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A Comparison between the Effects of Hydrophobic and Hydrophilic Statins on

Osteoclast Function In Vitro and Ovariectomy-Induced Bone Loss In Vivo

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Calcif Tissue Int (2007) 81:403–413
DOI 10.1007/s00223-007-9078-1
A Comparison between the Effects of Hydrophobic and
Hydrophilic Statins on Osteoclast Function In Vitro
and Ovariectomy-Induced Bone Loss In Vivo
Alun Hughes Æ Michael J. Rogers Æ Aymen I. Idris Æ
Julie C. Crockett
Received: 4 April 2007 / Accepted: 22 September 2007 / Published online: 3 November 2007

Springer Science+Business Media, LLC 2007
Abstract Statins potently inhibit 3-hydroxy-3-methyl-
glutaryl-coenzyme A reductase, blocking downstream
biosynthesis of isoprenoid lipids and causing inhibition of
protein prenylation. Prenylated signaling molecules are
essential for osteoclast function, consistent with our pre-
vious observation that mevastatin can inhibit osteoclast
activity in vitro. Several reports suggest that statins may
also have an anabolic effect on bone and stimulate osteo-
blast differentiation. This study sought to determine the
effects of both hydrophobic and hydrophilic statins, par-
ticularly rosuvastatin (RSV), on osteoclast function in vitro
and in vivo. All statins tested (RSV, pravastatin [PRA],
cerivastatin [CER], and simvastatin [SIM]) caused accu-
mulation of unprenylated Rap-1A in rabbit osteoclast-like
cells and J774 macrophages in vitro and inhibited osteo-
clast-mediated resorption. The order of potency for
inhibiting prenylation in vitro (at concentrations of 0.01–
50 lM) was CER [ SIM [ RSV [ PRA. The most potent
hydrophilic statin (CER, 0.05 and 0.3 mg/kg) inhibited
prenylation in rabbit osteoclasts 24 hours after a single
subcutaneous (s.c.) injection more effectively than the most
potent hydrophobic statin (RSV, 20 mg/kg). However, in a
mouse model of osteoporosis, s.c. 0.05 mg/kg/day CER and
2 or 20 mg/kg/day RSV for 3 weeks only mildly prevented
loss of cortical and trabecular bone induced by
A. Hughes (&)
M. J. Rogers
A. I. Idris
J. C. Crockett
Department of Medicine and Therapeutics, University of
Aberdeen, Institute of Medical Sciences, Foresterhill,
Aberdeen AB25 2ZD, UK
e-mail: a.hughes@abdn.ac.uk
Present Address:
A. I. Idris
Molecular Medicine Centre, Rheumatology,
Western General Hospital, Edinburgh, UK
ovariectomy. No increase in bone formation rate was
observed with statin treatment, suggesting that this effect
was due to inhibition of osteoclast-mediated resorption
rather than increased bone formation.
Keywords Osteoclast
Bone resorption
Statin

