-TCTTCATGGTGCTAGGAGCCA-3Ј, antisense primer, solution of alizarin red S at pH 4.0 (Sigma Chemical Co.),
Ј-CCTAAGGCCAACCGTGAAAAG-3Ј) was used as an washed with water, and air-dried. The surface area covered
(31)
internal control.
The expected sizes were 456 base pairs with dark red stain, representing mineralized nodules, was
(
bp) for iNOS and 632 bp for ␤-actin. The PCR products measured using an imaging software program (National
were electrophoresed on a 1% agarose gel containing 0.5 Institutes of Health NIH Image 1.62) on a Macintosh com-
(
33,34)
␮g/ml of ethidium bromide.
puter (Apple Computer, Inc., Cupertino, CA, USA).

1
018
WATANUKI ET AL.
Immunohistochemistry and enzyme histochemistry
Both tibias were fixed in a solution of 4% PFA in 0.1 M
of PB, decalcified with 0.2 M of EDTA in 0.1 M of PB
containing 10% sucrose at 4°C, dehydrated through a
graded series of alcohol baths and embedded in paraffin.
Five-micrometer-thick serial sections were cut on a mic-
rotome. For immunohistochemistry of iNOS, deparaffinized
and hydrated sections were briefly rinsed in phosphate-
buffered saline (PBS) at room temperature and incubated
with methanol containing 0.3% H O for 20 minutes at
2
2
room temperature to remove endogenous peroxidase activ- FIG. 1. RT-PCR expression of iNOS mRNA extracted from the
ϩ/ϩ
ϩ/Ϫ
Ϫ/Ϫ
ity. Nonspecific immunoglobulin binding was blocked by femur in iNOS , iNOS , and iNOS
incubation with PBS containing 5% normal goat serum for
mice. ␤-actin mRNA was
used as an internal control.
1
5 minutes at room temperature. Them, sections were in-
cubated with rabbit anti-iNOS polyclonal antibody (Wako)
at a dilution of 1:500 for 12 h at 4°C. After a brief rinse with
PBS, sections were incubated with the biotinylated second-
ary antibody, coupled to the labeled streptavidin-biotin
complex (LSAB Kit; Dako, Carpinteria, CA, USA). The
peroxidase complex was visualized by treatment with a
freshly prepared diaminobenzidine tetrahydrochloride (0.1
at 21 days. In addition, the mean values were not different
among mice of the AG-treatment group and the NG-
treatment group (Tables 1, 2, and 3).
Trabecular BV and local turnover of proximal tibia
during unloading and reloading
(35)
mg/ml) solution with 0.01% H O for 5 minutes.
For
2
2
ALP activity, after deparaffinization and hydration, sections
were rinsed briefly in Tris-buffered saline (pH 7.4) for 5
minutes at room temperature and stained using a bromo-
chloro indolylphosphate-Nitro blue tetrazolium (BCIP-NBT)
solution kit for ALP stain (Nacalai) for 60 minutes at room
In all three genotypes, trabecular BV (BV/TV) after tail
suspension was significantly lower than that of normal-
loading control on day 7 of the experiment (Table 1 and Fig.
2
). dLS/BS, MAR, and BFR/BS, after tail suspension, were
significantly lower, and trabecular Oc.S/BS was signifi-
cantly greater than the respective parameters of normal-
loading control.
(
35)
temperature.
Statistical analysis
On day 21 of the experiment, BV/TV, MAR, BFR/BS,
ϩ/ϩ
and Oc.S/BS of iNOS
mice, after reloading, were similar
Results were expressed as the mean Ϯ SEM. At each time
point, differences between the treatment group and the
control group in the same genotype were examined for
statistical significance using the Student’s t-test and Mann-
Whitney’s U test. Additionally, to compare the changes
between different time points in the treatment scheme,
Tukey-Kramer post hoc test after one-way analysis of vari-
ance (ANOVA) and Mann-Whitney’s U test were used. A
value of p Ͻ 0.05 denoted the presence of a statistically
significant difference.
to those of the normal-loading control (Table 2), whereas
dLS/BS was significantly higher than the control group.
ϩ/Ϫ
Furthermore, BV/TV of iNOS
mice after reloading was
significantly smaller than that of the normal-loading control.
Other parameters were not significantly different between
Ϫ/Ϫ
the two groups. In iNOS
mice, BV/TV, dLS/BS, MAR,
and BFR/BS were significantly lower and Oc.S/BS was
significantly greater than the respective parameters of the
control group.
