Absence of ERRalpha in female mice confers resistance to bone loss induced by age or estrogen-deficiency.
ABSTRACT ERRalpha is an orphan member of the nuclear hormone receptor superfamily, which acts as a transcription factor and is involved in various metabolic processes. ERRalpha is also highly expressed in ossification zones during mouse development as well as in human bones and cell lines. Previous data have shown that this receptor up-modulates the expression of osteopontin, which acts as an inhibitor of bone mineralization and whose absence results in resistance to ovariectomy-induced bone loss. Altogether this suggests that ERRalpha may negatively regulate bone mass and could impact on bone fragility that occurs in the absence of estrogens.
In this report, we have determined the in vivo effect of ERRalpha on bone, using knock-out mice. Relative to wild type animals, female ERRalphaKO bones do not age and are resistant to bone loss induced by estrogen-withdrawal. Strikingly male ERRalphaKO mice are indistinguishable from their wild type counterparts, both at the unchallenged or gonadectomized state. Using primary cell cultures originating from ERRalphaKO bone marrow, we also show that ERRalpha acts as an inhibitor of osteoblast differentiation.
Down-regulating ERRalpha could thus be beneficial against osteoporosis.
- SourceAvailable from: Gary Kunkel[show abstract] [hide abstract]
ABSTRACT: We have identified a novel zinc finger-containing transcription factor, called Osterix (Osx), that is specifically expressed in all developing bones. In Osx null mice, no bone formation occurs. In endochondral skeletal elements of Osx null mice, mesenchymal cells, together with osteoclasts and blood vessels, invade the mineralized cartilage matrix. However, the mesenchymal cells do not deposit bone matrix. Similarly, cells in the periosteum and in the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. These cells do, however, express Runx2/Cbfa1, another transcription factor required for bone formation. In contrast, Osx is not expressed in Runx2/Cbfa1 null mice. Thus, Osx acts downstream of Runx2/Cbfa1. Because Osx null preosteoblasts express typical chondrocyte marker genes, we propose that Runx2/Cbfa1-expressing preosteoblasts are still bipotential cells.Cell 02/2002; 108(1):17-29. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Runx2, osterix, and beta-catenin are essential for osteoblast differentiation. Runx2 directs multipotent mesenchymal cells to an osteoblastic lineage, and inhibits them from differentiating into the adipocytic and chondrocytic lineages. After differentiating to preosteoblasts, beta-catenin, osterix, and Runx2 direct them to immature osteoblasts, which produce bone matrix proteins, blocking their potential to differentiate into the chondrocytic lineage. Runx2 inhibits osteoblast maturation and the transition into osteocytes, keeping osteoblasts in an immature stage. Other transcription factors including Msx1, Msx2, Dlx5, Dlx6, Twist, AP1(Fos/Jun), Knox-20, Sp3, and ATF4 are also involved in osteoblast differentiation. To gain an understanding of bone development, it is important to position these transcription factors to the right places in the processes of osteoblast differentiation.Journal of Cellular Biochemistry 01/2007; 99(5):1233-9. · 3.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) are dominant regulators of bone resorption. Many hormones, cytokines and growth factors mediate bone resorption by altering the ratio of RANKL to OPG. RANKL and OPG expression is also altered in numerous bone diseases, and these changes can reflect disease etiology or compensatory responses to disease. RANKL stimulates osteoclast formation, function and survival, and each of these effects is inhibited by OPG. OPG suppresses bone resorption and increases the density, area and strength of both cancellous and cortical bone. Denosumab (AMG 162), a fully human monoclonal antibody to RANKL, shares the pharmacologic attributes of OPG but has a significantly longer half-life that allows less frequent administration.Current Opinion in Pharmacology 01/2006; 5(6):618-25. · 5.44 Impact Factor
Absence of ERRa in Female Mice Confers Resistance to
Bone Loss Induced by Age or Estrogen-Deficiency
Catherine Teyssier1., Marle `ne Gallet1., Be ´ne ´dicte Rabier2, Laurent Monfoulet2, Julien Dine2, Claire