Prenylation
Anabolic
Statins are potent inhibitors of 3-hydroxy-3-methylgluta-
ryl-coenzyme A reductase (HMG-CoAR), the proximal
and rate-limiting enzyme of the mevalonate pathway [1, 2].
Inhibition of HMG-CoAR prevents the production of
cholesterol (hence the effective use of statins for the
treatment of hypercholesterolemia) but also prevents the
synthesis of isoprenoid lipids necessary for the prenylation
of small guanosine triphosphatases (GTPases), critical
signaling molecules that require the addition of an iso-
prenoid lipid tail to direct them to cell membranes [3].
Nitrogen-containing bisphosphonates (N-BPs) are another
class of drugs that, like the statins, prevent the prenylation
of GTPases but by inhibiting farnesyl diphosphate syn-
thase, an enzyme downstream of HMG-CoAR in the
mevalonate pathway [4–7]. N-BPs are potent inhibitors of
osteoclast function and are used extensively in the treat-
ment of metabolic bone diseases including hypercalcemia
of malignancy, tumor-induced osteolysis, Paget’s disease,
and postmenopausal osteoporosis [8]. We and others have
shown that statins can also inhibit bone resorption in vitro
through inhibition of the mevalonate pathway, thereby
inhibiting osteoclast function in a similar fashion to N-BPs
[9]. However, it has also been suggested that statins may
act as bone anabolic agents, stimulating the activity of
osteoblasts both in vitro and in vivo. Mundy et al. [10]
showed that simvastatin (SIM) could induce the expression
123
404
of bone morphogenetic protein 2, a member of the trans-
forming growth factor b superfamily and a key regulator of
bone morphogenesis. The anabolic effects of the statins can
be abolished in vitro by restoring protein prenylation with
the addition of downstream products of HMG-CoAR [11].
Statins could therefore affect bone metabolism via both
antiresorptive and anabolic mechanisms, each mediated by
loss of prenylated proteins.
Statins can be subgrouped according to their hydro-
phobicity or hydrophilicity. Hydrophobic statins (e.g.,
SIM) enter the liver via the hepatic portal vein, easily
diffusing through the cell membrane in their inactive lac-
tone form [12, 13]. Once in the liver cells, the lactone ring
is hydrolyzed and the free-acid form of the drug remains
abundant in the liver prior to excretion. The hydrophilic
statins (e.g., rosuvastatin [RSV] and pravastatin [PRA])
require active transport into cells via transporters pre-
dominantly expressed in the liver. Both hydrophobic and
hydrophilic statins are therefore liver-specific, with rela-
tively little perfusion to distal tissues [12, 13]. Despite this,
numerous beneficial, pleiotropic effects (e.g., on the car-
diovascular and immune systems) have been reported with
clinical use of statins [14], suggesting that sufficient con-
centrations are present outside the liver to cause effects in
more distal tissues. RSV is a potent and highly efficacious
hydrophilic statin capable of inhibiting HMG-CoAR at
lower concentrations than SIM, cerivastatin (CER), and the
comparably hydrophilic PRA [15]. Displaying high hepatic
selectivity, it is expected that RSV would display a lower
incidence of pleiotropic effects than the hydrophobic stat-
ins. Given that statins have been demonstrated to affect
bone cells [4, 9, 10], we sought to compare the effects of
hydrophobic statins (CER and SIM) and hydrophilic statins
(RSV and PRA) on osteoclasts in vitro and on bone turn-
over in ovariectomized mice.
Materials and Methods
Reagents
RSV, CER, PRA, and SIM were provided by AstraZeneca
(Macclesfield, UK). All statins were used in the free-acid
form. Stock solutions were prepared in dimethyl sulfoxide
(DMSO), diluted in culture medium, and filter-sterilized
prior to use. Alendronate (ALN) was obtained from Sigma
(Poole, UK) and prepared as a 10 mM stock solution in
phosphate-buffered saline (PBS) as described previously
[4]. Stock solutions of all-trans geranylgeraniol (GGOH,
Sigma) and mevalonic acid (MVA, Sigma) were prepared
in ethanol. Dulbecco’s modified Eagle medium (DMEM),
a-minimum essential medium (a-MEM), fetal calf serum,
(FCS), penicillin, and streptomycin were from Invitrogen
123
A. Hughes et al.: Antiresorptive Effects of Statins
(Paisley, UK); and tissue culture plates were from Costar
(Cambridge, MA). All other reagents were from Sigma,
unless indicated otherwise.
Animals
Balb-C mice and New Zealand white rabbits were housed
in a designated animal facility and given ad libitum access
to food and water. All experiments with animals were
approved under U.K. Home Office regulations.
J774.2 cells
J774.2 mouse macrophage-like cells were cultured in
DMEM containing 100 U/mL penicillin, 100 lg/mL
streptomycin, 1 mM glutamine, and 10% (vol/vol) FCS. For
Western blot analysis, the cells were seeded into six-well
plates at a density of 5.5 · 105/well in 1.5 mL of medium.
After 24 hours, the medium was replaced with medium
containing statin, ALN, or an equivalent volume of DMSO
for 24 hours. In additional experiments, cells were treated as
above together with 20 lM GGOH or 100 lM MVA.
Generation of Osteoclast-Like Cells from Rabbit Bone
Marrow
Osteoclast-like cells were generated by culturing bone
marrow from New Zealand white rabbits 2–4 days old with
1,25-dihydroxyvitamin D3 (1,25[OH]2D3), as previously
described [16]. Ten nanomoles of 1,25(OH)2D3 was added
every 3 days until cultures consisted of [90% multinu-
cleated cells (*10 days), after which medium was replaced
with medium containing statin or ALN ( ± 20 lM GGOH
or 100 lM MVA) or an equivalent volume of DMSO and
incubated for a further 24 hours.
Effect of Statins on Rap1A Prenylation
Lysates of J774 cells or rabbit osteoclast-like cells were
prepared in radioimmunoprecipitation assay (RIPA) buffer
