AMD3100 improves ovariectomy-induced osteoporosis in mice by facilitating mobilization of hematopoietic stem/progenitor cells

Abstract

Inhibition of an increase of osteoclasts has become the most important treatment for osteoporosis. The CXCR4 antagonist, AMD3100, plays an important role in mobilization of osteoclast precursors within bone marrow (BM). However, the actual therapeutic impact of AMD3100 in osteoporosis has not yet been ascertained. Here we demonstrate the therapeutic effect of AMD3100 in treatment of ovariectomy-induced osteoporosis in mice. We found that treatment with AMD3100 resulted in direct induction of release of SDF-1 from BM to blood and mobilization of hematopoietic stem/progenitor cells (HSPCs) in an osteoporosis model. AMD3100 prevented bone density loss after ovariectomy by mobilization of HSPCs, suggesting a therapeutic strategy to reduce the number of osteoclasts on bone surfaces. These findings support the hypothesis that treatment with AMD3100 can result in efficient mobilization of HSPCs into blood through direct blockade of the SDF-1/CXCR4 interaction in BM and can be considered as a potential new therapeutic intervention for osteoporosis.

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BMB
Re
p
orts
BMB Rep. 2014; 47(8): 439-444
www.bmbreports.org
*Corresponding author. Tel: +82-53-420-4815; Fax: +82-53-424-
3349; E-mail: jsbae@knu.ac.kr
#
These authors contributed equally to this work.
http://dx.doi.org/10.5483/BMBRep.2014.47.8.159
Received 10 July 2013, Revised 22 July 2013,
Accepted 19 November 2013
Keywords: AMD3100, Hematopoietic stem/Progenitor cell,
Mobilization, Osteoclast, Osteoporosis
ISSN: 1976-670X (electronic edition)
Copyright ⓒ 2014 by the The Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/li-
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AMD3100 improves ovariectomy-induced osteoporosis in mice by
facilitating mobilization of hematopoietic stem/progenitor cells
Jin Young Im
1,2,3,#
, Woo-Kie Min
4,#
, Min Hee Park
1,2,3
, NamOh Kim
1,2,3
, Jong Kil Lee
1,2,3
, Hee Kyung Jin
1,5
, Je-Yong Choi
3,6
,
Shin-Yoon Kim
4
& Jae-sung Bae
1,2,3,
*
1
Stem Cell Neuroplasticity Research Group, Kyungpook National University,
2
Department of Physiology, Cell and Matrix Research
Institute, School of Medicine, Kyungpook National University,
3
Department of Biomedical Science, BK21 Plus KNU Biomedical
Convergence Program, Kyungpook National University, Daegu 700-842,
4
Department of Orthopaedic Surgery, Kyungpook National
University Hospital, Daegu 700-721,
5
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Kyungpook National
University, Daegu 700-721,
6
Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu
702-701, Korea
Inhibition of an increase of osteoclasts has become the most
important treatment for osteoporosis. The CXCR4 antagonist,
AMD3100, plays an important role in the mobilization of
osteoclast precursors within bone marrow (BM). However, the
actual therapeutic impact of AMD3100 in osteoporosis has not
yet been ascertained. Here we demonstrate the therapeutic
effect of AMD3100 in the treatment of ovariectomy-induced
osteoporosis in mice. We found that treatment with AMD3100
resulted in direct induction of release of SDF-1 from BM to
blood and mobilization of hematopoietic stem/progenitor cells
(HSPCs) in an osteoporosis model. AMD3100 prevented bone
density loss after ovariectomy by mobilization of HSPCs,
suggesting a therapeutic strategy to reduce the number of
osteoclasts on bone surfaces. These findings support the
hypothesis that treatment with AMD3100 can result in efficient
mobilization of HSPCs into blood through direct blockade of
the SDF-1/CXCR4 interaction in BM and can be considered as
a potential new therapeutic intervention for osteoporosis.
[BMB Reports 2014; 47(8): 439-444]
INTRODUCTION
Bone remodeling is required for balance between bone-form-
ing osteoblasts and bone resorbing osteoclasts (1, 2). Destruc-
tion of this balance may lead to the development of bone dis-
ease, such as osteoporosis, which is characterized clinically by
low bone density and a consequent increase in the risk of frac-
ture, and pathologically by a decrease in bone mineral density
(BMD) and an increase in osteoclastic bone resorption (3, 4).
