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ARTHRITIS & RHEUMATISM
Vol. 63, No. 12, December 2011, pp 3908–3917
DOI 10.1002/art.30646
© 2011, American College of Rheumatology
Distinguishing the Proapoptotic and Antiresorptive Functions of
Risedronate in Murine Osteoclasts
Role of the Akt Pathway and the ERK/Bim Axis
Takumi Matsumoto, Yuichi Nagase, Mitsuyasu Iwasawa, Tetsuro Yasui, Hironari Masuda,
Yuho Kadono, Kozo Nakamura, and Sakae Tanaka
Objective. Nitrogen-containing bisphosphonates
are one of the most successful therapeutics for osteoporosis. The aim of this study was to elucidate the
functional mechanism of one of the typical nitrogencontaining bisphosphonates, risedronate.
Methods. Osteoclasts generated from murine
bone marrow macrophages were treated with risedronate in vitro, and its effects on apoptosis and boneresorbing activity were examined. The mechanism of
action of risedronate was examined by gene induction of
constitutively active Akt-1 and constitutively active
MEK-1, and by gene deletion of Bim. Bimⴚ/ⴚ mice, in
which osteoclasts were resistant to apoptosis, were
treated with risedronate and analyzed radiographically,
biochemically, and histologically.
Results. Risedronate induced osteoclast apoptosis
through the mitochondria-dependent pathway with an
increased expression of Bim, and the proapoptotic effect
of risedronate was suppressed by Bim deletion and
constitutively active MEK-1 introduction. In contrast,
the risedronate-induced suppression of bone resorption
was completely reversed by inducing constitutively active Akt-1, but not by Bim deletion or constitutively
active MEK-1 introduction. These results suggested
that apoptosis and bone-resorbing activity of osteoclasts
were regulated through the ERK/Bim axis and the Akt
pathway, respectively, both of which were suppressed by
risedronate. Although osteoclast apoptosis in response
to risedronate administration was suppressed in the
Bimⴚ/ⴚ mice, risedronate treatment increased bone
mineral density in Bimⴚ/ⴚ mice at a level equivalent to
that in wild-type mice.
Conclusion. Our findings indicate that the antiresorptive effect of risedronate in vivo is mainly mediated by the suppression of the bone-resorbing activity of
osteoclasts and not by the induction of osteoclast apoptosis.
Bisphosphonates, stable analogs of pyrophosphate, strongly inhibit bone resorption and have been
used to treat various diseases driven by increased bone
resorption, such as postmenopausal osteoporosis, Paget’s disease, and tumor bone metastases (1). Although
bisphosphonates are poorly absorbed from the intestine,
they are quickly deposited on the bone surface once
absorbed (2). The acidic environment produced by
osteoclasts reduces the ability of bisphosphonates to
chelate Ca2⫹ and releases bisphosphonates from the
bone surface, and bisphosphonates are then ingested
into osteoclasts by endocytosis (3,4). It was speculated
that the concentration of bisphosphonates reaches as
high as 0.1–1 mM in the resorption lacuna (3).
Bisphosphonates are divided into 2 groups according to the structure of the side chains, a nitrogen-
Supported by grants-in-aid from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan, by Health Science
research grants (21591932 to Dr. Kadono and 20249024 and 21659350
to Dr. Tanaka) from the Ministry of Health, Labor, and Welfare of
Japan, and in part by funding for the Global COE Program, Medical
System Innovation on Multidisciplinary Integration, from the Ministry
of Education, Culture, Sports, Science and Technology of Japan.
Takumi Matsumoto, MD, Yuichi Nagase, MD, PhD, Mitsuyasu Iwasawa, MD, PhD, Tetsuro Yasui, MD, PhD, Hironari
Masuda, MD, Yuho Kadono, MD, PhD, Kozo Nakamura, MD, PhD,
Sakae Tanaka, MD, PhD: University of Tokyo, Tokyo, Japan.
Drs. Nakamura and Tanaka have received consulting fees,
speaking fees, and/or honoraria from Eisai Pharmaceuticals (less than
$10,000).
Address correspondence to Sakae Tanaka, MD, PhD, Department of Orthopaedic Surgery, Faculty of Medicine, University of
Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail:
[email protected]
Submitted for publication January 16, 2011; accepted in
revised form August 18, 2011.
3908
DISTINGUISHING THE FUNCTIONS OF RISEDRONATE
containing type and a non–nitrogen-containing type.
The difference in the structures results in different
bisphosphonate mechanisms of action for antiresorbing
activity (1). Non–nitrogen-containing bisphosphonates
are reported to act through the intracellular accumulation of nonhydrolyzable ATP analogs that exert cytotoxic effects on osteoclasts (5), while nitrogencontaining bisphosphonates inhibit the mevalonate
pathway and prevent the posttranslational prenylation of
small GTP-binding proteins such as Ras, Rho, Rac, and
Cdc42 (6). Previous studies have demonstrated that
small G proteins control the differentiation, apoptosis,
cytoskeletal organization, and vesicular trafficking of
osteoclasts (7–10). Therefore, the antiresorptive properties of nitrogen-containing bisphosphonates can be exhibited by inducing osteoclast apoptosis (11,12), altering
the osteoclast cytoskeleton (3,13), or affecting osteoclast
vesicular and membrane trafficking (14). Recently, it
was reported that nitrogen-containing bisphosphonates
suppress the bone-resorbing activity of osteoclasts independently of apoptosis (15). In addition, Weinstein et al
demonstrated that bone biopsy specimens from patients
with long-term alendronate administration exhibited an
increased osteoclast number (OcN) in proportion to the
accumulated amount of alendronate (16). These observations have led to the notion that the antiresorptive
function of nitrogen-containing bisphosphonates is regulated through a signaling pathway different from that of
their proapoptotic function.