(1% [v/v] Nonidet P-40, 0.1% [w/v] sodium dodecyl sul-
fate [SDS], 0.5% [w/v] sodium deoxycholate in PBS, plus
1% [v/v] protease inhibitor cocktail [Sigma]). Twenty
micrograms of J774 cell lysate or 50 lg of rabbit osteoclast
lysate was then electrophoresed on 12% polyacrylamide-
SDS gels (Criterion XP System; Bio-Rad, Hemel Hemp-
stead, UK) under reducing conditions. Following
electrophoresis, proteins were transferred to polyvinylidene
difluoride membrane and then hybridized with 0.2 lg/mL
A. Hughes et al.: Antiresorptive Effects of Statins
goat polyclonal anti-Rap1A antibody (sc1482; Santa Cruz
Biotechnology, Santa Cruz, CA), which recognizes only
the unprenylated form of Rap1A [17, 18], followed by
1 lg/mL anti-goat immunoglobulin G–horseradish per-
oxidase conjugate (Merck, Darmstadt, Germany).
Chemiluminescent bands were visualized using Supersig-
nal West Dura reagent (Pierce, Rockford, IL) and a Bio-
Rad Fluor-S Max MultiImager (Bio-Rad).
Effect of Statins on Osteoclast-Mediated Bone
Resorption In Vitro
Mature osteoclasts were isolated from New Zealand white
rabbits, 2–4 days old, as described previously [19]. Rabbit
pups were killed in halothane, and the limbs were removed.
Osteoclasts from the minced long bones of one rabbit were
vortexed and resuspended in 25 mL a-MEM (10% [v/v]
FCS, 100 U/mL penicillin, 100 lg/mL streptomycin, and 1
mM glutamine) and seeded (125 lL of cell suspension/well
in a 96-well tissue culture plate) onto 5-mm-diameter discs
of polished dentine. Osteoclasts were allowed to adhere for
2 hours before nonadherent cells were washed away in PBS.
Cultures were then incubated in fresh medium containing
statin or ALN (± 20 lM GGOH or 100 lM MVA) or an
equivalent volume of DMSO for 48 hours, after which the
cells on dentine slices were fixed in 4% formaldehyde.
Tartrate-resistant acid phosphatase (TRAP) staining was
performed as previously described [20]. TRAP-positive
multinucleated cells with three or more nuclei were con-
sidered to be osteoclasts. The number of osteoclasts per
dentine disc was determined, the cells were then removed
from the dentine, and resorption pits on the dentine surface
were quantified by reflected light microscopy and custom
image analysis software developed in-house using Aph-
elion ActiveX objects (ADCIS, Herouville-Saint-Clair,
France) as previously described [21].
Immunomagnetic Bead Isolation of Osteoclasts
following Statin Treatment In Vivo
Four-day-old New Zealand white rabbits were injected
subcutaneously with 2 or 20 mg/kg RSV, 0.05 or 0.3 mg/kg
CER, 1 mg/kg ALN, or an equivalent volume of PBS. After
24 hours, rabbits were killed with halothane. Mature
osteoclasts were isolated from the long bones based on the
high level of expression of avb3 integrin (vitronectin
receptor [VNR]) in osteoclasts by magnetic bead separa-
tion with 23c6 hybridoma supernatant (a kind gift from
Prof. Michael Horton, Rayne Institute, University College
London, London, UK) as described previously [18]. VNR-
positive and VNR-negative cells were separated using a
405
magnetic particle concentrator (Dynal, Oslo, Norway).
Fifty micrograms of cell lysate (in RIPA buffer) from each
fraction were used for Western blot determination of un-
prenylated Rap1A, as described above.
Analysis of the Effect of Statin Treatment on Bone
Parameters In Vivo
Fifty-six 8-week old female Balb-C mice (weighing 18.5–
20.5 g) were anesthetized and ovariectomized (OVX,
n = 28) or sham-operated (n = 28). Mice were subdivided
into groups of seven, with one subgroup from both the OVX
and sham-operated groups exposed to daily subcutaneous
(s.c.) injections of vehicle (PBS), 0.05 mg/kg CER, and 2 or
20 mg/kg RSV for 21 days. Intraperitoneal (i.p.) injections
of 20 mg/kg calcein green were performed on days 15 and
19. On day 21, mice were killed and the hind limbs, spleen,
and uterus harvested from each mouse and fixed in 4%
buffered formalin/saline (pH 7.4). Bone formation rate
(BFR) was measured by analysis of unstained decalcified
sections of the proximal right tibia using fluorescence
microscopy of calcein-stained surfaces for mineralized
perimeter (M.Pm, length of single- and double-labeled
surfaces) and mineral apposition rate (MAR, width between
double labels per day). Bone perimeter (B.Pm) was
obtained from dark field imaging of the same sections used
for measuring calcein-labeled perimeter and width.
Volumetric bone mineral content (BMC) and bone
mineral density (BMD) were measured ex vivo using
peripheral quantitative computed tomography (pQCT) on
an XCT Research M bone densitometer (Stratec Medizin-
technik, Pforzheim, Germany). A quality-assurance check
was performed daily with a Plexiglas-coated (PVC) phan-
tom according to the manufacturer’s instructions. Three
transverse sections of the proximal tibial metaphysis (0.9
mm distal to the growth plate, 0.4 mm between each sec-
tion) plus a midshaft cortical section (5.7 mm distal to the
growth plate) were measured with a voxel size of 70 lm
and analyzed with Stratec pQCT software version 5.1.4, as
previously described [22].
Three-dimensional analysis of proximal tibial metaph-
ysis trabecular structural parameters was carried out on a
SkyScan-1072 high-resolution desktop l-CT system
(Skyscan, Aartselaar, Belgium). The left tibial bone from
each animal was scanned through 180 (in 0.9 increments,
average of two frames per position) at 100 Kv (98 lA)
with a pixel size of 5.05 lm using a 0.5-mm aluminum
filter. Data set reconstruction with postalignment correction
was carried out using cone-beam reconstruction software
(Cone Rec V.2.23b, Skyscan). Two hundred slices of tra-
becular region (corresponding to a depth of 2 mm) were
selected distal to the epiphyseal growth plate, and cortical
123
406 A. Hughes et al.: Antiresorptive Effects of Statins