BV/TV and BFR/BS of iNOS
ϩ/ϩ
mice significantly in-
creased on day 21 compared with day 7 (Figs. 3A and 3B).
Ϫ/Ϫ
In iNOS
mice, BV/TV and BFR/BS values on day 7 and
RESULTS
day 21 were not significantly different (Figs. 3D and 3E).
ϩ/ϩ
Compared with values at 7 days, Oc.S/BS of iNOS
mice
iNOS mRNA expression
were significantly lower on day 21 of the experiment (Fig.
Ϫ/Ϫ
Expression of iNOS mRNA was not detected in RNA 3C). However, Oc.S/BS on day 21 in iNOS
extracted from the femur of iNOS
expression level in femurs from iNOS
mediate between those of iNOS
mice was
Ϫ/Ϫ
mice. iNOS mRNA not different from that on day 7 (Fig. 3F).
ϩ/Ϫ
mice was inter-
ϩ/ϩ
Ϫ/Ϫ
and iNOS
mice
ϩ/ϩ
Ϫ/Ϫ
Effects of AG in iNOS
mice on reloading
ϩ/ϩ
mice and NG in iNOS
(Fig. 1).
In iNOS
mice treated with AG on day 21, BV/TV and
Body weight and size of femur
MAR of the unloading-reloading group were significantly
No significant differences were observed in body weight smaller, whereas Oc.S/BS was significantly larger, than the
gain or length of femur among mice of the three genotypes respective parameters of the normal-loading control group
Ϫ/Ϫ
in the unloading group and the normal-loading control (Table 3). Treatment of iNOS
mice with NG did not
group at 7 days or in those of the subsequent reloading result in any significant change between the reloading group
group after unloading and the normal-loading control group and the control group.

NOS IN SKELETAL ADAPTATION TO ACUTE INCREASES IN MECHANICAL LOADING
1019
␮
␮
␮
␮

1
020
WATANUKI ET AL.
ϩ/ϩ
TABLE 3. EFFECTS OF AG IN INOS
Ϫ/Ϫ
MICE AND THOSE OF NG IN INOS
MICE ON THE HISTOMORPHOMETRIC
PARAMETERS OF TRABECULAR BONE IN THE PROXIMAL TIBIA
ϩ/ϩ
Ϫ/Ϫ
AG effects in iNOS
Group 5
mice
NG effects in iNOS
Group 7
mice
Normal
reloading after
tail suspension
Group 6
Normal loading
control
Normal
reloading after
tail suspension
Group 8
Normal loading
control
Weight at day 0 (g)
Weight at day 21 (g)
BV/TV (%)
22.56 Ϯ 0.27
23.64 Ϯ 0.37
8.21 Ϯ 0.50**
15.29 Ϯ 1.48
1.11 Ϯ 0.05*
14.53 Ϯ 0.85
19.21 Ϯ 1.11**
22.18 Ϯ 0.19
23.84 Ϯ 0.32
12.97 Ϯ 0.87
11.77 Ϯ 1.77
1.41 Ϯ 0.08
16.46 Ϯ 2.15
13.86 Ϯ 0.71
22.36 Ϯ 0.62
24.00 Ϯ 0.85
14.76 Ϯ 0.65
21.96 Ϯ 2.57
1.72 Ϯ 0.10
37.00 Ϯ 3.04
12.67 Ϯ 1.40
22.42 Ϯ 0.49
24.88 Ϯ 0.56
15.97 Ϯ 0.77
22.64 Ϯ 1.87
1.75 Ϯ 0.03
39.52 Ϯ 3.20
13.07 Ϯ 0.91
,
†
dLS/BS (%)
,
†
MAR (␮m/day)
BFR/BS (%-␮m/day)
Oc.S/BS (%)
,
†
Data are mean Ϯ SEM values of six mice.
p Ͻ 0.05; **p Ͻ 0.01; significantly different from group 6 in the same genotype mice (Student’s t-test); p Ͻ 0.01; significantly
†
*
different from group 6 in the same genotype mice (Mann-Whitney U tests).
ϩ/ϩ
Because of the treatment with AG in iNOS
reloading were cuboidal and densely immunopositive for iNOS (Fig.
mice, BV/TV, BFR/BS, and Oc.S/BS did not significantly 5B). Immunoreactivity for iNOS was negative in specimens
Ϫ/Ϫ
differ from those of the unloading group (Figs. 3A–3C). from iNOS
mice (Fig. 5C). ALP activity was identified
These BV/TV and BFR/BS values were significantly lower, in the osteoblastic layers in all specimens (Figs. 5D and 5E).
