ORIGINAL RESEARCH

Early Response of Bone Marrow Osteoprogenitors to SkeletalUnloading and Sclerostin Antibody

Mohammad Shahnazari • Thomas Wronski •

Vivian Chu • Alyssa Williams • Alicia Leeper •

Marina Stolina • Hua Zhu Ke • Bernard Halloran

Received: 24 February 2012 / Accepted: 16 April 2012 / Published online: 27 May 2012

� Springer Science+Business Media, LLC 2012

Abstract Sclerostin functions as an antagonist to Wnt

signaling and inhibits bone-forming activity. We studied

the effects of skeletal unloading and treatment with

sclerostin antibody (Scl-Ab) on mesenchymal stem cell,

osteoprogenitor and osteoclast precursor pools, and their

relationship to bone formation and resorption. Male

C57BL/6 mice (5-months-old) were hind limb unloaded for

1 week or allowed normal ambulation and treated with Scl-

Ab (25 mg/kg, s.c. injections on days 1 and 4) or placebo.

Unloading decreased the serum concentration of bone

formation marker P1NP (-35 %), number of colony-

forming units (CFU) (-38 %), alkaline phosphatase–

positive CFUs (CFU-AP?) (-51 %), and calcified nodules

(-35 %); and resulted in a fourfold increase in the number

of osteoclast precursors. The effects of Scl-Ab treatment on

unloaded and normally loaded mice were nearly identical;

Scl-Ab increased serum P1NP and the number of CFU,

CFU-AP?, and calcified nodules in ex vivo cultures; and

increased osteoblast and bone mineralizing surfaces in

vivo. Although the marrow-derived osteoclast precursor

population increased with Scl-Ab, the bone osteoclast

surface did not change, and the serum concentration of

osteoclast activity marker TRACP5b decreased. Our data

suggest that short-term Scl-Ab treatment can prevent the

decrease in osteoprogenitor population associated with

skeletal unloading and increase osteoblast surface and bone

mineralizing surface in unloaded animals. The anabolic

effects of Scl-Ab treatment on bone are preserved during

skeletal unloading. These findings suggest that Scl-Ab

treatment can both increase bone formation and decrease

bone resorption, and provide a new means for prevention

and treatment of disuse osteoporosis.

Keywords Bone � Osteoblast � Osteoclast � Sclerostin �Skeletal unloading

Targeting the Wnt signaling pathway to augment bone

formation has been the focus of numerous recent studies

[1–4]. Wnt pathways are involved in coordinating proper

bone development, formation, and growth, both before and

after birth [5, 6]. Signaling by Wnt proteins is antagonized

by sclerostin, which is expressed mainly by osteocytes and

functions to inhibit bone formation [7, 8]. Targeted dele-

tion of the sclerostin gene increases the osteoblast surface

(Ob.S/BS), mineralizing surface (MS/BS), bone formation

rate (BFR), and bone volume (BV/TV) [9]. Treatment of

adult rats with sclerostin antibody (Scl-Ab) for 3–5 weeks

is reported to increase MS/BS and BFR [10] and of

estrogen-deficient rats to increase Ob.S/BS, MS/BS, BFR,

and BV/TV [11]. These data overwhelmingly demonstrate

the anabolic effect of decreasing sclerostin activity. The

effects of blocking sclerostin activity on bone resorption

are less clear; the ratio of ratio of osteoclast surface to bone

Thomas Wronski received research funding from Amgen Inc. Marina

Stolina and Hua Zhu Ke are employed by Amgen. Marina Stolina,

Hua Zhu Ke, and Thomas Wronski have stock ownership in Amgen.

Other authors have stated that they have no conflict of interest.

M. Shahnazari � V. Chu � B. Halloran (&)

Division of Endocrinology, Veterans Affairs Medical Center,

University of California, San Francisco, CA 94121, USA

e-mail: bernard.halloran@ucsf.edu

T. Wronski � A. Williams � A. Leeper

Department of Physiological Sciences, College of Veterinary

Medicine, University of Florida, Gainesville, FL 32610-0125,

USA

M. Stolina � H. Z. Ke

Department of Metabolic Disorders, Amgen Inc., One Amgen

Center Drive, Thousand Oaks, CA 91320, USA

123

Calcif Tissue Int (2012) 91:50–58

DOI 10.1007/s00223-012-9610-9

surface (Oc.S/BS) has been reported to decrease [11] or

remain unchanged [9]. In postmenopausal women, Scl-Ab

was shown to increase bone formation markers and bone

mineral density with a concurrent reduction in serum type I

collagen C-telopeptides, a marker of bone resorption [4].