Osteoclasts are somatic cells that differentiate from hema-
topoietic stem/progenitor cells (HSPCs) (5). Previous study has
shown that sphingosine-1-phosphate (S1P), a lipid mediator
enhanced in blood, regulates the recruitment of osteoclast pre-
cursors from bone marrow (BM) into blood. Treatment with
the S1P agonist resulted in relief of bone loss through the re-
duction of osteoclast deposition onto bone surfaces (6). This
study led us to speculate that a more effective approach to the
recruitment of endogenous HSPCs into blood in patients with
osteoporosis could be via an important therapy involving the
decline of osteoclasts differentiated from HSPCs in BM.
Chemokine stromal cell-derived factor-1 (SDF-1, also termed
CXCL12), the most powerful chemoattractant of both human
and murine HSPCs, and its major receptor, CXCR4, are key
players in HSPC mobilization (6, 7). The number of circulating
stem cells can be significantly increased by mobilizing them
from the BM to the peripheral blood with cytokines, chemo-
kines, or small molecule inhibitors (8). Due to its potency, gran-
ulocyte colony-stimulating factor (G-CSF) is currently the most
widely used agent for induction of HSPC mobilization (9).
However, mobilization of HSPCs by G-CSF occurs through sup-
pression of osteoblast cells and decreasing expression of SDF-1
on osteoblasts, resulting in major impairment of HSC retention
in BM and reduced bone formation (9, 10). It is also associated
with a clinically significant osteopenia, characterized by an in-
crease in osteoclast activity and a decrease in BMD (11-13). In
order to overcome these obstacles, we used AMD3100, which
inhibits SDF-1-mediated mobilization through direct blockade
of the chemokine binding to its receptor, CXCR4, without im-
pairment of osteoblast function (10, 14, 15) in a model of post-
menopausal osteoporosis. AMD3100 has recently been re-
ported to enhance recruitment of CXCR4-dependent HSPCs by
inducing an increase in the level of SDF-1 in blood secreted

HSPCs mobilization by AMD3100 in osteoporosis
J
in Young Im, et al.
440 BMB Reports http://bmbreports.org
Fig. 1. SDF-1 level increased in blood and did not affect
osteoblasts. (A) Timeline of the experimental design of this study.
Twelve-week-old C57BL/6 mice (n = 40) were divided into fou
r
groups (Sham/PBS group [n = 10]; Sham/AMD3100 group [n =
10]; OVX/PBS group [n = 10]; OVX/AMD3100 group [n = 10]).
(B, C) Steady-state homeostasis fold change in levels of SDF-1
was evaluated in mouse plasma and BM supernatants after admin-
istration of PBS or AMD3100. AMD3100 induced release of func-
tional SDF-1 to plasma. (D) Expression of Osteocalcin, PTHR1,
Osterix, and Runx2 was analyzed by Real-Time PCR from BM o
f
OVX and sham mice. Data represent mean ± SEM (Student’s
t-test. n = 4-5 per group). *P ＜ 0.05 compared with AMD3100
treated mice or matched control.
Fig. 2. AMD3100 mobilized HSPCs to blood in an OVX model. (A)
We analyzed the effects of AMD3100 on HSPCs mobilization by
CFU assay in blood. (B) Flow cytometry analysis of Lin
-
Sca-1
+
c-kit
+
cells in BM of PBS-treated OVX and AMD3100-treated OVX mice.
Data represent mean ± SEM (Student’s t-test. n = 4-5 per group). *P
＜ 0.05 compared with AMD3100 treated mice or matched control.
from BM stromal cells (15, 16).
Based on these concepts and findings, this study was de-
signed to determine whether AMD3100 has the capacity to
mobilize HSPCs, resulting in a decrease in the number of os-
teoclasts in BM. If so, use of this molecule might have a benefi-
cial effect on ovariectomy (OVX)-induced osteoporosis in mice.