Macari1, Julie Espallergues3, Be ´atrice Horard4, Vincent Gigue `re5, Martine Cohen-Solal6, Olivier
Chassande1,2, Jean-Marc Vanacker1*
1Institut de Ge ´nomique Fonctionnelle de Lyon, Universite ´ de Lyon, Universite ´ Lyon 1, Centre National de la Recherche Scientifique, Institut National de la Recherche
Agronomique, Ecole Normale Supe ´rieure de Lyon, Lyon, France, 2Institut National de la Sante ´ et de la Recherche Me ´dicale U 577, Universite ´ Victor Segalen Bordeaux II,
Bordeaux, France, 3Institut National de la Sante ´ et de la Recherche Me ´dicale U710, Universite ´ de Montpellier II, Montpellier, France, 4Laboratoire de Biologie Mole ´culaire
de la Cellule, CNRS UMR5239, Ecole Normale Supe ´rieure de Lyon, Villeurbanne, France, 5The Rosalind and Morris Goodman Cancer Centre, Montre ´al, Canada, 6Institut
National de la Sante ´ et de la Recherche Me ´dicale U606, Ho ˆpital Lariboisie `re, Paris, France
Background: ERRa is an orphan member of the nuclear hormone receptor superfamily, which acts as a transcription factor
and is involved in various metabolic processes. ERRa is also highly expressed in ossification zones during mouse
development as well as in human bones and cell lines. Previous data have shown that this receptor up-modulates the
expression of osteopontin, which acts as an inhibitor of bone mineralization and whose absence results in resistance to
ovariectomy-induced bone loss. Altogether this suggests that ERRa may negatively regulate bone mass and could impact
on bone fragility that occurs in the absence of estrogens.
Methods/Principal Findings: In this report, we have determined the in vivo effect of ERRa on bone, using knock-out mice.
Relative to wild type animals, female ERRaKO bones do not age and are resistant to bone loss induced by estrogen-
withdrawal. Strikingly male ERRaKO mice are indistinguishable from their wild type counterparts, both at the unchallenged
or gonadectomized state. Using primary cell cultures originating from ERRaKO bone marrow, we also show that ERRa acts
as an inhibitor of osteoblast differentiation.
Conclusion/Significance: Down-regulating ERRa could thus be beneficial against osteoporosis.
Citation: Teyssier C, Gallet M, Rabier B, Monfoulet L, Dine J, et al. (2009) Absence of ERRa in Female Mice Confers Resistance to Bone Loss Induced by Age or
Estrogen-Deficiency. PLoS ONE 4(11): e7942. doi:10.1371/journal.pone.0007942
Editor: Sudha Agarwal, Ohio State University, United States of America
Received September 7, 2009; Accepted October 13, 2009; Published November 20, 2009
Copyright: ? 2009 Teyssier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Inserm (Programme National de Recherche sur les maladies osteo-articulaires), Agence Nationale pour la Recherche (grant
ANR-08-GENOPAT-012). C.T. has been funded by Fondation pour la Recherche Medicale. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Bone is a highly dynamic tissue subjected to active remodeling,
an equilibrium between construction by osteoblasts and resorption
by osteoclasts. Osteoblast are derived from mesenchymal stem
cells (MSCs) and their differentiation is promoted by various
factors such as the transcription factors Osterix and Runx2 [1–3].
Mature osteoblasts express receptor activator of nuclear factor-kB
ligand (RANKL), a protein that will signal through RANK, a
member of the tumor necrosis factor receptor superfamily present
at the surface of pre-osteoclasts, and induce differentiation of these
cells [4–7]. On another hand, osteoblasts, as well as other cell
types, also express and secrete osteoprotegerin (opg), a decoy
receptor that traps RANKL in the extracellular milieu, preventing
it from acting on pre-osteoclasts, and thus inhibiting the
differentiation of osteoclasts [8,9].
Stability of bone remodeling also requires other diffusible factors
among which estrogens are instrumental. This is particularly
illustrated after menopause in aging women, when the fall of
circulating level of estrogens leads to enhanced bone remodeling
with an excessive resorption [10,11]. The resulting osteoporosis
syndrome is associated with an enhanced fracture risk. Various
means are currently available to prevent and treat osteoporosis,
most of which mainly aim at inhibiting the excess of bone
resorption. These include for example the use of estrogens (natural
or synthetic) or of bisphosphonate to reduce osteoclast differen-
tiation or induce their apoptosis, respectively .