bone was excluded from the region of interest. Three-

dimensional analysis of bone morphometric parameters
(trabecular bone volume [BV/TV] and trabecular number
[Tb.N]) was performed using CTanalyzer software (Sky-
scan, V1.4.1.3).

Statistical Analysis

One-way analysis of variance (ANOVA) followed by

Bonferroni’s or Fischer’s least significant difference (LSD)
post hoc test were used to statistically analyze results using
SPSS version 13.0 software (SPSS, Chicago, IL). P £ 0.05
was considered statistically significant.

Results

Statins Dose-Dependently Inhibit Osteoclast-Mediated

Bone Resorption In Vitro by Inhibiting the Mevalonate
Pathway

Rabbit osteoclasts were seeded onto dentine discs and

treated with statins for 48 hours, after which the number of
osteoclasts and the area of resorbed dentine were deter-
mined (Fig. 1a). Osteoclast numbers did not decrease after
48 hours of treatment with 0.01–50 lM statin (with the
exception of 50 lM CER, which reduced the number of
TRAP-positive cells by 38%; data not shown). However,
the area of dentine resorbed per osteoclast decreased in a
concentration-dependent manner with each statin tested,
with the order of potency CER [ SIM [ RSV [ PRA
(Fig. 1a). PRA only inhibited bone resorption at concen-
trations ‡50 lM (not shown).
To determine whether inhibition of osteoclastic bone
resorption was mediated by inhibition of the mevalonate
pathway, we treated osteoclasts on dentine with 10 lM
CER, 10 lM SIM, or 50 lM RSV, with or without two
products of the mevalonate pathway: GGOH (a cell-per-
meable form of the isoprenoid pyrophosphate GGPP) and
MVA (Fig. 1b). Both GGOH and MVA restored osteo-
clast-mediated resorption in the presence of 10 lM SIM,
10 lM CER, or 50 lM RSV.
Statins Cause the Accumulation of Unprenylated
Rap1A in Osteoclasts and Macrophages
Inhibition of protein prenylation by statins was assessed
using an antibody specific for the unprenylated form of
Rap1A, a member of the Ras superfamily of small GTP-
binding proteins [17, 18]. Prenylation was inhibited in J774
mouse macrophage-like cells after 24-hour treatment with
Fig. 1 The effects of statins on rabbit osteoclast-mediated bone
resorption. (a) Mature rabbit osteoclasts seeded on dentine discs were
treated for 48 hours with 0.01–50 lM CER, RSV, or SIM or 100 lM
ALN prior to quantification of TRAP-positive osteoclast numbers and
area of mineral surface resorbed. (b) Mature rabbit osteoclasts seeded
on dentine discs were treated for 48 hours with 10 lM CER, 10 lM
SIM, or 50 lM RSV in the absence or presence of either 20 lM
GGOH or 100 lM MVA prior to quantification of TRAP-positive
osteoclasts and area of mineral surface resorbed. Data are expressed
as means ± standard error of the mean from at least three individual
experiments (three replicates per experiment), expressed as a
percentage of the area of mineral resorbed per osteoclast in untreated
cultures. *P \ 0.05, **P \ 0.01, and ***P \ 0.01 values are signif-
icantly different from untreated control.
P \ 0.05,

P \ 0.01, and




P \ 0.001 values are significantly different from statin treatment
alone (ANOVA, Bonferroni post hoc)
0.01–10 lM statin (Fig. 2a), with a similar order of
potency to that observed for inhibition of bone resorption.
Accumulation of unprenylated Rap1A could be detected in
J774 cell lysates after treatment with 0.1 lM CER, 1 lM
SIM, or 10 lM RSV, while PRA barely affected Rap1A
prenylation even at 10 lM (Fig. 2a). Similar effects were
observed in rabbit osteoclast-like cells (Fig. 2c). Concen-
trations of 0.1 lM CER, 1 lM SIM, and 10 lM RSV
caused the accumulation of unprenylated Rap1A, while
PRA had little effect on Rap1A prenylation at concentra-
tions up to 10 lM (Fig. 2c). The effect of statins on Rap1A
prenylation was rapid; 10 lM RSV caused detectable