The present study aimed to distinguish the molecular mechanisms regulating the proapoptotic function
and antiresorptive function of nitrogen-containing bisphosphonates. We previously reported that the proapoptotic Bcl-2 homology 3 (BH3)–only domain protein
Bim plays a crucial role in the osteoclast apoptosis
induced by cytokine deprivation (17). In the present
study, we showed that risedronate-induced osteoclast
apoptosis was markedly suppressed in Bim⫺/⫺ mice,
while risedronate increased bone volume (BV) in the
mice to a level similar to that in wild-type (WT) mice.
We also demonstrated that the cell viability and boneresorbing function of osteoclasts were regulated by the
ERK/Bim pathway and the Akt pathway, respectively.
MATERIALS AND METHODS
Animals. Newborn and 8-week-old male ddY and
C57BL/6J mice were purchased from Sankyo Labo Service Co.
Bim⫺/⫺ mice (on a C57BL/6 genetic background) were kindly
provided by Dr. Philippe Bouillet and Dr. Andreas Strasser
(Walter and Elisa Hall Institute, Melbourne, Victoria, Australia). The breeding and genotyping of Bim⫺/⫺ mice were
3909
performed as previously described (18). All animals were
housed under specific pathogen–free conditions and treated
with humane care under the approval of the Animal Care and
Use Committee of the University of Tokyo.
Generation of osteoclasts and survival/bone resorption
assay. Osteoclasts were generated using a coculture system
previously described (7). Briefly, when murine osteoblastic
cells and bone marrow cells were cultured on collagen gel–
coated dishes in the presence of 10 nM 1␣,25-dihydroxyvitamin
D3 (1␣,25[OH]2D3) and 1 ␮M prostaglandin E2 (PGE2), osteoclasts were differentiated on day 6 of culture. The cells were
dispersed by treatment with 0.1% bacterial collagenase (Wako
Pure Chemical) for 10 minutes and then used for the pit
formation assay and survival assay as follows.
For the bone resorption assay, the cells were resuspended in ␣-minimum essential medium (␣-MEM) containing
10% fetal bovine serum (FBS), replated on dentin slices, and
cultured for 24 hours with or without the indicated concentrations of risedronate. After cells were removed by treating the
dentin slices with 1M NH4OH, the resorption areas were
visualized by staining with 1% toluidine blue. The resorption
pit area was quantified using an image analyzing system
(Microanalyzer; Japan Poladigital). For the survival assay, the
resuspended cells were placed on culture dishes. After 5 hours
of incubation, the cultures were treated with ␣-MEM containing 0.1% collagenase and 0.2% Dispase for 10 minutes to
remove osteoblastic cells and purify osteoclasts, and the remaining osteoclasts were further cultured in ␣-MEM containing 10% FBS with or without the indicated concentrations of
risedronate. For the survival assay using caspase inhibitors, the
osteoclasts purified in the way described above were cultured
in the presence or absence of 30 ␮M risedronate with DMSO,
30 ␮M caspase 8 inhibitor (Z-IETD-FMK), or 30 ␮M caspase
9 inhibitor (Z-LEHD-FMK). Cell viability/survival rate was
expressed as the percentage of morphologically intact tartrateresistant acid phosphatase (TRAP)–positive multinucleated
cells. TRAP staining was performed at pH 5.0 in the presence
of L(⫹)-tartaric acid using naphthol-AS-MX phosphate in
N,N-dimethylformamide as the substrate. The number of
TRAP-positive osteoclasts remaining at the different time
points is shown as a percentage of the cells at time zero.
Sealing zone formation on dentin slices was analyzed
as follows. The cells were cultured on dentin slices as described
above. After 16 hours, the cells were fixed in phosphate
buffered saline (PBS) containing 4% paraformaldehyde for 10
minutes and then stained by TRAP. Cells were then incubated
for 30 minutes with rhodamine-conjugated phalloidin solution
(Molecular Probes) to visualize F-actin. Sealing zone formation on dentin slices was observed using fluorescence microscopy (Biozero; Keyence). The sealing zone formation rate is
represented as the proportion of the cells with an uninterrupted ring-like F-actin structure among the TRAP-positive
multinucleated cells.
Western blotting and enzyme-linked immunosorbent
assay (ELISA). All extraction procedures were performed at
4oC or on ice. Cells were washed with ice-cold PBS, and
proteins were extracted with Tris–NaCl–EDTA buffer (1%
Nonidet P40, 10 mM Tris HCl [pH 7.8], 150 mM NaCl, 1 mM
EDTA, 2 mM Na3VO4, 10 mM NaF, and 10 ␮g/ml aprotinin).