That sclerostin regulates bone resorption is supported by

studies that suggest that sclerostin may direct hematopoi-

etic cell lineage allocation, stimulate osteoclast recruit-

ment, and increase activity [12–16].

Skeletal disuse or loss of weight bearing inhibits bone

formation and induces osteopenia in both humans and

animals [17–21]. These effects are associated with a

reduction in Ob.S/BS and MS/BS [18, 22–27]. In animal

models, hind limb unloading increases sclerostin expres-

sion in bone, suggesting a mechanism by which skeletal

unloading down-regulates osteogenesis and decreases bone

formation [28]. In vitro mechanical loading of osteoblastic

cell lines has been shown to decrease expression of scle-

rostin mRNA and protein [29, 30]. The effects of skeletal

unloading on bone resorption are less clear, with reports

indicating that unloading in mice and rats increases

osteoclast number and bone resorption [18, 31, 32] or has

no effect [22, 24–26, 33–35]. In the adult human, resorp-

tion markers are increased significantly with loss of weight

bearing [17, 36–38].

Data on the effects of Scl-Ab treatment during skeletal

unloading are limited. Tian et al. [39] reported that long-

term treatment with Scl-Ab (twice per week for 4 weeks)

increased bone mass by stimulating bone formation and

inhibiting bone resorption in an immobilized female rat

model. However, the early response of bone to unloading

and Scl-Ab treatment has received little attention, and the

effects of unloading and Scl-Ab on the osteoprogenitor and

osteoclast precursor populations have not been studied. We

studied the effects of unloading and treatment with a

neutralizing Scl-Ab on the mesenchymal stem cell, osteo-

progenitor, and osteoclast precursor pools, as well as their

relationship to bone formation and resorption using the

mouse hind limb unloading model. Our results suggest that

treatment with Scl-Ab greatly increases osteoprogenitor

number and bone formation, causes an unexpected increase

in the size of the osteoclast precursor pool, but greatly

suppresses bone resorption.

Materials and Methods

Animal Protocol

Thirty-two male C57BL/6 mice, 5-months-old (n = 8/

experimental group), were obtained from Jackson Labo-

ratories (Sacramento, CA). The animals were housed in air-

filtered, humidity- and temperature-controlled rooms with

equal 12 h light–12 h dark cycles and fed a standard mouse

diet. They were allowed to acclimate in our animal care

facility for 1 week before the experiment. The animal

protocol for the study was in accordance with the National

Institutes of Health’s Guide for the Care and Use of Lab-

oratory Animals and approved by the Animal Care and Use

Committee at the Veterans Affairs Medical Center, San

Francisco. Skeletal unloading was induced using the hind

limb elevation or tail suspension model [22, 40]. Animals

were divided into four groups of eight animals each. They

were either normally loaded (L) or unloaded (UL)

for 1 week and treated with either vehicle or Scl-Ab

(25 mg/kg, days 1 and 4) at a dose known to increase bone

formation [10, 11, 39]. The mice were injected subcuta-

neously with calcein (10 mg/kg) and demeclocycline

(20 mg/kg) 7 and 2 days before euthanasia, respectively, to

label bone mineralizing surfaces and measure bone for-

mation. At the time of euthanasia, serum samples were

collected, and tibiae and femurs were dissected for bone

marrow cell culture and histomorphometry.

In separate studies, mice were loaded or unloaded for 1,

2, and 4 weeks to establish the time course of the effects of

unloading on the osteoprogenitor and osteoclast precursor

pools. One week of unloading induced the greatest changes

in osteoprogenitor and osteoclast precursor number (data

not shown), causing us to chose this time to study the

effects of Scl-Ab treatment on the response of bone to

unloading.

Measurement of Bone Biomarkers in Serum

The bone formation marker N-terminal propeptide of type

1 collage (P1NP) and the bone resorption marker tartrate-

resistant acid phosphatase 5b (TRACP5b) were measured

in serum by enzyme immunoassay using commercial kits

provided by IDS (Fountain Hills, AZ). Both assays were

performed in duplicate and in accordance with the manu-

facturer’s instructions. Serum was separated and stored at

-80 �C until processed.