123
A. Hughes et al.: Antiresorptive Effects of Statins
Fig. 2 The effect of statins on accumulation of unprenylated Rap1A.
J774 cells were treated with (a) 0.01–10 lM CER, PRA, RSV, or
SIM or 100 lM ALN for 24 hours or (b) 10 lM CER, PRA, RSV, or
SIM for 24 hours in the presence or absence of either 20 lM GGOH
or 100 lM MVA. 1,25(OH)2D3-generated rabbit osteoclast-like cells
were treated with (c) 0.01–10 lM CER, PRA, RSV, or SIM or 100
accumulation of unprenylated Rap1A in J774 and osteo-
clast-like cells within 30 min of treatment (data not
shown).
Addition of 100 lM MVA completely prevented the
accumulation of unprenylated Rap1A in J774 cells treated
with CER, SIM, or RSV, while 20 lM GGOH partially
prevented the accumulation of unprenylated Rap1A
(Fig. 2b). In osteoclast-like cells, MVA and GGOH
appeared to be equally effective at preventing the accu-
mulation of unprenylated Rap1A in the presence of 10 lM
CER, SIM, or RSV (Fig. 2d).
Single Injections of RSV and CER Are Sufficient to
Inhibit Prenylation in Bone Marrow Cells
To determine whether sufficient concentrations of the
hydrophilic RSV could reach bone tissue to inhibit protein
407
lM ALN for 24 hours or (d) 10 lM CER, PRA, RSV, or SIM for 24
hours in the presence or absence of either 20 lM GGOH or 100 lM
MVA. Western blot analysis was then performed on cell lysates
using an antibody that specifically recognizes 21-kDa unprenylated
Rap1A. An antibody against b-actin was used as a loading control.
Data are representative of at least three independent experiments
prenylation and thereby inhibit bone resorption in vivo, we
injected 4-day-old rabbits with 0.05 or 0.3 mg/kg CER,
doses previously demonstrated to inhibit prenylation in
bone cells in vivo [9], or with 2 or 20 mg/kg RSV. Twenty-
four hours after s.c. injection with statin or PBS, rabbits
were killed, long bones were minced, and cell fractions
were separated based on the expression of VNR (avb3
integrin). VNR+ cells isolated by antibody selection and
magnetic bead separation were verified as osteoclasts by
light microscopy (large, multinucleated, and stained posi-
tive for TRAP), while the VNR– fraction contained the
remainder of the bone marrow cells. The hydrophobic CER
strongly inhibited Rap1A prenylation in vivo at both 0.05
and 0.3 mg/kg doses in both VNR+ and VNR– fractions
(Fig. 3a). RSV had no detectable effect on Rap1A prenyl-
ation in either cell fraction at a dose of 5 mg/kg. However, a
clear band of unprenylated Rap1A could be detected in
lysates of cells from both the VNR+ and VNR– fractions
123
408
Fig. 3 Inhibition of Rap1A prenylation by statins in vivo. Four-day-
old New Zealand white rabbits were injected with (a) 2 or 20 mg/kg
RSV or 0.03 or 0.5 mg/kg CER or (b) 1 mg/kg ALN. Twenty-four
hours after injection, VNR+ cells (osteoclasts) and VNR– cells were
isolated from bone marrow, and cell lysates were analyzed by
after treatment with the 20 mg/kg dose. By contrast, as
expected, the bisphosphonate ALN (which is selectively
internalized by osteoclasts [23, 24]) caused the accumula-
tion of unprenylated Rap1A only in the VNR+ osteoclast
fraction, even at the very high dose of 1 mg/kg (Fig. 3b).
RSV and CER Partially Protect against Ovariectomy-
Induced Bone Loss in Mice
Having established that injections of high-dose RSV
caused mild but detectable inhibition of protein prenylation
in osteoclasts in vivo, we next addressed the effects of
long-term administration of RSV compared to CER in a
murine model of acute bone loss induced by ovariectomy.
Three days after surgery, daily injection with statin was
commenced. On termination of the experiment on day 21,
each mouse was weighed and killed and the hind limbs,
spleen, and uterus were harvested. Uterus weight decreased
significantly in OVX compared to sham-operated animals,
confirming the success of the OVX surgery. There was no
significant variance in spleen weight, indicating the lack of
postoperation infection in any of the mice. No deleterious
effects on weight were observed with daily statin treat-
ment; OVX groups tended to be heavier than sham groups
(by approximately 1 g, data not shown).
Ex vivo examination of the proximal tibia by pQCT
revealed protection of cortical BMD and BMC from OVX-
induced loss following 21 days of statin treatment (Fig. 4a,
123
A. Hughes et al.: Antiresorptive Effects of Statins
Western blot using an antibody that specifically recognizes 21-kDa
unprenylated Rap1A. An antibody against b-actin was used as a
loading control. Data are representative of at least three independent
experiments
b). Both CER and RSV significantly preserved cortical
BMD in OVX mice, with 20 mg/kg/day RSV providing the