The effects of estrogens are mediated by two estrogen receptors
(ERs) a and b, which act as ligand-dependent transcription factors
and belong to the nuclear receptor superfamily . This family
also comprises so-called orphan receptors, i.e. for which no ligand
has been identified to date . Estrogen-Receptor Related
receptor a (ERRa) was among the first orphan receptors isolated,
based on its sequence similarity to ERa [14,15]. Despite this close
proximity, ERRa does not bind estrogen nor any identified
natural ligand . ERRa is expressed in several tissues both
PLoS ONE | www.plosone.org1 November 2009 | Volume 4 | Issue 11 | e7942
during embryonic development and in the adult . A number of
studies have identified ERRa as a major actor in the regulation of
energy metabolism in oxidative tissues such as heart or slow-twitch
skeletal muscle [18, 19 for reviews]. This receptor is indeed
involved in the regulation of energy uptake, storage and
consumption as well as in mitochondrial biogenesis and function
and cardiac response to stress. Most of these effects are thought to
depend on the PGC-1a coactivator, with which ERRa physically
interacts. High expression of ERRa in various human tumors
(originating from such organ as breast, colon and ovary) correlates
with a poor prognosis [reviewed in 20]. Proliferation of various
cancer cell lines can be inhibited by a synthesis modulator of
ERRa, but not by its mere siRNA-mediated knocking-down .
ERRa is highly expressed in all ossification zones during mouse
development, as well as in osteoblastic lines and normal human
bones . Furthermore, a polymorphic variant of the human
ERRa promoter has been associated with variable bone mineral
density (BMD) in premenopausal women . Although the
molecular functions of ERRa in bone cells have not been
determined, osteopontin (opn), an inhibitor of bone mineralization
[24–30], has been identified as a positive ERRa transcriptional
Here, we show that ERRaKO female mice resist to age-induced
bone loss and present a more elevated bone formation rate as
compared to wild type animals. Remarkably, ERRa deficiency
also conferred resistance to bone loss induced by estrogen
withdrawal. Noteworthy, no phenotype was observed in the bones
of ERRa-deficient males. ERRaKO originating MSCs displayed
enhanced capacities to differentiate into osteoblasts, together with
a lower opn expression, as compared to wt MSCs. Altogether our
results reveal the role of ERRa in the control of bone density and
suggest important cross talks between ERRa and hormonal
signalling pathways in bone physiology.
Results and Discussion
ERRa Deficiency Affects Bone Aging in Female, but Not in
The structural parameters of cortical and trabecular bone were
measured in the femur diaphysis and metaphysis, respectively, of
wild type (wt) and ERRa knockout female mice at 14 and 24
weeks. Cortical bone mineral density (BMD) and thickness
augmented with age in a similar manner in both genotypes
(Fig. 1A and B). The trabecular mineral density (TMD) was
slightly more elevated (but not statistically significant) in wt
animals as compared to ERRaKO, but significantly increased
with time in the latter genotype (Fig. 1C). Lower trabecular bone
volume (BV/TV; Fig. 1D) and trabecular number (TbN; Fig. 1E)
were observed in ERRaKO animals relative to wt ones at 14 wk.
Interestingly whereas these two parameters were significantly
reduced at 24 wk in wt animals as an effect of age, no significant
variation occurred in ERRaKO mice. These results indicate that
ERRaKO female mice, although deficient in bone trabeculae at
maturity (14 wk), are resistant to age-related bone loss. Strikingly
no difference was observed in any of the structural parameters of
both cortical and trabecular bone between wild type and mutant
male mice (Table 1), indicating a gender-dependent effect of the
absence of ERRa.
The evolution of bone between wild type and ERRaKO
females prompted us to examine the parameters of bone
resorption and bone formation at 14 wk of age (i.e. before a
significant difference could be observed between genotypes). Bone
formation rate, which represent the activity of osteoblasts, was
more elevated in ERRaKO females than in wt animals (Fig. 2A).