The lysates were clarified by centrifugation at 12,000g for 10
minutes. For Western blotting analysis, lysates were subjected
3910
to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis with a 7.5–15% Tris glycine gradient gel or 15% Tris
glycine gel and transferred onto nitrocellulose membranes
(Bio-Rad). After blocking with 6% milk/Tris buffered saline–
Tween, membranes were incubated with primary antibodies to
cleaved caspase 3, caspase 8, cleaved caspase 9, Bcl-xL, Bcl-2,
phospho-Akt at Ser473, Akt, phospho-ERK, ERK (all from Cell
Signaling Technology), Bim (Santa Cruz Biotechnology), cytochrome c oxidase 4, cytochrome c (both from BD Biosciences),
or ␤ -actin (Sigma-Aldrich), followed by horseradish
peroxidase–conjugated goat anti-mouse IgG and goat antirabbit IgG (both from Promega).
Immunoreactive bands were visualized with ECL Plus
(Amersham) according to the manufacturer’s instructions. The
blots were stripped by incubation for 20 minutes in stripping
buffer (2% SDS, 100 mM 2-mercaptoethanol, and 62.5 mM
Tris HCl [pH 6.7]) at 50°C and reprobed with additional
antibodies. Separation of the mitochondrial fraction and cytosolic fraction was performed using an ApoAlert cell fractionation kit (Clontech) according to the manufacturer’s instructions. Phospho–Akt-1 and phospho–p44/42 MAPK were also
quantified using a PathScan Phospho–Akt-1 (Ser473) Sandwich
ELISA kit and PathScan Phospho–p44/42 MAPK (Thr202/
Tyr204) Sandwich ELISA kit (Cell Signaling Technology) according to the manufacturer’s instructions.
Real-time polymerase chain reaction (PCR). Messenger RNA (mRNA) was isolated from osteoclasts using
Isogen (Wako Pure Chemical), and an aliquot (1 ␮g) was
reverse-transcribed using a QuantiTect Reverse Transcription
kit (Qiagen) to make single-stranded complementary DNA.
PCR was performed using a ABI Prism 7000 Sequence Detection System (Applied Biosystems) using QuantiTect SYBR
Green PCR Master Mix (Qiagen) according to the manufacturer’s protocol. All reactions were run in triplicate. After data
collection, the relative mRNA copy number of a specific gene
was calculated with a standard curve generated with serially
diluted plasmids containing PCR amplicon sequences, and
normalized to rodent total RNA with mouse ␤-actin serving
as the internal control. Standard plasmids were synthesized
with a TOPO TA cloning kit (Invitrogen) according to the
manufacturer’s instructions. The primers we used to detect
the common form of all Bim splice variants were 5⬘CTTCCATACGACAGTCTC-3⬘ and 5⬘-AACCATTTGAGGGTGGTCTTC-3⬘.
Expression constructs and gene transduction. The
adenovirus vectors used in the experiments, and the genes
carried by the vectors, were as follows: AxGFP (green fluorescence protein [GFP] gene), AxMekCA (constitutively active
MEK-1 gene), and AxAktCA (constitutively active Akt-1 gene).
AxGFP was used as a control vector. AxMekCA and AxAktCA
were provided by Dr. Hideki Katagiri (Tohoku University).
Constitutively active MEK-1 was generated by an exchange of
the 2 Raf-1 phosphorylation sites on Ser218 and Ser222 for Glu218
and Glu222, and constitutively active Akt-1 was generated by
adding a myristoylation signal sequence to its N-terminus.
Viral titers were determined by the end point dilution assay,
and the viruses were used at 50 multiplicities of infection
(MOI). Infection of osteoclasts by adenovirus vectors was
carried out following the method previously described (19).
Briefly, on day 4 of culture, when osteoclasts began to appear,
mouse cocultures were incubated for 1 hour at 37°C with a
small amount of ␣-MEM containing the recombinant adeno-
MATSUMOTO ET AL
viruses at the desired MOI. Cells were then washed twice with
PBS and further incubated at 37°C in ␣-MEM containing 10%
FBS, 10 nM 1␣,25(OH)2D3, and 1 ␮M PGE2. Experiments
were performed 36 hours after the infection. Retrovirus packaging was performed by transfection of the pMx vectors into
BOSC cells. Retrovirus construction of Bcl-2 and Bcl-xL and
infection of the osteoclast precursors were carried out as
previously described (20).
In vivo risedronate treatment. For in vivo analysis,
normal saline or 0.01 mg risedronate/kg body weight was
subcutaneously injected daily for 14 days into 14-week-old
Bim⫺/⫺ and 14-week-old WT mice (3 animals/group). The day
after the final injection, blood samples were collected retroorbitally under anesthesia from Bim⫺/⫺ mice and WT mice with
or without risedronate treatment immediately prior to killing.
Sera were obtained using a capillary blood collection tube
with serum separator (Becton Dickinson). The serum concentration of C-terminal crosslinking telopeptide of type I collagen (CTX-I) was measured by RatLaps ELISA (Nordic Bioscience). Plain radiographs were obtained using a soft x-ray
apparatus (CMB-2; Softex), and the bone mineral density
(BMD) was measured by dual-energy x-ray absorptiometry
using a bone mineral analyzer (PIXImus Densitometer; GE
Medical Systems).