greatest protection (11.4% decrease in BMD with OVX
compared to sham control vs. 3.6% loss with 20 mg/kg/day
RSV) (Fig. 4a). To a lesser extent, cortical BMC was also
protected by CER and RSV treatment, with OVX causing a
45.0% decrease in BMC compared to sham controls vs. a
decrease of only 33.9% in 20 mg/kg/day RSV–treated mice
(Fig. 4b). In addition to protective effects on BMD and
BMC, a significant (P [ 0.05) increase of 4.2% in cortical
BMD was observed in sham-operated mice with 2 mg/kg/
day RSV, a trend that was also observed (but did not reach
significance) with 0.05 mg/kg CER and 20 mg/kg/day RSV
treatment (Fig. 4a).
Trabecular bone parameters were measured by lCT
analysis of the proximal tibia (Fig. 4c, d). OVX caused a
54.3% decrease in BV/TV compared to sham animals vs. a
loss of 39.5% with 2 mg/kg/day RSV (Fig. 4c). A similar
magnitude of protection for Tb.N was exhibited with 2 mg/
kg/day RSV treatment, decreasing the loss of Tb.N from
49.1% in control OVX to 37.3% with RSV treatment
(Fig. 4d). No further benefit was observed with 20 mg/kg/
day RSV in either parameter. In concordance with BV/TV
and Tb.N, we observed no difference in trabecular separation
(Tb.Sp) in sham animals following statin treatment, but
OVX increased Tb.Sp in control animals by 57.6%
(P \ 0.001, data not shown). OVX mice treated with CER
and 20 mg/kg/day RSV partially restored Tb.Sp to 33.6%
(P \ 0.01) and 37.3% (P \ 0.05), respectively, compared to
A. Hughes et al.: Antiresorptive Effects of Statins
Fig. 4 The effects of repeated administration of RSV and CER on
physical bone parameters. OVX or sham-operated 8-week-old
female Balb-C mice were treated for 21 days with 0.05 mg/kg
CER, 2 or 20 mg/kg RSV, or PBS control by s.c. injection. Proximal
tibial cortical (a) BMD and (b) BMC were obtained by pQCT 0.9
mm distal to the growth plate. Three-dimensional analysis of (c)
trabecular bone volume and (d) trabecular number was obtained
OVX control, while 2 mg/kg/day RSV did not cause a sig-
nificant decrease in Tb.Sp induced by OVX (data not shown).
Representative three-dimensional reconstructions of tra-
becular bone in the proximal tibia are shown in Figure 5.
Statins Cause a Decrease in BFR In Vivo
The width between calcein labels injected 4 days apart was
used to calculate MAR and BFR. In control animals, BFR
increased significantly following OVX (P \ 0.01)
(Table 1), consistent with an increase in bone turnover. In
sham animals, no significant difference was observed in
either MAR (data not shown) or BFR (Table 1) following
either CER or RSV treatment. However, in OVX animals
CER and both doses of RSV significantly decreased BFR
compared to untreated OVX animals to levels that were not
significantly different from those in sham animals.
Discussion
The aims of this study were to compare the effects of
hydrophilic and hydrophobic statins on osteoclasts in vitro
409
from the tibial metaphysis by lCT. Data are displayed as percentage
change from untreated sham average, open bars denote sham animals
and closed bars represent OVX animals. Values are the average of
seven animals per group ± standard error of the mean. *P \ 0.05,
**P \ 0.01, and ***P \ 0.001 values are significantly different
from OVX control.
P \ 0.05 values are significantly different from
sham control (ANOVA, LSD post hoc test)
and on bone turnover in vivo. Statins inhibit HMG-CoAR,
a proximal enzyme in the mevalonate pathway. As a result
of inhibition of HMG-CoAR, statins decrease the biosyn-
thesis of cholesterol and have been shown to inhibit the
synthesis of prenyl groups that are important for membrane
targeting of small GTPase proteins involved in osteoclast
function [1, 3, 6, 19].
Previous work in our laboratory demonstrated that
mevastatin potently inhibits osteoclast-mediated resorption
in murine calvaria in vitro [4]. In this present study, in
rabbit osteoclasts, all four statins tested caused an accu-
mulation of unprenylated proteins that corresponded to
their ability to inhibit osteoclast-mediated resorption in
vitro. The order of potency (CER [ SIM [ RSV [ PRA)
for inhibiting prenylation was similar in J774 macrophage
cells. Protein geranylgeranylation has been shown to be
essential for osteoclast function and requires the synthesis
of geranylgeranyl diphosphate (GGPP) that occurs down-
stream from HMG-CoAR in the mevalonate pathway.
GGOH, the cell-permeable form of GGPP, can be used to
replenish the pool of GGPP that is depleted by HMG-
CoAR inhibition and, hence, restore geranylgeranylation of
signaling molecules (including Rap1A) [6, 25–28]. GGOH
partially restored osteoclast activity and at least partially
123
410 A. Hughes et al.: Antiresorptive Effects of Statins