ϩ
and the mean Oc.S/BS value was significantly higher than Thickening of the ALP layer was noted at the trabecular
the corresponding values of the AG untreated reloading and endocortical surfaces of mice killed 12 h after reloading
Ϫ/Ϫ
group. Because of the treatment with NG in iNOS
re- (Fig. 5E). ALP activities and height of cuboidal osteoblasts
loading mice, BV/TV and BFR/BS were significantly subsequently decreased to normal levels in specimens from
higher, whereas Oc.S/BS was significantly lower compared later periods after reloading.
with those of the unloading group (Figs. 3D–3F). The same
parameters also were significantly different from those of
the NG untreated reloading group.
DISCUSSION
Our study indicated that trabecular bone mass in the
Bone marrow cell cultures for nodule formation
ϩ/ϩ
proximal tibia of iNOS
mice diminished during a 7-day
ϩ/ϩ
In iNOS
mice, the bone nodule area after tail- period of tail suspension and then recovered to the initial
suspension was significantly smaller compared with base- level during subsequent 14-day normal reloading. The in-
line value and normal-loading ground controls at 7 days crease in BV induced by reloading was associated with
(
Fig. 4A). However, the corresponding values after reload- increased BFRs and reduced Oc.S. Mineralized nodule for-
ing were not significantly different from controls on day 21 mation from bone marrow cells recovered to the initial
Ϫ/Ϫ
Ϫ/Ϫ
or the baseline values. In iNOS
mice, the bone nodule levels. However, in iNOS
mice trabecular BV, reduced
area after tail suspension also was lower on day 7 relative to by tail suspension, failed to recover. Bone formation did not
the baseline (Fig. 4B). However, the area after reloading increase and neither did the mineralized nodule formation.
remained lower compared with the control on day 21 and In iNOSϩ/Ϫ mice, the parameters of bone turnover and
baseline value. When the bone nodule areas of tail- volume partially improved during reloading. Administration
ϩ/ϩ
suspended mice of the two genotypes were compared, the of AG in iNOS
mice inhibited the increase in BV, and
ϩ/ϩ
value on day 21 of iNOS
mice was significantly greater suppressed recovery of bone formation. Furthermore, der-
Ϫ/Ϫ
Ϫ/Ϫ
than that of iNOS
mice (Figs. 4C and 4D).
mal application of NG tape in iNOS
mice increased BV,
improving turnover to the level of normal-loading controls.
Unloading by tail suspension reduced bone formation and
Immunoreactivity for iNOS and ALP enzyme activity
increased bone resorption, consequently leading to de-
ϩ/ϩ
ϩ/ϩ
In iNOS
mice, immunoreactivity for iNOS was de- creased trabecular bone mass in both iNOS
tected in osteoblasts and osteocytes in the trabecular and iNOS
and
Ϫ/Ϫ
mice. The changes in bone turnover and mineral-
endocortical surfaces in all specimens throughout the ex- ized nodule formation from bone marrow cells in these
perimental period. The osteoblasts were apparently flattened C57BL/6J mice were compatible with previous observa-
(13)
Ϫ/Ϫ
and their immunoreactivity for iNOS was positive in spec- tions in ddy mice.
However, in iNOS
mice, the re-
imens harvested on day 7 after tail suspension (Fig. 5A). sponse of bone and bone marrow cells to subsequent re-
Twelve hours after subsequent reloading, the osteoblasts loading was blunted, resulting in persistently reduced bone

NOS IN SKELETAL ADAPTATION TO ACUTE INCREASES IN MECHANICAL LOADING
1021
ϩ/ϩ
ϩ/Ϫ
Ϫ/Ϫ
FIG. 2. Trabecular bone in the proximal tibia of iNOS , iNOS , and iNOS
magnification ϫ10).
mice. Undecalcified sections under dark-field illumination
(
formation and increased bone resorption. The responses of crease in ALP activity of osteoblasts after 12 h of reloading
ϩ/ϩ
bone formation and resorption to reloading were blunted in iNOS
also in iNOS
mice. Immunoreactivity to iNOS was densely
ϩ/ϩ
mice that were treated with AG, which positive in osteoblasts and osteocytes. Although the reduc-
exerts inhibitory effects on all NOS isoforms and other tions in Oc.S were similar in iNOS
L-arginine–dependent metabolic pathways distinct from trabecular bone formation and BV in iNOS
ϩ/ϩ
ϩ/Ϫ
and iNOS
mice,
ϩ/Ϫ
mice was at
(
36)
Ϫ/Ϫ
ϩ/ϩ
NOS.