Osteoblast Culture

Tibiae and femurs were cleaned of adherent tissues and

diaphyseal bone marrow stromal cells were collected and

plated at 1.3 9 105 nucleated cell/cm2 in a 100-mm plate

as previously described [41]. On day 2, nonadherent cells

were removed and cultured under osteoclast induction

conditions as described under below. The culture medium

in the adherent cells was changed to secondary medium

(aMEM supplemented with 10 % fetal bovine serum, 1 %

antibiotics, 0.1 % Fungizone, 50 lg/100 ml L-ascorbic

acid, and 3 mM b-glycerophosphate) on day 5 to induce

mesenchymal cells to form osteoblasts. Subsequent media

M. Shahnazari et al.: Early Response of Bone Marrow Osteoprogenitors 51

123

changes were performed every 2 days for 28 days. The

number of colony-forming units (CFU) and alkaline

phosphatase–positive CFU (CFU-AP?) with diameter

greater than 3-mm were quantified on day 14 of culture as

previously described [42–44]. Mineralized calcium nodule

formation was assessed at day 28 as previously described

[42–44]. Briefly, plates were stained with 2 % alizarin red

(Sigma-Aldrich, St. Louis, MO) for 10 min and rinsed five

times with distilled water to remove loosely bound stain.

The number of distinct alizarin red–stained colonies with

diameters greater than 1-mm was quantified.

Osteoclast Culture

To assess the effects of Scl-Ab on the number of preoste-

oclasts in the bone marrow, the nonadherent cell fraction

from the bone marrow cell cultures was removed and

washed with phosphate-buffered saline (PBS). The cells

were suspended in PBS, counted with a hemocytometer,

and seeded into 24-well tissue culture plates at 1 9 106

cells per well. The cultures were carried for 6 days in the

stromal cell culture medium supplemented with RANK-L

(40 ng/ml) and M-CSF (10 ng/ml). Medium was changed

every 2 days, and on the sixth day, cells were washed twice

with PBS and stained for tartrate-resistant acid phosphatase

(TRAP) using a commercial kit from Sigma-Aldrich. Dark

reddish-purple multinucleated cells ([3 nuclei) were

counted as TRAP-positive osteoclasts.

Bone Histomorphometry

The left distal femur was obtained for bone histomorpho-

metric measurements. The bones were fixed in 10 % phos-

phate-buffered formalin for 24 h, dehydrated in increasing

concentrations of ethanol, and embedded undecalcified in

methyl methacrylate. Sections at thicknesses of 4 and 8 lm

were cut with a microtome (Leica RM 2065, Germany). The

thinner sections were stained according to the Von Kossa

method with a tetrachrome counterstain (Polysciences,

Warrington, PA), whereas the thicker sections remained

unstained for fluorochrome-based measurements of bone

formation. Cancellous bone volume (BV/TV), osteoblast

and osteoclast surfaces (Ob.S/BS and Oc.S/BS), mineraliz-

ing surface (MS/BS), mineral apposition rate (MAR), and

bone formation rate (BFR/BS, ratio of bone formation rate

to surface referent) were measured in the secondary

spongiosa of the distal femoral metaphysis with an image

analysis program (Bioquant Image Analysis, Nashville, TN)

as previously described [22].

Data Analysis

All data are reported as mean ± standard error of the mean

and were analyzed by one-way analysis of variance and the

Tukey test. Significance was assumed for p \ 0.05.

Results

Body weights among the groups at the beginning of the

experiment were not different. At day 7, L and UL animals

weighed 31 ± 0.75 g and 29.3 ± 0.75, respectively, and

weights did not change significantly during the course of

P1NP

ng

/ml

0

20

40

60

80

†

TRACP5b

L L+Scl.Ab UL UL+Scl.AbL L+Scl.Ab UL UL+Scl.Ab

U/L

0

5

10

15

20

25

30

**

**

****

Fig. 1 Bone formation and resorption markers in serum in 5-month-old

C57BL/6 male mice normally loaded (L) or unloaded (UL) and treated

with vehicle or sclerostin-neutralizing antibody, L ? Scl-Ab and UL ?