Histologic analyses. Tissues were fixed in 4%
paraformaldehyde/PBS, decalcified in 10% EDTA for 2 weeks
at 4oC, embedded in paraffin, and cut into 3 ␮m–thick sections.
Hematoxylin and eosin staining was performed according to
the standard procedure. Histomorphometric analysis was performed in the primary and secondary spongiosa of the proximal tibia beginning from the lowest point of the growth plate
to a point 1.0 mm distally. Osteoclasts were identified by
TRAP staining. The total number of osteoclasts was expressed
as the number per millimeter at the cancellous bone perimeter
(BPm). Giant osteoclasts were defined as cells that had more
than 8 nuclear profiles (2-dimensional images of a section) and
that were detached from bone. Cells undergoing apoptosis
were identified by means of the TUNEL method, which
specifically labels the 3⬘-hydroxyl terminal DNA strand breaks.
For the TUNEL procedure, all agents, including buffers, were
part of a kit (In Situ Cell Death Detection kit, peroxidase;
Roche Applied Science); the staining procedure was carried
out according to the manufacturer’s recommendation. Apoptotic cells were defined as being TUNEL positive and having
apoptotic morphologic features of chromatin condensation,
nuclear fragmentation, and cytoplasmic contraction or fragmentation.
Statistical analysis. The in vitro experiments were
repeated at least 3 times, and the representative data from 1
experiment were used for statistical analysis. Results are
expressed as the mean ⫾ SD of data obtained from 6 independent culture dishes in 1 typical experiment. In the in vivo
experiments, the data obtained from 3 different animals were
used. Statistical analyses were performed using Student’s unpaired 2-tailed t-test or analysis of variance.
RESULTS
Risedronate induces osteoclast apoptosis through
the mitochondrial pathway. When the osteoclasts
generated in vitro were treated with risedronate, the
DISTINGUISHING THE FUNCTIONS OF RISEDRONATE
Figure 1. A, Dose-dependent reduction by risedronate (RIS) of the
number of tartrate-resistant acid phosphatase (TRAP)–positive osteoclasts (OCs), shown as a percentage of the cells at time zero. ⴱ ⫽ P ⬍
0.01 versus untreated cells (control). B, Risedronate-induced expression of cleaved caspase 3, caspase 8, and cleaved caspase 9 in
osteoclasts. Positive control (PC) cell lysates for cleaved caspase 8
immunoblotting (IB) were obtained from spleen cells stimulated with
400 ng/ml FasL for 16 hours. C, Risedronate-induced release of
cytochrome c from the mitochondrial fraction (M) into the cytosolic
fraction (C) in osteoclasts. Cytochrome c oxidase 4 (Cox4) is a marker
of the mitochondrial fraction. D, Effects of caspase 8 inhibitor
(Z-IETD-FMK) and caspase 9 inhibitor (Z-LEHD-FMK). Top, TRAP
staining of representative cultures. Bars ⫽ 100 ␮m. Bottom, Number
of TRAP-positive osteoclasts shown as a percentage of the cells at time
zero. ⴱ ⫽ P ⬍ 0.01 versus cultures without risedronate treatment. E,
Effects of retroviral overexpression of Bcl-2 (RxBcl-2) and Bcl-xL
(RxBcl-xL). Top, Overexpression of Bcl-2 and Bcl-xL confirmed by
Western blotting. Bottom, Number of TRAP-positive osteoclasts
shown as a percentage of the cells at time zero. ⴱ ⫽ P ⬍ 0.01 versus
cultures without risedronate treatment. In A, D, and E, experiments
were repeated at least 3 times, and results are expressed as the mean ⫾
SD of data obtained from 6 independent culture dishes in 1 typical
experiment.
number of osteoclasts was reduced in a dose-dependent
manner, and risedronate at concentrations of ⱖ3 ␮M
induced a significant reduction in the OcN after 24 hours
of treatment (Figure 1A). Osteoclasts treated with risedronate exhibited condensation and segmentation of the
nuclei by Hoechst 33342 fluorescence microscopic ana-
3911
lysis, morphologic features which are reminiscent of
apoptosis (Figure 2). The 17-kd and 19-kd forms of
cleaved caspase 3, active fragments generated from
procaspase 3, were increased after 16 hours of risedronate treatment, as determined by Western blot analysis
(Figure 1B), further confirming that risedronate induces
apoptosis in osteoclasts. Interestingly, cleaved caspase 3
was observed immediately after purification (time zero)
and decreased 8 hours after purification in control
osteoclasts. We think that this is because the purification
maneuver using collagenase and Dispase may somehow
be harmful and temporarily stimulate caspase 3.