The surface occupied by osteoblasts was identical in both
genotypes (Fig. 2B). Serum markers of bone resorption (C-
terminal fragment of collagen 1 [Ctx], indicative of osteoclast
activity) were not different between wild type and mutant females,
indicating that bone resorption was not affected by the ERRa
deficiency (Fig. 2C). Consistently, a comparable number of
osteoclasts (Fig. 2D), spaning an identical relative surface
(Fig. 2E) were detected in both strains.
Altogether this suggests that the resistance of ERRaKO animals
to age-induced bone loss could be due to an enhanced osteoblastic
activity, and not to variation in osteoclast-related parameters.
Figure 1. Bone phenotype of ERRaKO females mice. Cortical
bone mineral density (BMD; A), cortical thickness (B), trabecular mineral
density (TMD; C), bone volume fraction (BV/TV; D) and trabecular
number (TbN; E) were determined at 14 (white bars) and 24 wk (black
bars) in the femur of female wt and ERRaKO mice (n=6 to 9). Errors bars
represent s.e.m. ns: not significant; *:p,0.05; *** p,0.005.
ERRa and Bone Loss
PLoS ONE | www.plosone.org2 November 2009 | Volume 4 | Issue 11 | e7942
ERRa Negatively Regulates Osteoblast Differentiation
We thus analyzed the capacities of ERRaKO bone marrow
mesenchymal cells to differentiate into osteoblasts ex vivo. After
seven days in the appropriate medium, ERRa mutant cells
produced a greater number of differentiation foci as estimated by
alkaline phosphatase (ALP) staining (Fig. 3A). This was accompa-
nied by a higher mineralization activity, as revealed by von Kossa
staining. As measured by quantitative PCR, the expression of
various osteoblast differentiation markers, such as Runx-2, ALP or
osteocalcin was found more elevated in ERRaKO cells by
quantitative PCR. In contrast, opn, an ERRa target gene [31–
33], was less expressed in ERRaKO cells than in wild type cells.
We also analyzed the kinetics of ALP expression during osteoblast
differentiation (Fig. 3B). In ERRaKO cells, ALP expression
followed the same profile as in wild-type cells but was more
elevated at each time point. The phenotype displayed by mutant
cells was rescued by infection with an ERRa-encoding adenovirus
(AdERRa; Fig. 3C). Indeed, ALP staining was reduced by
AdERRa infection as compared to cells infected with a GFP-
encoding adenovirus. AdERRa also reduced the mRNA expres-
sion of Runx-2, ALP and osteocalcin. This repression was not a
general, non-specific phenomenon since osteopontin expression
was enhanced by AdERRa, as expected. This suggests that the
capacities of MSCs to differentiate into osteoblasts are enhanced in
the absence of ERRa and that the number of precursor cells is not
modified. The results above may reflect an effect of the absence of
ERRa on osteoblast proliferation rather than on differentiation.
To evaluate this hypothesis, we determined the growth capacities
of bone marrow cells (Fig. 3D). ERRaKO cells displayed identical
growth rate as wild-type cells, as estimated by determining cell
number after 3 and 7 days in culture. However, under these
conditions, ERRaKO cells still displayed a higher ALP expression
as measured by staining and mRNA expression. We thus
concluded that the absence of ERRa enhanced the osteoblastic
differentiation of bone marrow cells without modifying their
proliferation capacities. This is in agreement with recent results
showing that the inhibition of ERRa expression in mammary cells
does not modify proliferation . Our results are in contrast with
previous data suggesting that ERRa promotes osteoblast differen-
tiation . However, it is worth noting that our study analyzed
femoral mesenchymal cells (which form endochondral bone, i.e.