Scl-Ab, respectively (mean ± SE, n = 8). P1NP N-terminal propeptide

of type 1 procollagen, TRACP5b tartrate-resistant acid phosphatase 5b.

** indicates a difference between L versus L ? Scl-Ab or UL versus

UL ? Scl-Ab at p B 0.001. � indicates a difference between L and UL

groups at p B 0.05

Fig. 2 Treatment of normally loaded (L) and unloaded (UL) mice

with sclerostin-neutralizing antibody (Scl-Ab), L ? Scl-Ab and

UL ? Scl-Ab, respectively. Bone marrow stromal cells were cultured

and the numbers of colony-forming units (CFU), CFU positive for

alkaline phosphatase (CFU-AP?), and mineralizing nodules were

quantified (mean ± SE, n = 8). Representative images are shown for

each group. * and ** indicate a difference between L versus L ? Scl-

Ab or UL versus UL ? Scl-Ab at p B 0.05 and p B 0.001, respec-

tively. � and � indicate a difference between L and UL groups at

p B 0.05 and p B 0.001, respectively

c

52 M. Shahnazari et al.: Early Response of Bone Marrow Osteoprogenitors

123

UL

L

L

UL

Vehicle-treated

L

CFUScl-Ab-treated

Nu

mb

er/ 1

00 m

m d

ish

0

5

10

15

20

25

30N

um

ber

/ 100

mm

dis

h

0

10

20

30

40

50

60

L

CFU-AP+

‡

CFU

L+Scl.Ab U

‡

UL UL+Scl.AAb

L L+Scl.Ab UUL UL+Scl.AAb

L L+Scl.Ab UUL UL+Scl.AAb

†

Mineralizing nodules

L

UL

Mineralizing Nodules

Nu

mb

er/ 1

00 m

m d

ish

0

2

4

6

8

10

*

*

†

**

**

**

*

CFU-AP+

M. Shahnazari et al.: Early Response of Bone Marrow Osteoprogenitors 53

123

the experiment. L ? Scl-Ab and UL ? Scl-Ab animals

weighed 31.4 ± 0.57 and 30 ± 0.76 g, respectively, and

weights between these groups did not differ during the

course of the experiment.

The serum concentration of the bone formation marker

P1NP was 35 % lower in UL mice than in L mice

(p \ 0.05), whereas the serum concentration of the bone

resorption marker TRACP5b was not affected after 1 week

of unloading (Fig. 1). Treatment with Scl-Ab increased the

serum concentration of P1NP (twofold, p \ 0.0001) and

decreased the serum concentration of TRACP5b (50 %,

p \ 0.0001) in both UL and L mice.

One week of skeletal unloading induced a decrease in

CFU (-38 %, p \ 0.05), CFU-AP? (-51 %, p \ 0.001),

and calcified nodules (-35 %, p \ 0.05); and resulted in a

fourfold increase in the number of osteoclasts formed from

the nonadherent population of marrow stromal cells

(osteoclast precursors) (Figs. 2, 3). Treatment of unloaded

animals with Scl-Ab resulted in twofold increases in CFU,

CFU-AP?, and calcified nodules. Similar increases in

these variables were observed in L mice receiving Scl-Ab.

Scl-Ab treatment had no effect on osteoclast precursor

number in UL mice but increased the number in the L

group by more than twofold (Fig. 3).

The BV/TV, trabecular number, and trabecular separa-

tion were not significantly affected by unloading or Scl-Ab

treatment (Table 1). Although trabecular thickness did not

change significantly in response to Scl-Ab in normally L

mice, it increased in UL mice.

Osteoclast surface/bone surface was increased by 55 %

with 1 week of unloading, but the difference did not reach

significance. No difference in Ob.S/BS was observed

between L and UL groups (Table 1). The means of the

MAR and BFR/BS were 31 and 36 % lower in UL when

compared to L mice, but these changes did not reach sig-

nificance. Treatment of UL mice with Scl-Ab resulted in

more than a fourfold increase in Ob.S/BS (p and le; 0.001)

and a twofold increase in MS/BS (p \ 0.05). The BFR/BS

tended to increase ([twofold), but not significantly.

Treatment of L mice with Scl-Ab increased Ob.S/BS

(p \ 0.001) but did not significantly affect the MS/BS or

BFR/BS. Oc.S/BS did not differ among the groups. No

effects of either unloading or treatment with Scl-Ab were

observed for MAR.