There are 2 distinct apoptosis pathways in mammals. One is the death receptor pathway, which is
initiated by death receptors, such as the tumor necrosis
factor receptor, which contain the intracellular death
domain. Upon ligand binding, intracellular signaling is
propagated through FADD adaptor protein–mediated
activation of caspase 8. The other is the mitochondrial
pathway, which is regulated by the pro- and antiapoptotic Bcl-2 family members and induces the release of
cytochrome c from mitochondria, leading to Apaf-1
adaptor–mediated activation of caspase 9. After 16
hours of risedronate treatment, cytochrome c was released into the cytosolic fraction more abundantly in
osteoclasts receiving risedronate treatment than in control osteoclasts (Figure 1C). The cleaved form of caspase
9 was clearly increased in osteoclasts, while the 18-kd
active fragment of caspase 8 was hardly observed (Figure
1B). The risedronate-induced reduction of the number
of osteoclasts was fully reversed by a caspase 9 inhibitor
(Z-LEHD-FMK) but only partially by a caspase 8 inhibitor (Z-IETD-FMK) (Figure 1D). The reduced viability
of osteoclasts receiving risedronate treatment was completely recovered by retrovirus vector–mediated overex-
Figure 2. Risedronate-induced nuclear condensation and segmentation in osteoclasts. Osteoclasts generated in vitro were left untreated
or were treated with 3 ␮M risedronate for 24 hours, and nuclear
condensation and segmentation were examined by Hoechst 33342
fluorescence microscopic analysis. Bars ⫽ 20 ␮m. See Figure 1 for
definitions. Color figure can be viewed in the online issue, which is
available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)
1529-0131.
3912
pression of the antiapoptotic Bcl-2 family members
Bcl-2 or Bcl-xL (Figure 1E). These results indicate that
risedronate mainly induces osteoclast apoptosis through
the mitochondria-dependent pathway.
Resistance of Bimⴚ/ⴚ mouse osteoclasts to
risedronate-induced apoptosis. We then examined the
effect of risedronate on the expression levels of pro- and
antiapoptotic Bcl-2 family members. Immunoblot analysis revealed an increase in Bim expression within 8 hours
of risedronate addition, while the expression levels of
the antiapoptotic Bcl-2 family members, Bcl-2 and BclxL, remained unchanged (Figure 3A). Among the 3
isoforms of Bim generated by alternative splicing, BimEL
was mainly induced by risedronate treatment in osteoclasts. BimL was also up-regulated in response to risedronate treatment (Figure 3A). The expression of other
proapoptotic family members, such as Bax or Bad, was
also not altered by risedronate treatment (Figure 3A).
No significant difference in the Bim mRNA level was
observed by real-time PCR between osteoclasts cultured
with and those cultured without risedronate, suggesting
that the changes in the Bim protein levels are due to
posttranslational mechanisms (Figure 3B).
We then examined the effect of risedronate on
the osteoclasts generated from Bim⫺/⫺ mouse bone
marrow cells. Risedronate markedly reduced the number of WT mouse osteoclasts, and ⬍20% of the cells
remained after 24 hours of treatment, while risedronate
had no significant effect on Bim⫺/⫺ mouse osteoclasts
(P ⫽ 0.329) (Figure 3C). We previously reported that
Bim⫺/⫺ mouse osteoclasts exhibit less bone-resorbing
activity than WT mouse osteoclasts. Interestingly, in
spite of the prolonged survival of Bim⫺/⫺ mouse osteoclasts compared to WT mouse osteoclasts, pit formation was similarly and almost completely inhibited by
risedronate in both WT mouse osteoclasts and Bim⫺/⫺
mouse osteoclasts (Figure 3D). The mean ⫾ SD reduction rates were 85.3 ⫾ 5.2% in WT mouse osteoclasts
and 90.2 ⫾ 5.1% in Bim⫺/⫺ mouse osteoclasts, with no
significant difference (P ⫽ 0.15).
Suppression of osteoclast apoptosis in Bimⴚ/ⴚ
mice does not alter the antiresorptive effect of risedronate in vivo. To examine the impact of Bim deficiency in
risedronate-induced osteoclast apoptosis in vivo, risedronate (0.01 mg/kg body weight) was administered to
the Bim⫺/⫺ mice and WT mice once a day for 14 days.
We previously reported that Bim⫺/⫺ mice have mild
osteosclerosis due to the decreased bone-resorbing activity of osteoclasts, although the number of osteoclasts
increased in Bim⫺/⫺ mice (17). Radiographic analysis
showed that risedronate treatment increased the radio-
MATSUMOTO ET AL
Figure 3. A, Western blotting showing effects of risedronate on the
expression of Bcl-2 family proteins. B, Transcriptional regulation of
Bim in osteoclasts as determined by real-time polymerase chain
reaction. Results are expressed as the mean ⫾ SD of 3 independent
experiments. C, Resistance of Bim ⫺/⫺ mouse osteoclasts to
risedronate-induced cell reduction. Osteoclasts generated from bone
marrow cells of wild-type (WT) or Bim⫺/⫺ mice were cultured in the
presence or absence of 30 mM risedronate. Top, TRAP staining of
representative cultures. Bars ⫽ 100 ␮m. Bottom, Number of TRAPpositive osteoclasts shown as a percentage of the cells at time zero.
D, Suppression of bone resorption by risedronate in both WT and
Bim⫺/⫺ mouse osteoclasts. Top, Resorption pits of representative
cultures. Bars ⫽ 100 ␮m. Bottom, Pit area per cell. In C and D,
experiments were repeated at least 3 times, and results are expressed
as the mean ⫾ SD of data obtained from 6 independent culture dishes
in 1 typical experiment. ⴱ ⫽ P ⬍ 0.01 versus cultures without
risedronate. See Figure 1 for other definitions.
opacity, especially in the distal femur, and increased
BMD in the Bim⫺/⫺ mice at a level equivalent to that in
the WT mice (Figures 4A and B). Risedronate treatment reduced the mean level of serum CTX-I, a measure of resorption breakdown products, to approximately half in WT and in Bim⫺/⫺ mice (Figure 4C).