RESULTS
To determine the effect of AMD3100 on OVX mice, we in-
jected OVX mice with AMD3100. The injection protocol is de-
scribed in Fig. 1A. Recent studies have demonstrated direct in-
duction of release of SDF-1 into the circulation and an in-
crease in mobilization of HSPCs by treatment with AMD3100
(14). Therefore, we performed an analysis in order to de-
termine whether treatment with AMD3100 could induce re-
lease of SDF-1 and whether it is associated with recruitment of
HSPCs. Consistent with previous results (14), we found that
levels of functional SDF-1 decreased in BM and increased in
plasma of the AMD3100-treated group (Fig. 1B and C). These
results indicate that treatment with AMD3100 resulted in an
increase in SDF-1 in blood and might affect HSPCs mobi-
lization from BM to blood. Next, we investigated osteoblast
lineage-specific genes, including Osteocalcin, PTHR1, Osterix,
and Runx2 in order to determine whether AMD3100 has an
effect on osteoblast. However, no significant differences in the
levels of osteoblast lineage-specific genes were observed be-
tween the groups (Fig. 1D). These results confirmed that
AMD3100 induced an increase in levels of SDF-1 in blood
and did not alter osteoblasts of OVX mice.
To determine whether AMD3100 can mobilize HSPCs from
BM in OVX mice, we administered AMD3100 or phos-
phate-buffered saline (PBS) to OVX for 21 days. As shown in
Fig. 2A, the number of hematopoietic colony-forming unit
(CFU) cells showed a significant increase in AMD3100-treated
OVX mice compared to PBS-treated OVX mice. Flow cyto-
metric analysis was performed for evaluation of the percentage
of HSPCs in BM. As expected, the percentage of Lin
-
Sca-1
+
c-Kit
+
(LSK) cells showed a decrease in AMD3100-treated
OVX mice, compared with PBS-treated OVX mice in BM, al-
though this did not reach statistical significance (Fig. 2B).
These data indicate that AMD3100 induces mobilization of
HSPCs from BM to blood in a model of OVX-induced
osteoporosis.
To investigate the effect of AMD3100 on different bone pa-

HSPCs mobilization by AMD3100 in osteoporosis
Jin Young Im, et al.
441http://bmbreports.org BMB Reports
Fig. 3. AMD3100 relieves OVX-induced bone loss through reduction of osteoclast number onto the bone surface. (A) Representative mi-
cro-CT images of the distal femurs in each group are shown. (B) Each graph represents BMD, BVF, BMC, TMD, Tb.N., Tb.Sp., Cr.BMD
and Cr.BMC in total analysis of cortical and trabecular bone. (C) The effects of AMD3100 on the expression of genes associated with os-
teoclast differentiation by quantitative real-time PCR. Total RNA was extracted from BM cultures treated with or without RANKL in the pres-
ence or absence of 25 μg/ml of AMD3100 for 3 days. Data represent mean ± SEM (Anova, Tukey’s HSD test. n = 3 per group). *P ＜
0.05 compared with AMD3100-treated media or matched control. (D) Representative pictures showing osteoclasts in the trabecular region o
f
OVX mice. Reduction of multi-nucleated TRAP
+
osteoclasts was detected in BM. Arrowheads indicate active TRAP
+
osteoclasts stained in
red (original magnification, ×20, Scale bar 100 μm). (E) Histogram representing the osteoclast number/bone surface [N.Oc/BS (/mm)] and (F)
the osteoblast number/bone surface [N.Ob/BS (/mm)]. Data indicate mean ± SEM (Student’s t-test. n= 4-5 per group). *P ＜ 0.05 compared
with PBS-treated OVX mice.
rameters, we performed micro-CT analysis for the assessment
of BMD, bone mineral content (BMC), bone volume fraction
(BVF), tissue mineral density (TMD), trabecular number
(Tb.N.), trabecular separation (Tb.Sp.), cortical bone mineral
density (Cr.BMD) and cortical bone mineral content (Cr.BMC).
Increased bone density was observed in AMD3100-treated
OVX mice compared with PBS-treated OVX mice (Fig. 3A).
BMD showed an increase in AMD3100-treated OVX mice
compared with PBS-treated OVX mice; however, BVF, BMC,
TMD, Tb.N., Tb.Sp., Cr.BMD and Cr.BMC were similar be-
tween the groups (Fig. 3B).