Discussion

Our goal was to examine the early effects of Scl-Ab

treatment on the mesenchymal stem cell, osteoprogenitor,

and osteoclast precursor pools, as well as their relationship

to bone formation and resorption in normally loaded and

unloaded mice. The results suggest that Scl-Ab treatment

has rapid and profound effects on the osteoprogenitor and

osteoclast precursor populations in both loaded and

unloaded mice, and that the early anabolic response to Scl-

Ab is preserved during skeletal unloading, a finding con-

sistent with previous reports [45].

Osteoclast

L

UL

Osteoclast

L L+Scl.Ab UL UL+Scl.Ab

Nu

mb

er/ w

ell

0

5

10

15

20

25

*

‡

Vehicle-treated Scl-Ab-treated

Fig. 3 Treatment of normally loaded (L) and unloaded (UL) mice

with sclerostin-neutralizing antibody, L ? Scl-Ab and UL ? Scl-Ab,

respectively. Bone marrow nonadherent cell populations were

cultured and osteoclast formation (TRAP ? multinucleated cells,

n [ 5 nuclei) were quantified ex vivo (mean ± SE, n = 8). Repre-

sentative images are shown for each group. * indicates a difference

between L vs. L ? Scl-Ab at p B 0.05. � indicates a difference

between L and UL groups at p B 0.001

54 M. Shahnazari et al.: Early Response of Bone Marrow Osteoprogenitors

123

Hind limb unloading is accompanied by a reduction in

the number of osteoblast progenitors in the bone marrow,

Ob.S/BS, MS/BS, BFR, and BV/TV [18, 22, 33, 35, 46]. In

our study, we also found a reduction in the number of

osteoprogenitors and bone formation as judged by the

serum concentration of P1NP, but we did not observe

changes in Ob.S/BS, MS/BS, BFR, or BV/TV. The absence

of significant changes in these variables may in part reflect

the short duration of our study. Our data show that

unloading induces rapid changes in the mesenchymal stem

cells (MSC) and osteoprogenitor populations. The MSC

serves as a pool of cells for recruitment to the osteoblast

lineage. To change the bone surface populations, cells must

be recruited from the MSC pool, stimulated to migrate to

active bone surfaces, and induced to mature into func-

tioning osteoblasts. Data from our model are consistent

with the notion that although the MSC and osteoprogenitor

populations have changed by 7 days, longer periods of

unloading may be required to detect significant changes in

the Ob.S/BS, MS/BS, BFR, and BV/TV.

Skeletal unloading is also reported to be accompanied

by an increase in Oc.S/BS [31, 32], although other inves-

tigators found no change in Oc.S/BS [34, 35]. Whether a

change in Oc.S/BS is observed depends on the length of

time of unloading and the model used. 1 week of skeletal

unloading in mice has been reported to increase Oc.S/BS

and numbers of TRAP? multinucleated cells from bone

marrow cultures [31], whereas 2 weeks of unloading in

growing rats [22] or 5 weeks of unloading in adult

rats [24] did not change the osteoclast surface. In our

study, we also found an increase in TRAP? multinu-

cleated cells from cultured marrow, but we did not

observe significant changes in either Oc.S/BS or serum

TRACP5b.

Like the Ob.S/BS, this may reflect the short duration of

our study. By 7 days, the osteoclast precursor population

may have changed, but not enough time may have elapsed

to significantly alter the Oc.S/BS. The findings that there

are rapid changes in the MSC, osteoprogenitor, and

osteoclast precursor cell populations after skeletal unload-

ing suggests that there are regulatory pathways linking

bone strain and osteocyte activity to the stem and precur-

sor cell populations that give rise to osteoblasts and

osteoclasts.

Hind limb unloading is associated with an increase in

expression of sclerostin and a decrease in expression of

Wnt target genes [47]. Our data show that Scl-Ab treatment

in vivo greatly increases the number of MSC, CFU-AP?,

and calcified nodules in both normally loaded and unloaded

mice. Loss of weight bearing does not impair Scl-Ab-

induced augmentation of the MSC and osteoprogenitor

populations. These data suggest that sclerostin may not

only regulate recruitment of cells into the osteoblast line-

age, but also may regulate the MSC pool size.