DISTINGUISHING THE FUNCTIONS OF RISEDRONATE
3913
Figure 4. A, Representative radiographic images of the distal femur of 14-week-old male Bim⫺/⫺ and wild-type (WT) mice that were
left untreated or were treated with risedronate for 14 days. B, Bone mineral density of the distal femur in Bim⫺/⫺ and WT mice that
were left untreated or were treated with risedronate. ⴱ ⫽ P ⬍ 0.05 versus untreated WT mice; ⴱⴱ ⫽ P ⬍ 0.05 versus untreated
Bim⫺/⫺ mice. C, Serum concentration of C-terminal crosslinking telopeptide of type I collagen (CTX-I) in Bim⫺/⫺ and WT mice.
ⴱ ⫽ P ⬍ 0.01 versus untreated mice of the same genotype. D, TRAP staining of the distal femur of WT and Bim⫺/⫺ mice. Bars ⫽ 100 ␮m.
E, Osteoclast number per bone perimeter (OcN/BPm) in Bim⫺/⫺ and WT mice. ⴱ ⫽ P ⬍ 0.01 versus untreated WT mice.
F, Risedronate-induced osteoclast apoptosis in Bim⫺/⫺ and WT mice. ⴱ ⫽ P ⬍ 0.01 versus untreated mice of the same genotype.
G, TRAP staining of normal osteoclasts in untreated WT mice (left) and giant osteoclasts in risedronate-treated WT mice (right).
Bars ⫽ 10 ␮m. H, Proportion of giant osteoclasts in Bim⫺/⫺ and WT mice. ⴱ ⫽ P ⬍ 0.01 versus untreated mice of the same genotype.
In B, C, E, F, and H, results are expressed as the mean ⫾ SD of 3 samples obtained from different animals. See Figure 1 for other
definitions.
OcN/BPm was increased by risedronate treatment by ⬃50% in WT mice, but not in Bim⫺/⫺ mice
(Figures 4D and E). The mean ratio of apoptotic
osteoclasts to total osteoclasts was ⬃12% and ⬃2% in
risedronate-treated WT mice and risedronate-treated
Bim⫺/⫺ mice, respectively (Figure 4F). These results
demonstrate that Bim⫺/⫺ mouse osteoclasts are resistant to risedronate-induced apoptosis in vivo as well
as in vitro, but the suppression of osteoclast apoptosis
does not affect the in vivo effect of risedronate increasing bone mass. Interestingly, risedronate treatment induced an ⬃5-fold increase of giant osteoclasts compared
to untreated controls in both WT and Bim⫺/⫺ mice
(Figures 4G and H).
Effect of risedronate on ERK and Akt activity in
osteoclasts. It was previously reported that the suppression of bone resorption by alendronate and risedronate
is independent of their effects on apoptosis (15). As
shown in Figure 5A, risedronate significantly suppressed
pit formation at a concentration of 0.03 ␮M, a much
lower concentration than that required to induce osteoclast apoptosis (Figure 1A). To address the possible
underlying molecular mechanisms, we examined the
effect of risedronate on ERK and Akt activity in osteoclasts. Risedronate suppressed both Akt and ERK activity in osteoclasts in a time- and dose-dependent manner
(Figures 5B and C). To compare the effect of risedronate on ERK and Akt activity in further detail, we
performed ELISA analysis. Interestingly, risedronate
suppressed ERK activity in osteoclasts at no less than 3
␮M, while it significantly suppressed Akt activity at 0.03
␮M (Figure 5D).
Regulation of osteoclast apoptosis and activity by
the ERK and Akt pathways, respectively. To analyze the
role of ERK and Akt signals in osteoclasts, we separately
activated these pathways in osteoclasts by infecting them
with AxAktCA or AxMekCA. As shown in Figure 6A,
these adenoviruses efficiently activated the Akt and
ERK pathways in osteoclasts as shown by phospho-Akt
or phospho-ERK immunoblotting. The mean ⫾ SD
risedronate-induced reduction rate of OcN was 70.0 ⫾
12.0% with empty vector, 40.2 ⫾ 8.5% with AxAktCA,
and 8.4 ⫾ 12.1% with AxMekCA. Both AxAktCA and
AxMekCA infection significantly reversed the effect of
3914
MATSUMOTO ET AL
risedronate compared to empty vector infection (P ⬍
0.05 for empty vector versus AxAktCA, P ⬍ 0.01 for
empty vector versus AxMekCA), although risedronate
still significantly reduced the number of osteoclasts
infected with AxAktCA. The effect of AxMekCA was
stronger than that of AxAktCA (8.4 ⫾ 12.1% versus
40.2 ⫾ 8.5%; P ⬍ 0.05) (Figure 6B).