Osteoporosis is likely the result of osteoclastic deposition
rather than osteoblastic defects (3, 4). Therefore, in order to
determine whether mobilization of AMD3100 affected osteo-
clast differentiation, we first examined HSPCs differentiation
into mature functional osteoclasts by treatment with
AMD3100 (17). According to the result, AMD3100 did not af-
fect osteoclast differentiation (Fig. 3C). We also performed tar-
trate resistant acid phosphatase (TRAP) staining for detection of
osteoclasts in the trabecular region of OVX mice. The number
and size of TRAP
+
active osteoclasts (black arrowhead)
showed a decrease in the trabecular region in AMD3100-treat-
ed OVX mice (Fig. 3D). Histomorphometric analysis was per-
formed for determination of the number of osteoclasts. A de-
crease in the number of osteoclasts was observed in
AMD3100-treated OVX mice compared with PBS-treated OVX
mice (Fig. 3E). Next, we performed H&E staining in order to
determine the effect of AMD3100 on the number of
osteoblasts. Our findings showed that osteoblast numbers did
not change significantly among the AMD treatment groups
(Fig. 3F). Taken together, the data presented here demonstrate
that treatment with AMD3100 induced mobilization of BM-de-
rived HSPCs into blood, leading to amelioration of bone loss
in a model of OVX-induced osteoporosis by reducing the num-
ber of osteoclasts attached to the bone surface.
DISCUSSION
Previous studies have demonstrated a unique role of
AMD3100 in the rapid mobilization of osteoclast precursors,
HSPCs from BM to blood (9, 10). However, the actual ther-
apeutic impact of AMD3100 on pathology of osteoporosis and
its mechanism of action have not been determined.
AMD3100, a small bicyclam derivative, selectively inhibits
binding of SDF-1 in osteoblasts to its receptor, CXCR4, in
HSPCs, resulting in rapid mobilization of HSPCs (14).
AMD3100-induced levels of SDF-1 can also induce mobi-
lization of HSPCs (18). The data presented here suggest that

HSPCs mobilization by AMD3100 in osteoporosis
J
in Young Im, et al.
442 BMB Reports http://bmbreports.org
AMD3100 can improve bone loss in an OVX mice model by
decreasing the number of osteoclasts differentiated from
BM-derived HSPCs.
We found that administration of AMD3100 resulted in an
increase in the functional levels of SDF-1 in plasma compared
with BM. This result suggests that release of HSPCs from BM to
blood occurred in response to increasing levels of plasma
SDF-1 in blood following administration of AMD3100. In ad-
dition, no changes in the levels of osteoblast markers were de-
tected after treatment with AMD3100. These findings indicate
that mobilization of HSPCs by AMD3100 occurs through di-
rect blockade of CXCR4-mediated sensing of the SDF-1 che-
motactic gradient in BM and by promoting the release of
SDF-1 from BM stromal cells into the circulation, similar to
previous reports (10, 19).
Notably, we demonstrated that the total number of CFUs in
blood was increased by treatment with AMD3100 in Sham
and OVX mice. We also confirmed that the LSK compartment
was decreased by administration of AMD3100 in BM of OVX
mice. Consistent with these results, micro-CT analyses con-
firmed an increase in bone thickness as a result of the in-
hibition of osteoclastic bone resorption. These results indicate
that AMD3100 prevented bone loss in OVX mice through re-
duction of osteoclast number onto bone surfaces. However, al-
though mobilization and levels of SDF-1 were increased in the
plasma of Sham mice, treatment with AMD3100 did not result
in significant change of BMD. This is because AMD3100 has
been shown to very rapidly mobilize HSPCs into blood, with-
out altering BM niche function, with restoration to normal
steady state within hours (9, 15). These results suggest that
AMD3100 had no effect on bone density under normal
steady-state BM environment not in osteoporosis.
In summary, results of this study demonstrated that
AMD3100, a novel small-molecule antagonist of CXCR4, had
no influence on osteoblasts and directly targeted distribution
of the interaction of CXCL12/CXCR4, resulting in rapid mobi-
lization of HSPCs into the blood circulation and prevented
bone loss induced by osteoporosis. In this study, for the first
time, we link bone remodeling with the regulation of
AMD3100-induced mobilization of HPSCs and propose that
the delicate balance of osteoblasts and osteoclasts is also a ma-
jor regulator of hematopoiesis. Therefore, we suggest the po-
tential for use of AMD3100 as an alternative therapy for treat-
ment of osteoporosis.