Previous rat studies have reported that Scl-Ab treatment

increases Ob.S/BS, MS/BS, MAR, BFR, and BV/TV

[10, 11]. The changes in BFR seem to be primarily linked

to an increase in MS/BS. The results of our studies are

similar with respect to osteoblast and MS/BS. Osteoblast

surface was greatly increased in both unloaded and nor-

mally loaded mice treated with Scl-Ab, and MS/BS was

either increased (unloaded mice) or tended to be increased

(normally loaded mice). Unlike previous studies where the

MAR was measured after 5 weeks of treatment [10], the

MAR in our study after 1 week of treatment seemed to be

unresponsive. The reason for this is not clear, but it likely

reflects the relatively short duration of our study. Normally

a relatively long time is required to change BV/TV, and we

Table 1 Bone histomorphometric parameters from distal femoral metaphysis in mice treated with Scl-Ab (mean ± SE, n = 8)

Femoral metaphyses Experimental group

L L ? Scl-Ab UL UL ? Scl-Ab

BV/TV (%) 7.6 ± 0.4 8.6 ± 0.6 8.0 ± 0.8 9.0 ± 0.6

Tb.Th. (lm) 16.7 ± 1 17.9 ± 0.9 16.5 ± 0.4 19.6 ± 0.7*

Tb.N. (1/mm) 5.4 ± 0.1 5.6 ± 0.2 5.6 ± 0.3 5.5 ± 0.3

Tb.Sp. (lm) 172 ± 4.6 163 ± 6 170 ± 14 171 ± 10

MS/BS (%) 12.3 ± 2.6 20.1 ± 3.4 11.4 ± 2.6 24.0 ± 4.9*

MAR (lm/d) 1.44 ± 0.30 1.70 ± 0.20 1.0 ± 0.17 1.16 ± 0.19

BFR/BS (nm3/nm2/d) 22.6 ± 8.0 35.6 ± 8.5 14.5 ± 5.0 32.3 ± 9.2

Oc.S/BS (%) 0.67 ± 0.2 0.43 ± 0.1 1.0 ± 0.3 0.8 ± 0.2

Ob.S/BS (%) 15.1 ± 3.2 46.7 ± 4.5** 12.6 ± 4.4 57.0 ± 6.9**

L loaded, UL unloaded, Scl-Ab sclerostin neutralizing antibody, BV/TV ratio of bone volume to total volume, Tb.Th. trabecular thickness, Tb.N.trabecular number, Tb.Sp. trabecular separation, MS/BS ratio of mineralizing surface to bone surface, MAR mineral apposition rate, BFR/BS ratio

of bone formation rate to surface referent, Oc.S/BS ratio of osteoclast surface to bone surface, Ob.S/BS ratio of osteoblast surface to bone surface

Difference between L versus L ? Scl-Ab or UL versus UL ? Scl-Ab is shown for * p B 0.05 and ** p B 0.001

M. Shahnazari et al.: Early Response of Bone Marrow Osteoprogenitors 55

123

did not expect to find a Scl-Ab effect after only 1 week of

treatment.

The changes in CFU-AP? and calcium nodule number

induced by Scl-Ab treatment and unloading were propor-

tional to the changes in CFU or MSC number, suggesting

that the increase in osteoprogenitor number in treated mice

may reflect the increase in the MSC number. Scl-Ab

treatment seems to increase the MSC pool size but to have

less of an effect on the propensity of these cells to form

osteoblasts in vitro. In vivo, however, our data do not

exclude the possibility that neutralization of sclerostin

activity may promote recruitment of osteoprogenitors into

the osteoblast lineage. It may also stimulate migration of

osteoblasts to the bone surface and bone-forming activity.

Scl-Ab treatment of normally loaded and unloaded mice

also increased the osteoclast precursor population. This

unexpected result suggests that Wnt signaling may play an

important role in regulation of the osteoclast precursor pool

[13]. Although limited, there are data directly linking

LRP5/6 expression and hematopoietic stem cell function

[48, 49], and canonical Wnt signaling has been shown to

alter the differentiation potential of Lin-CD34?CD1a-

human thymic progenitors [14]. Sclerostin may also act

indirectly to regulate the osteoclast precursor pool.