We then examined the effect of the Akt and
Figure 5. A, Dose-dependent suppression of pit formation by risedronate. B and C, Time-dependent (B) and dose-dependent (C) effects
of risedronate on Akt and ERK activity in osteoclasts shown by
Western blotting with anti–phospho-ERK antibody and anti–phosphoAkt antibody. The blots were reprobed with anti-ERK antibody
and anti-Akt antibody, respectively. D, Quantitative enzyme-linked
immunosorbent assay for the measurement of phospho-Akt and
phospho-ERK in osteoclasts treated with increasing concentrations of
risedronate for 12 hours. Cultures stimulated with macrophage colonystimulating factor were used as the positive control. In A and D,
experiments were repeated at least 3 times, and results are expressed
as the mean ⫾ SD of data obtained from 6 independent culture wells in
1 typical experiment. ⴱ ⫽ P ⬍ 0.01 versus cultures without risedronate
treatment. See Figure 1 for definitions.
Figure 6. A, Western blotting shows adenovirus vector–mediated
expression of constitutively active MEK-1 and Akt-1 in osteoclasts.
B, Risedronate-induced reduction of osteoclast number was reversed
by AxMekCA infection. AxAktCA only partially reversed the effect of
risedronate. Left, TRAP staining of representative cultures. Bars ⫽
100 ␮m. Right, Number of TRAP-positive osteoclasts shown as a
percentage of the cells at time zero. C, Risedronate-induced suppression of bone resorption was completely reversed by AxAktCA infection
but not by AxMekCA infection. Left, Resorption pits of representative
cultures. Bars ⫽ 100 ␮m. Right, Pit area per cell. D, Risedronateinduced disruption of sealing zone formation was completely reversed by AxAktCA infection but not by AxMekCA infection. Left,
Rhodamine-labeled phalloidin staining of representative cultures.
Bars ⫽ 50 ␮m. Right, Proportion of osteoclasts with sealing zone.
In B–D, ⴱ ⫽ P ⬍ 0.01 versus the corresponding cultures without
risedronate. Experiments were performed at least 3 times, and results
are expressed as the mean ⫾ SD of data obtained from 6 independent
culture dishes in 1 typical experiment. See Figure 1 for definitions.
ERK pathways on bone resorption. The pit-forming
activity of osteoclasts infected with the control vector
and AxMekCA was almost completely suppressed by
30 ␮M risedronate. In contrast, osteoclasts infected with
DISTINGUISHING THE FUNCTIONS OF RISEDRONATE
AxAktCA exhibited unaltered pit-forming activity in the
presence of risedronate (Figure 6C). To obtain insight
into the mechanism by which Akt activation cancelled
the inhibitory effect of risedronate on bone resorption,
we examined the sealing zone formation of osteoclasts
cultured on dentin slices by rhodamine-labeled phalloidin staining. Sealing zone formation on the dentin slices
was reduced to ⬃50% by risedronate treatment in
osteoclasts infected with control vector and AxMekCA.
In contrast, sealing zone formation was not affected by
risedronate treatment in osteoclasts infected with
AxAktCA (Figure 6D). These results suggest that the
antiresorptive effect of risedronate is due to the disruption of cytoskeletal organization, which is caused by
reduced Akt activity.
DISCUSSION
The nitrogen-containing bisphosphonates, including risedronate and alendronate, are among the
most effective antiosteoporosis drugs, and clinical data
have been accumulating in support of their effectiveness
in reducing osteoporotic fractures. However, despite
such clinical evidence, their exact mechanism of action
remains to be elucidated. Although the nitrogencontaining bisphosphonates induce osteoclast apoptosis,
recent studies indicate that they suppress the boneresorbing activity of osteoclasts at 10-fold lower doses
than those required to induce osteoclast apoptosis (15),
suggesting an antiresorptive mechanism of action other
than osteoclast apoptosis.
Nitrogen-containing bisphosphonates are known
to inhibit the mevalonate pathway and reduce the prenylation of small GTP-binding proteins, such as Ras,
Rho, Rac, and Cdc42 (6), and a critical role of the
small GTP-binding proteins in osteoclast function has
recently been reported (7–10). However, the upstream
and downstream signaling pathways of small G proteins in osteoclasts have not been fully determined (21).
In particular, it remains elusive whether nitrogencontaining bisphosphonate–induced osteoclast apoptosis
and suppression of bone resorption are independently
regulated or are controlled through the same mechanism. In this study, an effort was made to distinguish the
molecular mechanisms underlying the proapoptotic and
the antiresorptive effects of risedronate, and it was
found that the ERK/Bim axis mainly regulates osteoclast
apoptosis and that the Akt pathway regulates bone
resorption. In addition, the in vivo effect of risedronate
was elucidated using apoptosis-defective Bim⫺/⫺ mice.
We first investigated the risedronate-induced os-
3915
teoclast apoptosis signaling pathway in vitro, and we
found that the osteoclast apoptosis induced by risedronate occurs via the mitochondrial pathway, which is
mediated by Bcl-2 family proteins. We previously reported that the proapoptotic BH3-only domain protein
Bim plays a crucial role in mitochondria-mediated osteoclast apoptosis, and that the Bim expression level is
posttranslationally regulated by the ERK-induced
ubiquitin/proteasome pathway (17). Risedronate treatment increased Bim expression at the protein level, and
Bim⫺/⫺ mouse osteoclasts were resistant to risedronateinduced apoptosis as compared to WT mouse osteoclasts, indicating an essential role of Bim in
risedronate-induced osteoclast apoptosis. However,
bone-resorbing activity was almost completely abolished
by risedronate treatment even in Bim⫺/⫺ mouse osteoclasts as well as in WT mouse osteoclasts. In addition,
mandatory activation of ERK pathways by adenovirus
vector–mediated overexpression of constitutively active
MEK-1 significantly reversed risedronate-induced reduction of OcN even in the presence of risedronate, but
was unable to recover the bone-resorbing activity of
osteoclasts suppressed by risedronate. These results
clearly demonstrate that the ERK/Bim axis critically
regulates the proapoptotic but not the antiresorptive
effect of risedronate.