In iNOS
mice, trabecular bone formation ap- an intermediate level between that observed in iNOS
Ϫ/Ϫ
parently recovered after administration of NG, a donor of and iNOS
mice. These results suggest that the degree of
increase in bone formation depends on the amount of NO
(37)
NO through a metabolic pathway independent of NOS.
These data indicate that NO generated through iNOS played generated in bone cells by acute overloading, such as me-
a critical role in recovery of bone turnover and bone mass chanical compression and reloading after bone mass was
during the period of reloading after tail suspension.
The role of NO in stimulating bone formation induced by
decreased by tail suspension.
Recently, we have shown that the numbers of adherent
Chow et stromal cells and colony-forming unit fibroblastic (CFU-f)
(37,38)
mechanical loading has been observed in rats.
(
37)
al.
showed that acute compression of the coccygeal ver- colonies were similar in tail-suspended mice and control
(13)
tebral body increased the mineralized surface and trabecular mice during 7-day tail suspension.
In contrast, reloading
bone formation; both parameters further increased by for 14 days increased total bone marrow cells, adherent
administration of an NO donor, S-nitroso-N-acetyl-D, stromal cells, and mineralized nodule formation in tibias.
L-penicillamine, or S-nitroso-glutathione. In this rat model, These findings indicated that the effect of unloading on
the increase in trabecular bone formation was suppressed by osteogenic cells in bone marrow apparently differed from
G
pretreatment with L-N -monomethyl-arginine, an NO inhib- that of subsequent reloading. This study suggested that
itor, 15 minutes before application of the compressive force iNOS had no impact on reduction of bone formation or
(
38)
to the bone.
In this study, we confirmed the rapid in- increased bone resorption during tail suspension. Because

1
022
WATANUKI ET AL.
FIG. 3. Changes in iNOS genotypes and ef-
ϩ/ϩ
fects of AG in iNOS
iNOS
mice and NG in
Ϫ/Ϫ
mice on histomorphometric parame-
ters of trabecular bone in the proximal tibia
during normal reloading after tail suspension. (A
and D) BV/TV, (B and E) BFR/BS, and (C and
F) Oc.S/BS. Data are presented as mean Ϯ SEM.
*
p Ͻ 0.01, compared with day 7 unloading mice
in same genotype by Tukey-Kramer post hoc test
†
after one-way ANOVA and p Ͻ 0.01 by Mann-
‡
§
Whitney U test, respectively. p Ͻ 0.05; p Ͻ
.01, between AG or NG-treated and untreated
reloading mice by Tukey-Kramer post hoc test
0
͉
͉
¶
after one-way ANOVA and p Ͻ 0.05 and p Ͻ
0
.01 by Mann-Whitney U test.
ϩ/ϩ
Ϫ/Ϫ
FIG. 4. Changes in bone nodule area in bone marrow cultures in (A) iNOS
and (B) iNOS
mice during unloading and subsequent
ϩ/ϩ
Ϫ/Ϫ
reloading experiments. The values of bone nodules were compared between unloading-reloading iNOS
presented as mean Ϯ SEM (n ϭ 4). *p Ͻ 0.01 and p Ͻ 0.05 compared with the controls of the same genotype at baseline by Tukey-Kramer post
hoc test after one-way ANOVA and Mann-Whitney U test, respectively. p Ͻ 0.05 and p Ͻ 0.01 compared with the controls in the same genotype
during the same experimental period by Student’s t-test, and p Ͻ 0.05 by Mann-Whitney U test. p Ͻ 0.01 and p Ͻ 0.05 between
and iNOS
mice (C). Data are
†
‡
§
͉
͉
¶
#
ϩ/ϩ
Ϫ/Ϫ
unloading-reloading iNOS
represent mineralized nodules.
mice and iNOS
mice by Student’s t-test and Mann-Whitney U test, respectively. (D) Dark red–stained areas
the development of osteoclasts is regulated by osteogenic
cells, it is reasonable to suggest that the increase in bone calization of eNOS was detected in flat bone lining cells,
resorption during unloading and its reduction during subse- osteocytes,
Previous studies have indicated that although immunolo-
(
39)
(40)
(41)
and cuboidal osteoblasts,
iNOS expres-
quent reloading are both secondary to the effects on the sion was not detected in any bone cells of osteoblastic
(40,41)
development of osteoblasts.
lineage in normal adult rats.