MATERIALS AND METHODS
Animals and treatments
Mouse studies were approved by the Kyungpook National
University Institutional Animal Care and Use Committee
(IACUC). Female C57BL/6 mice were purchased from Jackson
Laboratory (Bar Harbor, ME). Mice were housed in an air-con-
ditioned room with a 12 hr light/dark cycle at a temperature of
22 ± 2
o
C and humidity of 45-65% and given free access to
food and tap water. Mice underwent either sham surgery or
OVX at 12 weeks of age and were sacrificed at 16 weeks of
age. One week after surgery, sham and OVX mice received an
intraperitoneal injection with AMD3100 (Sigma-Aldrich
#A5602, St. Louis, MO, Sigma Aldrich.com) (5 mg/kg/day) or
PBS for 21 days. At the end of treatment, the mice were sacri-
ficed, and blood samples were collected by cardiac puncture
for the CFU assay. Femora were removed, fixed with 4% paraf-
ormaldehyde in PBS solution (pH 7.4) for 16 hr, and then stor-
ed (4
o
C) at 80% ethanol for measurement of bone density.
Quantitative Real-Time PCR
RNA samples were extracted from whole BM cells of four in-
dividual animals per group and isolated from the cultured cells
using the RNeasy Mini kit (Qiagen, Hilden, Germany), and the
concentration was determined using a Nanodrop ND-1000
spectrophotometer. A total of 5 μg of each RNA was converted
to cDNA using the sprint RT complete-oligo (dT) 18 (Clontech,
MountainView, CA) according to the manufacturer’s guide.
The cDNA was quantified using the QuantiTect SYBR Green
PCR Kit (Qiagen). For each investigated transcript, a mixture of
the following reaction components was prepared to the in-
dicated end-concentration: forward primer (5 pM), reverse pri-
mer (5 pM), and QuantiTect SYBR Green PCR Master mix. The
10 μl master-mix was added to a 0.1 ml tube, and 5 μl vol-
ume, containing 100 ng reverse transcribed total RNA, was
added as polymerase chain reaction (PCR) template. The tubes
were closed, centrifuged, and placed into the Corbett research
RG-6000 real-time PCR machine (Corbett LifeScience, Sydney,
Australia). The following primers were used: Osteocalcin
(Forward 5’-GGGCAATAAGGTAGTGAACAG-3’, Reverse 5’-G
CAGCACAGGTCCTAAATAGT-3’), Osterix (Forward 5’-GCGT
ATGGCTTCTTTGTGCCT-3’, Reverse 5’-AGCTCACTATGGCT
CCAGTCC-3’), Runt-related transcription factor 2 (RUNX2)
(Forward 5’-ATACTGGGATGAGGAATGCG-3’, Reverse 5’-CC
AAGAAGGCACAGACAGAA-3’), parathyroid receptor-1 (PTHR1)
(Forward: 5’-GATTCTGGTGGAGGGACTGT-3’, Reverse 5’-GGA
TGATCCACTTCTTGTGC-3’), Atp6v0d2 (Forward 5’-CGGAAA
AGAACTCGTGAAGA-3’, Reverse 5’-CTGGAAGCCCAGTAAA
CAGA-3’), NFATc-1 (Forward 5’-AGGTGACACTAGGGGACA
CA-3’, Reverse 5’-AGTCCCTTCCAAGTTTCCAC-3’), TRAP
(Forward 5’-ACTTCCCCAGCCCTTACTAC-3’, Reverse 5’-TCA
GCACATAGCCCACACCG-3’).
Enzyme-Linked Immunosorbent Assay (ELISA)
Murine plasma was collected by cardiac puncture in tubes (a 1
ml syringe containing 50 μl of 100 mM EDTA), and BM was
flushed with PBS. After centrifugation, plasma and BM super-
natants were collected, and used for detection of SDF-1 pro-
tein by ELISA (R&D Systems, Minneapolis, MN, USA).