Fig. 5 Three-dimensional reconstructions of regions of interest data set used to calculate the lCT data in Figure 4e and f. Each
measured by lCT. Images are top–down views of trabecular reconstruction is of a tibia closest to the group median for BV/TV
structure in the proximal tibial metaphysis, reconstructed from the and Tb.N

Table 1 Effects of repeated administration of RSV and CER on BFR prevented the accumulation of unprenylated Rap1A (a
2
BFR (lm /lm/day) surrogate marker of protein prenylation) in J774 and
osteoclast-like cells following statin treatment. MVA also
Sham OVX
partially restored osteoclast activity but fully restored (at
Control 0.53 ± 0/07** 0.75 ± 0.06 least within the limit of detection by Western blotting) the
Cerivastatin, 0.05 mg/kg/day 0.61 ± 0.05* 0.50 ± 0.06*** prenylation of Rap1A in both cell types. The failure of
Rosuvastatin GGOH to fully prevent the loss of prenylated Rap1A was
2 mg/kg/day 0.59 ± 0.05* 0.58 ± 0.05* probably due to the sensitivity of cells to the cytotoxic
20 mg/kg/day 0.61 ± 0.06 0.60 ± 0.07* effects of GGOH (data not shown), limiting the maximal
concentration that could be used without detriment to the
OVX or sham-operated 8-week-old female Balb-C mice were treated
for 21 days with 0.05 mg/kg CER, 2 or 20 mg/kg RSV, or PBS control cells. Nonetheless, the recovery of prenylated Rap1A and
by daily s.c. injection. BFRs (lm2/um/day) were determined by the osteoclast-mediated bone resorption in the presence of
MAR (lm/day between calcein labels) multiplied by M.Pm (lm) GGOH and MVA is consistent with the conclusion that
divided by B.Pm (lm). Values are the average of seven animals per statins inhibit bone resorption in vitro by inhibiting the
group ± standard error of the mean. *P \ 0.05, **P \ 0.01, and
***P \ 0.001 are values significantly different from OVX control downstream formation of isoprenoids in the mevalonate
(ANOVA, LSD post hoc test) pathway that are required for protein prenylation.