We have detected iNOS

NOS IN SKELETAL ADAPTATION TO ACUTE INCREASES IN MECHANICAL LOADING
1023
FIG. 5. Immunoreactivity for iNOS and en-
zyme activity of ALP in the proximal tibia dur-
ing reloading after tail suspension. Adjacent sec-
tions (magnification ϫ65) obtained after (A and
D) 7 days of unloading and (B and E) 12 h after
subsequent reloading were (A and B) immuno-
stained with an anti-iNOS polyclonal antibody
(
1:500 dilution) and (D and E) stained with ALP.
iNOS was localized in osteocytes and osteo-
blasts. (C) Immunoreactivity for iNOS was neg-
Ϫ/Ϫ
ative in specimens from iNOS
mice. ALP
was localized in the endosteum and osteoblasts.
Note that endosteal osteoblasts exhibited cuboi-
dal shapes after reloading. Specific regions are
indicated as follows: Eo, endosteum; Cb, cortical
bone; Ob, osteoblast; Oc, osteocyte.
Ϫ/Ϫ
protein in osteoblasts and osteocytes during the unloading- Because of normal bone growth in iNOS
mice, we
reloading experimental period. In our study, it should be expect that this suppressed bone formation will recover
noted that iNOS expression was observed in the limited gradually if allowed to reload for a longer period.
conditions of unloading and reloading but not in the normal-
In conclusion, in this study we showed that unloading by
loading state. Some studies have observed iNOS expression tail suspension reduced bone formation and increased bone
(
40)
in bone cells by cytokine stimulation,
with experimental resorption, consequently leading to decreased trabecular
(
41)
(42)
ϩ/ϩ
Ϫ/Ϫ
colitis and in neonates. Furthermore, bone turnover in bone mass in both iNOS
and iNOS
mice the response of bone and bone marrow
mice exhibit marked reduction in trabecular cells to subsequent reloading was blunted, resulting in per-
mice. However,
Ϫ/Ϫ
Ϫ/Ϫ
Ϫ/Ϫ
iNOS
mice is apparently different from that in eNOS
in iNOS
Ϫ/Ϫ
mice. eNOS
(
26)
BV in the distal femur until the age of 20 weeks,
com- sistent reduction in bone formation and increased bone
ϩ/ϩ
(25)
Ϫ/Ϫ
pared with values in eNOS
mice.
Trabecular BFRs resorption. Administration of an NO donor to iNOS
ϩ/ϩ
and mineralized surfaces measured by fluorescence labeling mice and of an NO inhibitor to iNOS
mice simulated the
(
25)
are retarded until the age of 8 weeks. Mineralized nodule changes in bone mass and turnover during reloading. iNOS
formation from bone marrow cells of the hindlimbs was plays a critical role in adjusting bone turnover in response to
Ϫ/Ϫ
reduced also. However, in this study in iNOS
mice, the an acute increase in mechanical loading.
normal-loading controls at the ages of 9–11 weeks did not
exhibit any reduction in trabecular BV or bone formation. In
addition, mineralized nodule formation at 8 weeks of age
was not reduced. These may be speculated that although
eNOS contributes to the physiological control of bone turn-
ACKNOWLEDGMENTS
This work was supported in part by grants from the
(25)
over such as bone growth and the bone-conserving action Ministry of Health and Welfare of Japan (Comprehensive
(
26)
of estrogen,
iNOS activity plays a critical role in adjust- Research on Aging and Health to K.I. and T.N.) and the
ing bone turnover to acute increases in mechanical loading. Ministry of Education, Science, Sports, and Culture of Ja-

1
024
WATANUKI ET AL.
pan (Scientific Research B 11470315 to A.S. and Scientific
Research B 12470313 and Scientific Research on Priority
Areas B 12137210 to T.N.).
bearing after a period of skeletal unloading. Am J Physiol
2
57:E606–E610.
1
2
2
9. Matsumoto T, Nakayama K, Kodama Y, Fuse H, Nakamura T,
Fukumoto S 1998 Effect of mechanical unloading and reload-
ing on periosteal bone formation and gene expression in tail-
suspended rapidly growing rats. Bone 22:89S–93S.
0. Ralston SH, Todd D, Helfrich M, Menjamin N, Grabowski PS
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Address reprint requests to:
Makoto Watanuki, M.D.
Department of Orthopedic Surgery
University of Occupational and Environmental Health
1
–1 Iseigaoka, Yahatanishi-ku
Kitakyushu 807-8555, Japan
1
997 Osteoprotegerin: A novel secreted protein involved in the Received in original form August 13, 2001; in revised form De-
regulation of bone density. Cell 89:309–319.
cember 19, 2001; accepted January 28, 2002.