requiring a cartilage anlagen) derived from ERRaKO mice,
whereas Bonnelye et al.  used transiently transfected rat
calvarial cells (which form intramembranous bone). These
methodological differences might account for the apparent
discrepancies found between both studies. In contrast, our data
are in agreement with published results in which MSCs originating
from an independent ERRaKO strain were found more prone to
differentiate into osteoblasts . Interestingly this report also
show that ERRa concomitantly promotes adipocyte differentiation
of MSCs, suggesting that the receptor modulates an inverse
relationship between adipogenic and osteogenic commitment of
MSCs. In further support of our data, it is also worth noting that
the closely related ERRc receptor has recently been found to
inhibit BMP2-induced osteoblast differentiation in vitro as well as
BMP2-induced ectopic bone formation in vivo . Elevated
osteoblast differentiation ex vivo in the absence of ERRa is
furthermore consistent with the more elevated BFR found in vivo in
ERRaKO mice. Furthermore, opn has been shown to inhibit
bone mineralization [24–30]. A lower level of opn expression in
the absence of ERRa could thus result in higher mineralization as
observed in aging ERRaKO mice.
Table 1. Trabecular bone parameters of wt and ERRaKO male mice (n=8 to 10).
14 wk24 wk Orx1
wt ERRaKOwt ERRaKO wt ERRaKO
TMD2(mg/cc)752 +/2 43
19.3 +/2 1.9
7.7 +/2 0.6
770 +/2 33
19 +/2 0.7
7.5 +/2 0.4
731 +/2 8
9.4 +/2 0.8
4.7 +/2 0.3
694 +/2 7
10 +/2 0.4
4.5 +/2 0.5
598 +/2 14
5.9 +/2 0.2
3.6 +/2 0.3
570 +/2 19
5.5 +/2 0.5
3.9 +/2 0.3
1: orchidectomized animals.
2: trabecular mineral density.
3: bone volume/tissue volume.
4: trabecular number.
Figure 2. Dynamic parameters of ERRaKO female mice. Bone
formation rate (BFR; A) and mineralizing surface per trabecular bone
surface (MS/BS; B) were measured in the metaphysis of 14 wk old mice.
C) Concentration of the C-terminal fragments of collagen 1 (Ctx) were
measured in the serum of 14 wk old females. Osteoclast number per
bone perimeter (OCn/Bp; D) and osteoclast surface per bone surface
(OCs/Bs; E). n=8. White bars: wild type; black bars: ERRaKO mice. Error
bars represent s.e.m. ns: not significant; *:p,0.05.
ERRa and Bone Loss
PLoS ONE | www.plosone.org3November 2009 | Volume 4 | Issue 11 | e7942
ERRa Deficiency Protects Females from Bone Loss
Resulting from Sex Hormone Ablation
Normal aging causes a moderate bone loss as the result of a
progressive imbalance between bone formation and bone
resorption. This equilibrium is also disrupted in favor of bone
resorption upon sex hormone deficiency which leads to a severe
bone loss, as can be observed in women after menopause or in
mice after ovariectomy. Since bone formation is more elevated in
ERRaKO we hypothesized that this phenomenon might com-
pensate for the remodeling imbalance induced by sex-hormone
withdrawal. Gonadectomized wild type females showed an
important decrease of relative bone volume (Fig. 4A) and
trabecular number (Fig. 4B) four wks after surgery. Strikingly,
none of these parameters were significantly affected in ovariecto-
mized ERRaKO animals, indicating that cancellous bone was
preserved in mutant mice. Bone formation rate was enhanced in
wt females as marker of enhanced bone remodeling (Fig. 4C). In
contrast, this parameter did not vary in ERRaKO mice according
to the hormonal status. In males, orchidectomy resulted in similar
variations in mineral density, bone volume and trabecular number
in wild type and mutant mice (Table 1), indicating suggesting that
bone was equally affected, independently of the genotype. We thus
concluded that the absence of ERRa completely protects against
the bone loss induced by deficiency in sex hormone action in
females, but not in males.