Hematopoietic CFU assay
Single-cell suspensions of peripheral blood (PB) after ammo-
nium chloride lysis were plated into 35 mm dishes (3 × 10
5

HSPCs mobilization by AMD3100 in osteoporosis
Jin Young Im, et al.
443http://bmbreports.org BMB Reports
cells/plate) with MethoCult GF M3434 (StemCell Technolog-
ies). Hematopoietic colonies were counted and scored after in-
cubation for 12-14 days at 37
o
C, 5% CO
2
, as instructed by the
manufacturer.
Flow cytometry
BM cells from the femurs and tibias were collected by flushing
with 20 ml PBS passed through a 25-gauge needle. After cen-
trifugation at 1,300 rpm for 5 min, the supernatant was re-
moved and the cells were then washed by ammonium chlor-
ide lysis. Cells were incubated first using a Lineage Cell
Depletion Kit magnetic labeling system with the biotinylated
lineage antibody cocktail (CD5, CD45R [B220], CD11b, Gr-1
[Ly-6G/C], and Ter-119) for 10 min at 4
o
C and anti-biotin
MicroBeads (Milt-enyi Biotec) for an additional 20 min at 4
o
C.
Positive immunoselection was performed with PE/Cy7-conju-
gated anti-Sca-1 (BD Pharmingen), APC-conjugated anti-mouse
CD117 (c-Kit) (BD Pharmingen), SAV-PB, and a FACS Aria (BD
Biosciences) using a flow cytometer.
Micro-computed tomography
For micro-computed tomography (micro-CT) in vivo imaging,
we sacrificed and then scanned each group of mice at 8 μm
resolution using the eXplore Locus scanner (GE Healthcare). In
the femora, scanning regions were confined to the distal meta-
physis, extending proximally 1.7 mm from the proximal tip of
the primary spongiosa. BMD, BMC, BVF, TMD, Tb.N., Tb.Sp.,
Cr.BMD and Cr.BMC were applied for performance of quanti-
tative analysis using software provided with 2.0+ ABA
Micro-view of the micro-CT system.
Osteoclast differentiation
BM cells were prepared by removal from the femurs and tibias
of seven-week-old mice. The bone marrow suspension was
added to plates along with macrophage colony stimulating fac-
tor (M-CSF; 30 ng/ml). After culture for 24 hr, the non-adherent
cells were collected and resuspended in α-MEM containing
10% FBS. For the osteoclastogenesis experiments, BM-derived
macrophages were plated into 6-well plates at a density of 2 ×
10
6
cells/well in α-MEM with 10% FBS, receptor activator for
nuclear factor κB ligand (RANKL; 100 ng/ml) and M-CSF (30
ng/ml) in the presence or absence of AMD3100 (25 μg/ml) for
3 days.
Histological analysis
Femurs were fixed in 4% paraformaldehyde for 24 hr; the tis-
sues were then decalcified in 10% EDTA for one week, dehy-
drated in ethanol, embedded in paraffin, sectioned to 4-μm
thickness and stained with hematoxylin and eosin (H&E). For
TRAP staining, sections were stained with 225 μM Naphthol
AS-MX phosphate (Sigma-Aldrich, St. Louis, MO, USA), 0.84%
N, N-dimethylformamide (Sigma-Aldrich), and 1.33 mM Fast
Red Violet LB Salt (Sigma-Aldrich) in 50 mM sodium acetate
(pH 5.0) containing 50 mM sodium tartrate, and incubated for
30 min. After incubation, sections were washed in distilled
water and counterstained with 1% methyl green. We per-
formed histomorphometric analysis using the Bioquant
OSTEOII Program (BIOQUANT Image Analysis Corporation,
Nashville, TN, USA).
Statistical analysis
The Student’s t-test was used for comparison of two groups,
whereas Tukey’s HSD test and Repeated Measures Analysis of
Variance test were used for multi group comparisons accord-
ing to the SAS statistical package (Release 9.1; SAS Institute
Inc., Cary, NC). P ＜ 0.05 was considered significant.
ACKNOWLEDGEMENTS
This work was supported by the Basic Science Research
Program (2013R1A1A2008239) and Bio & Medical Technology
Development Program (2012M3A9C6049913) through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology, Republic of
Korea. Additional support for this work was provided by
Biomedical Research Institute grant, Kyungpook National
University Hospital (2012).
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