123
A. Hughes et al.: Antiresorptive Effects of Statins
Having shown that both hydrophobic and (to a lesser
extent) hydrophilic statins decrease protein prenylation in
vitro, we next investigated if both classes of statin could
inhibit prenylation in vivo. In accordance with the in vitro
data, we also found that RSV, and particularly CER,
inhibited protein prenylation in bone cells in vivo. Pre-
nylation was disrupted in VNR+ and VNR– fractions of
cells isolated from rabbit long bone homogenates 24 hours
after s.c. injection of either 20 mg/kg RSV or 0.05 mg/kg
CER. Since loss of prenylated proteins correlates well with
loss of osteoclast function in vitro (Figs. 1 and 2), this
indicates that sufficiently high concentrations of circulating
statins may reach the bone microenvironment and could
inhibit osteoclast function in vivo following treatment with
relatively high doses of statins compared to an equivalent
human dose. However, the effect of both RSV and CER on
prenylation in vivo was considerably less than that of a
dose of ALN and did not display the osteoclast specificity
of the bisphosphonate. No obvious inhibition of Rap1A
prenylation was detectable in bone cells 24 hours after
treatment with 5 mg/kg RSV, but given the long elimina-
tion half-life of RSV (20 hours in humans [29]), it was
considered plausible that, in further in vivo experiments,
daily dosing with 2 mg/kg RSV could cause a cumulative
effect on prenylation in bone cells.
In order to examine the in vivo effects of statin treat-
ment on bone, we used a mouse model of osteoporosis
induced by OVX. The 2 mg/kg dose of RSV and the 0.05
mg/kg dose of CER used in this in vivo study represent
approximately four to five times the maximal oral dose
currently or previously used clinically in humans. OVX
mice treated for 3 weeks with either CER (0.05 mg/kg/day)
or RSV (2 and 20 mg/kg/day) displayed mild preservation
of both cortical (measured by pQCT) and trabecular
(measured by lCT) bone compared to control mice. pQCT
measurement of the tibia revealed a significant increase in
BMD in sham-operated 2 mg/kg RSV–treated animals
compared to controls. However, no changes in either cor-
tical midshaft parameters (by pQCT, data not shown) were
observed in sham-operated mice following RSV treatment,
which suggests this increase is not due to an anabolic
effect. Aside from proximal tibia BMD, all of the signifi-
cant improvements in bone parameters were observed in
statin-treated OVX animals but not in sham-operated ani-
mals. No further improvement of any parameters measured
was observed with the increase of RSV dose from 2 to 20
mg/kg/day. The lack of a dose-dependent effect observed
with increasing concentration of RSV may be due to a
dose-saturation effect, possibly caused by drug distribution
or limited uptake into cells within the bone microenvi-
ronment due to the lack of specific transporters for the
hydrophilic RSV. Alternatively, the mild nature of the
changes observed may suggest that a substantially larger
411
dose than the tenfold difference we used may provide dose
dependence, but this would require the use of a clinically
disproportionate dose.
To determine whether the improvement in bone
parameters in statin-treated OVX mice was due to anabolic
or antiresorptive effects, we measured MAR and BFR, both
dynamic measures of bone turnover. As expected, the
osteoclast activity caused by OVX stimulated an increase
in MAR and BFR, indicating upregulation of bone turn-
over, with a significant loss of both trabecular and cortical
bone in OVX animals. Statin treatment abolished the OVX-
induced increase in BFR, indicating restoration of normal
bone turnover. Importantly, statin treatment did not
increase either MAR or BFR in sham or OVX mice,
indicating the lack of a bone anabolic effect. We therefore
conclude that the mild improvement in bone parameters in
statin-treated OVX mice is due to an antiresorptive, rather
than anabolic, effect.
There is an abundance of in vitro and in vivo data to
support the conclusion that statins have the potential to
affect bone remodeling, but the question remains as to
whether this translates into any clinical relevance in
humans. Retrospective analyses of case-control studies
tend to demonstrate a decreased incidence of fracture in
populations taking a wide range of statins [30–33], but this
trend is not replicated in small randomized controlled trials
[34–36]. There is no evidence to support PRA having any
positive effects on bone turnover in humans [33, 34, 36],
suggesting that the similarly hydrophilic RSV is also
unlikely to have marked effects on bone turnover in
humans at the doses currently used. Our data support this
hypothesis since only mild protective effects were seen in
OVX mice using very high doses of RSV.
Our results do not correlate with the observation origi-
nally made by Mundy et al. [10] that statins have an
anabolic effect on bone. Although there have been several
subsequent in vitro and in vivo studies supporting an
anabolic mechanism of action [11, 37–40], other studies
corroborate our own observations that statins exhibit an
antiresorptive effect [9, 41–44]. Choice of statin or route of
administration [37] may affect the response observed,
although in our in vivo study we used the most potent
hydrophilic and hydrophobic statins and observed no
increase in BFR following 3 weeks of daily s.c. adminis-
tration. Differing doses of SIM have been demonstrated to
differentially effect bone turnover [45], suggesting that the
dose may also influence the result observed, although we
did not see any difference in effect between the 2 and 20
mg/kg doses of RSV we used in vivo.
Given equal levels of cellular uptake, RSV would be
expected to be the most potent statin at inhibiting prenyl-
ation and osteoclast function, due to its lower half-maximal
inhibitory concentration for inhibiting purified HMG-CoA
123
412
reductase [46]. However, CER and SIM were clearly more
potent than RSV at inhibiting protein prenylation in
osteoclasts in vitro and in vivo, presumably since CER and
SIM do not require active transport and are therefore
internalized more efficiently than RSV, which (like PRA)
requires active transport [47]. Since the transport systems
for uptake of RSV and PRA appear to be liver-specific
[48], passive diffusion probably accounts for most of the
cellular uptake of RSV and PRA by osteoclasts observed in
our study. RSV was more effective than PRA on osteo-
clasts and protein prenylation in vitro, presumably
reflecting the much higher potency of RSV than PRA for
inhibiting HMG-CoAR.
In summary, we have demonstrated that hydrophobic and
hydrophilic statins can inhibit osteoclast function in vitro by
preventing the prenylation of small GTPases. Furthermore,
we have shown that high doses of statins can inhibit protein
prenylation in osteoclasts in vivo and that daily s.c. treat-
ment with high doses of hydrophobic CER or hydrophilic
RSV can mildly prevent the loss of bone caused by OVX in
mice, probably via an antiresorptive effect. However,
despite these findings, given the predominantly liver-spe-
cific targeting of orally administered statins, especially the
hydrophilic PRA and RSV [47], it appears unlikely that
sufficient circulating levels of statin (particularly hydro-
philic statins) would reach the bone microenvironment
following oral administration of normal doses of statins to
substantially affect bone remodeling in humans.
Acknowledgments This work was funded by a grant from
AstraZeneca.
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