The mechanisms responsible for this phenomenon are not
identified but it is tempting to speculate that i) the higher
differentiation capacities of MSCs in osteoblasts in the absence of
ERRa as well as ii) the low expression of opn in ERRaKO
osteoblasts could be involved in sparing bone under estrogen
Figure 3. Ex vivo differentiation of bone marrow osteoblast precursors originating from ERRaKO mice. A) Upper panels: Alkaline
phosphatase (ALP)- and von Kossa staining after 7 d and 21 d, respectively, in differentiation medium. Lower panel: expression of osteoblast
differentiation markers measured by QPCR after 7 d in differentiation medium. Data are expressed relative to wild type level. Ocn: osteocalcin; opn:
osteopontin. B) Time course of ALP expression after switch to differentation medium in wild type- (white bars) and ERRaKO- (black bars) originating
cells measured by QPCR. Data are expressed relative to wild type level at d0. C) Rescue of the differentiation phenotype by infection with an ERRa-
encoding vs a GFP-encoding adenovirus. Upper panel: ALP staining; lower panel: expression of osteoblast differentiation markers determined by
QPCR. Adenoviruses were added together with the differentiation medium and the experiment was stopped after 7 d. Data are expressed relative to
AdGFP infected cells. D) Proliferation of osteoblast precursors. Upper left panel: time course of cell number; lower left panel: time course of ALP
staining; right panel: time course of ALP enzymatic activity relative to protein content. Time is indicated after switch to differentiation medium. All
experiments were performed at least three times using female bone marrow. Staining views represent a single typical experiment; expression data
and cell counting represent a typical experiment performed in triplicate with error bars indicating S.D.
ERRa and Bone Loss
PLoS ONE | www.plosone.org4 November 2009 | Volume 4 | Issue 11 | e7942
deficiency challenge. Indeed, it has been shown that opn-deficient
mice are resistant to ovariectomy-induced bone loss .
In summary, our data show that the absence of ERRa leads to a
resistance in age- as well as estrogen-deficiency-dependent bone
loss exclusively in female mice. This finding may have important
consequences for the treatment of osteoporosis following meno-
pause, and will need careful investigation to unravel the
mechanisms through which ERRa acts in osteoblast differentia-
tion and bone mineralization in vivo.
Materials and Methods
ERRaKO animals have been described elsewhere . All
animal experiments were performed in the Plateau de Biologie
Experimentale de la Souris (PBES; ENS Lyon) under Animal care
procedures, conducted in accordance with the guidelines set by the
European Community Council Directives (86/609/EEC) and
approved by the local ethical committee. Animals were in
C57black6 background and, except were stated, had access to
food and water ad libitum. Mice were sacrificed by cervical
dislocation at 10 a.m.
For surgery, animals were anesthetized with sodium pentobar-
bital. Testes were ligatured and cut through an incision in the
scrotum. Ovaries were removed through an incision in the flanks.
Animals were sacrificied 4 wk after operation. Statistical signifi-
cance was analyzed using one-way ANOVA.
X-ray Micro-Computed Tomography Analysis
3D microarchitecture of the femur was evaluated using a high-
resolution (8 mm) microtomographic imaging system (eXplore
Locus, GE, USA). A 3D region within the secondary spongiosa in
the proximal metaphysis of the femur was reconstructed,
beginning 500 mm proximal to the growth plate and extending
to 1.5 mm. Cortical bone was reconstructed from a 1 mm thick
region of interest centered on the diaphysis, 5 mm distal from the
proximal growth plate. Morphometric parameters were computed
using the Advanced Bone Analysis Microview Software (GE).
For histological analyses, undecalcified bones were fixed,
deshydrated in ethanol solutions at 4uC and embedded in
methyl-methacrylate according to standard protocols. For each
femur, 7 mm thick longitudinal sections, parallel to the sagittal
plane, were prepared. Bone sections were stained with von Kossa/
van Giseon reagent, images were captured using a Nikon eclipse
80i microscope and analyzed using NIS Elements AR 2.30
software (Nikon). To determine bone formation parameters,
calcein (10 mg/kg body weight) was injected to mice i.p. 5 and
2 d before sacrifice. Bone formation rate was calculated as
interlabeled widths/interval time. To determine bone resorption
parameters, sections were stained for tartrate-resistant acid
phosphatase (TRAP). TRAP-positive multinucleated cells attached
to bone were scored as osteoclasts and results were expressed as
number of osteoclasts per trabecular bone perimeter. Bone-related
degradation products from Collagen 1 (Ctx) in serum were
measured by immunosorbent assay, using a RatLaps ELISA kit
from Nordic Bioscience Diagnostics A/S (Herlev, Denmark),
following the recommendations of the manufacturers. Measure-
ments were performed after overnight fasting.