Recruitment of cells into the hematopoietic lineages is a

dynamic process, and if the rate of recruitment from the

osteoclast precursor pool into the osteoclast lineage is

slowed, it is conceivable that this might result in an

abnormal accumulation of preosteoclasts. Stimulation of

Wnt/b-catenin signaling in osteoblasts has been shown to

decrease osteoclast formation by up-regulating osteopro-

tegerin (OPG) and down-regulating RANK-L [12, 13, 15,

16]. Our results are consistent with the idea that inhibition

of sclerostin action through treatment with the Scl-Ab

stimulates Wnt signaling in the osteoblast, thereby

increasing the OPG/RANK-L ratio and inhibiting osteo-

clastogenesis. Sclerostin may also influence migration to

and maintenance of the osteoclast population on the bone

surface. Spencer et al. [13] report that LRP6 is expressed in

mature osteoclasts. If sclerostin impairs migration, reduces

osteoclast stability on the bone surface, or both, it might

account, in part, for the apparent anomaly between osteo-

clast precursor number and Oc.S/BS.

Oc.S/BS has been reported to decrease [11] or remain

unchanged in response to blocking sclerostin [9]. Our cell

data are similar and suggest that Scl-Ab treatment for

1 week has little or no effect on Oc.S/BS. Serum

TRACP5b, however, was decreased, suggesting that

although osteoclast number may not change, osteoclast

activity per cell may decrease. Serum CTX (C-terminal

telopeptides of type I collagen, another marker of bone

resorption) has been reported to be unchanged after Scl-Ab

treatment, a finding consistent with the maintenance of a

normal Oc.S/BS in these studies [10]. The reason for

the discrepancy between the serum concentrations of

TRACP5b and CTX is not clear but may reflect the nature

of these variables. TRAP is expressed by cells other than

osteoclasts and may not in all instances accurately reflect

breakdown of bone.

The dose of Scl-Ab we chose was based on previous

animal studies showing efficacy on bone formation [10, 11,

39]. In future studies, it will be important to determine the

effects of lower and higher doses of Scl-Ab on osteoclast

formation and activity. Therapeutically, there may be

other doses that are more efficacious for regulating bone

resorption.

Collectively, our data suggest that sclerostin may func-

tion not only to regulate the osteoprogenitor pool size and

bone formation, but also to regulate the osteoclast precur-

sor pool size and osteoclast activity. Despite the increase in

osteoclast precursors, osteoclast number on the bone sur-

face remained unchanged and serum TRACP5b activity

clearly decreased, suggesting that Scl-Ab treatment may

act to block resorption by inhibiting recruitment of cells

from the osteoclast precursor population into the OC

lineage, impairing migration to the bone surface, sup-

pressing osteoclast fusion and activity, or causing an

increase in osteoclast turnover. These findings suggest a

new paradigm where the hematopoietic cell population and

lineage allocation are regulated by sclerostin. The benefi-

cial effects of Scl-Ab treatment in patients with low bone

mass may be mediated through increased formation and,

despite an increase in osteoclast precursors, decreased bone

resorption. Future studies are suggested to investigate the

effects of Scl-Ab on osteoclast formation in vitro to

examine the direct effects of inhibiting sclerostin on

osteoclast precursors in addition to the effects poten-

tially mediated through osteoblasts and their associated

cytokines.

In summary, our data suggest that skeletal unloading

causes a rapid decrease in the MSC and osteoprogenitor

populations, and a rapid increase in the osteoclast precursor

population in the bone marrow. Because of the short-term

nature of our studies (7 days), these changes may not be

reflected in changes in BFR/BS and bone volume. Scl-Ab

treatment in both unloaded and normally loaded mice

markedly increased MSC, osteoprogenitor, and osteoclast

precursor numbers and osteoblast surface, but did not affect

osteoclast surface. Serum concentrations of P1NP and

TRACP5b in unloaded and normally loaded, and treated

and untreated mice confirmed the pro-anabolic effects of

Scl-Ab treatment on bone and demonstrated that the ana-

bolic response of bone to Scl-Ab is preserved during

skeletal unloading or loss of weight bearing. These findings

suggest that Scl-Ab treatment may be useful in the pre-

vention and treatment of disuse osteoporosis.

56 M. Shahnazari et al.: Early Response of Bone Marrow Osteoprogenitors

123

Acknowledgments This work was supported by the Veterans

Affairs Merit Review program, NASA, and the Northern California

Institute for Research and Education.

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