This was further confirmed in vivo by analyzing
the effect of risedronate on Bim⫺/⫺ mice. Bim⫺/⫺ mouse
osteoclasts were resistant to risedronate-induced apoptosis in vivo as well; nonetheless, risedronate suppressed bone resorption in Bim⫺/⫺ mice as efficiently as
in WT mice, as determined by the decrease in serum
CTX-I and the increase in BV. These results indicate
that risedronate’s in vivo effect of increasing bone mass
is mainly caused by suppressing the bone-resorbing
activity of osteoclasts and not by inducing osteoclast
apoptosis.
Weinstein et al recently reported an increase in
the number of osteoclasts and the appearance of giant
osteoclasts in human bone biopsy samples after longterm oral alendronate treatment (16). They attributed
the presence of the giant osteoclasts to the enhanced
survival of osteoclasts, which enabled them to fuse with
other mononuclear cells. In our risedronate treatment
experiments, giant osteoclasts comprised ⬃5% of the
total OcN both in the risedronate-treated WT and
Bim⫺/⫺ mice as compared to 1% in the untreated mice.
This suggests that nitrogen-containing bisphosphonate–
induced giant osteoclast formation is independent of
ERK/Bim-induced osteoclast apoptosis. Formation of
giant osteoclasts by nitrogen-containing bisphospho-
3916
nates may need changes other than the enhancement of
survival, such as the detachment from bone surface, the
structure change of cytoskeleton, and the expression of
cell adhesion factors. Further study will be required to
clarify the exact mechanism of giant osteoclast formation.
We found that risedronate suppressed the boneresorbing activity of osteoclasts at concentrations equivalent to those required to suppress Akt activity, while
the induction of apoptosis and ERK suppression required much higher concentrations. These observations
led us to the hypothesis that the antiresorptive and
proapoptotic effects of risedronate are mediated by the
Akt and ERK pathways, respectively. This hypothesis
was confirmed by the fact that the risedronate-induced
suppression of bone resorption was completely reversed
by the activation of the Akt pathway by AxAktCA
infection, while bone resorption was markedly suppressed in control vector– or AxMekCA-infected osteoclasts.
After 16-hour incubation on dentin slices, a sealing zone was formed in 70–80% of the total osteoclasts
infected with the control vector, AxAkt CA , and
AxMekCA under the untreated condition. Risedronate
treatment reduced the intact sealing zone formation to
less than half in the osteoclasts infected with the control
vector and AxMekCA, but had no effect on the osteoclasts infected with AxAktCA. These results suggest that
risedronate-induced disruption of the sealing zone in
osteoclasts was caused by the suppression of the Akt
activity.
Our findings suggest that the ERK pathway
mainly regulates osteoclast survival and that the Akt
pathway regulates osteoclast activity. However, overexpression of AktCA in fact increased OcN at the basal
level and partially reversed risedronate-induced reduction of OcN (Figure 6B), indicating that the Akt pathway
also regulates osteoclast survival. This was consistent
with our previous observation that the overexpression of
dominant-negative Rac1, which suppressed macrophage
colony-stimulating factor–dependent activation of the
Akt pathway but not the ERK pathway, partially inhibited osteoclast survival (9). Further study is required to
segregate the roles of the ERK and Akt pathways in
osteoclast survival.
Akt is a downstream effector of phosphatidylinositol 3-kinase (PI3K) (22). It was reported that wortmannin, a specific inhibitor of PI3K, disrupted actin ring
formation in osteoclasts (23). In addition, a recent study
showed that deficiency of p85␣, a regulatory subunit of
class IA PI3K, resulted in reduced Akt activity and
MATSUMOTO ET AL
defective actin ring formation in osteoclasts (24). These
reports support our hypothesis that Akt critically regulates the cytoskeletal organization in osteoclasts. Further
study is required to fully understand the molecular
mechanisms underlying the Akt-mediated regulation of
the osteoclast cytoskeleton.
It should also be clarified in the future whether
nitrogen-containing bisphosphonates other than risedronate exert their effect through the same mechanism. In
addition, it remains unclear whether risedronate in fact
inhibits bone resorption by suppressing osteoclast Akt
activity in vivo. Further understanding of the functional
mechanism of risedronate and other nitrogen-containing
bisphosphonates would enable their optimal use in the
clinic.
ACKNOWLEDGMENTS
The authors thank Reiko Yamaguchi and Hajime
Kawahara (Department of Orthopaedic Surgery, University of
Tokyo), who provided expert technical assistance. Pacific Edit
reviewed the manuscript prior to submission.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Tanaka had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Matsumoto, Nagase, Tanaka.
Acquisition of data. Matsumoto, Nagase, Iwasawa, Yasui, Masuda.
Analysis and interpretation of data. Matsumoto, Kadono, Nakamura,
Tanaka.
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