Bone Marrow Cultures
Primary MSCs were collected from bone marrow of femur and
tibia of 12-week old mice and cultured in six-well dishes
(46106cells/well) in MEMa containing 10% fetal calf serum
with 100 U/ml penicillin G and 100 mg/ml streptomycin. For
osteoblastic differentiation, after 5 d of culture the medium was
supplemented with 50 mg/ml L-ascorbic acid and 10 mM b-
glycerophosphate and replaced every 2 to 3 days for 3, 7, 14 or 21
days. Osteoblastic differentiation was evaluated by ALP staining
and ALP enzymatic activity. For ALP staining, cells were rinsed
twice with PBS, and then fixed with 4% (v/v) formaldehyde for
5 min. After 3 washes with H2O, cells were incubated 30 min with
Fast Blue RR salt and Naphtol AS-MX Phosphate Alkaline
solution 0,25% (Sigma Aldrich) in the dark. For ALP enzymatic
activity measurement, cells were lysed with Lysis Buffer (50 mM
Tris, HCl pH 7,5; 0,1% Triton-X100; 0,9% NaCl). Cell lysates
were incubated with 0,2 M of Naphtol Alkaline solution (Sigma
Aldrich) and the conversion of p-nitrophenyl phosphate to p-
nitrophenol was spectrophotometrically determined and normal-
ized to cellular protein content by using detergent compatible
Bradford reagent (Pierce). For proliferation tests, viable cells were
visually counted after resuspension and trypan blue coloration.
Adenoviral vectors expressing GFP or ERRa have been previously
described  and kindly donated by Anastasia Kralli.
RNAs were purified using Guanidinium thiocyanate/phenol/
chloroforme extraction. Total RNAs were reverse transcribed
using Superscript II retrotranscription kit (Invitrogen). Quantita-
tive PCR were performed using the SYBR GREEN Jump Start kit
(Sigma Aldrich) in duplicate on an ABI apparatus using standard
procedure. Expression data were normalized to the expression of
the 36b4 housekeeping gene. Sequence of the primers used in this
36b4: 59-ACCTCCTTCTTCCAGGCTTT-39 and 59-CCC-
ACCTTGTCTCCAGTCTTT-39; ALP: 59-GCCCTCCAGAT-
CCTGACCAA-39 and 59- GCAGAGCCTGCTGGTCCTTA-
39; ERRa: 59-CAAACGCCTCTGCCTGGTCT-39 and 59-AC-
TCGATGCTCCCCTGGATG-39; OCN: 59-ACCTCACAGA-
TGCCAAGCCC-39 and 59-AGCGCCGGAGTCTGTTCACT-
39; OPN: 59-TCTCCTTGCGCCACAGAATG-39 and 59-TC-
GTCCATGTGGTCATGGCT-39; Runx-2: 59-GACGTGCC-
CAGGCGTATTTC-39 and 59-GGAACTGCCTGGGGTCT-
Figure 4. Bone structural parameters of ovariectomized
ERRaKO mice. Bone volume fraction (BV/TV; A), trabecular number
(TbN; B), and bone formation rate (BFR; C) were determined in the
femur 4 wk after surgery (performed at 10 wk of age) in sham (white
bars) or ovariectomized (black bars) wild type and ERRaKO mice. n: 8.
Errors bars represent s.e.m. ns: not significant; *:p,0.05; *** p,0.005.
ERRa and Bone Loss
PLoS ONE | www.plosone.org5November 2009 | Volume 4 | Issue 11 | e7942
We thank Anastasia Kralli for the gift of adenovirus vectors. We thank the
personnel of the PBES, ENS Lyon, for helping in performing animal
Conceived and designed the experiments: MCS OC JMV. Performed the
experiments: CT MG BR LM JD CM JE BH. Analyzed the data: MCS
OC JMV. Contributed reagents/materials/analysis tools: VG. Wrote the
paper: MCS OC JMV.
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ERRa and Bone Loss
PLoS ONE | www.plosone.org6 November 2009 | Volume 4 | Issue 11 | e7942