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: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: 1.7 million women were diagnosed with and 521,000 women died from breast cancer in 2012. This review considers first current treatment options: surgery; radiotherapy; and systemic endocrine, anti-biological, and cytotoxic therapies. Clinical management includes prevention, early detection by screening, treatment with curative intent, management of chronic disease, and palliative control of advanced breast cancer. Next, the potential of novel drugs that target DNA repair, growth factor dependence, intracellular and intercellular signal transduction, and cell cycle are considered. Estrogen-related receptor alpha has attracted attention as a therapeutic target in triple-negative breast cancers with de novo resistance to, and in breast cancers with acquired resistance to, endocrine therapies such as antiestrogens and aromatase inhibitors. Estrogen-related receptor alpha is an orphan receptor and transcription factor. Its activity is regulated by coregulator proteins and posttranslational modification. It is an energy sensor that controls adaptation to energy demand and may facilitate glycolytic metabolism and mitochondrial oxidative respiration in breast cancer cells. Estrogen-related receptor alpha increases breast cancer cell migration, proliferation, and tumor development. It is expressed at high levels in estrogen receptor-negative tumors, and is proposed to activate estrogen-responsive genes in endocrine-resistant tumors. The structures and functions of the ligand-binding domains of estrogen receptor alpha and estrogen-related receptor alpha, their ability to bind estrogens, phytoestrogens, and synthetic ligands, and the effects of ligand agonists, antagonists, and inverse agonists on biological activity, are evaluated. Synthetic ligands of estrogen-related receptor alpha have activity in preclinical models of metabolic disorders, diabetes, osteoporosis, and oncology. The clinical settings in which these novel drugs might have utility in the management of advanced breast cancer, and biomarkers for stratification of patients likely to benefit, are discussed. Finally, the potential side effects of the novel drugs on metabolism, osteoporosis, osteo-metastasis, and cachexia are considered.Cancer Management and Research 01/2014; 6:225-252.
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ABSTRACT: To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/- males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/- versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/- but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.PLoS ONE 01/2014; 9(10):e109592. · 3.53 Impact Factor
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ABSTRACT: During the last decade, our view on the skeleton as a mere solid physical support structure has been transformed, as bone emerged as a dynamic, constantly remodeling tissue with systemic regulatory functions including those of an endocrine organ. Reflecting this remarkable functional complexity, distinct classes of humoral and intracellular regulatory factors have been shown to control vital processes in the bone. Among these regulators, nuclear receptors (NRs) play fundamental roles in bone development, growth, and maintenance. NRs are DNA-binding transcription factors that act as intracellular transducers of the respective ligand signaling pathways through modulation of expression of specific sets of cognate target genes. Aberrant NR signaling caused by receptor or ligand deficiency may profoundly affect bone health and compromise skeletal functions. Ligand dependency of NR action underlies a major strategy of therapeutic intervention to correct aberrant NR signaling, and significant efforts have been made to design novel synthetic NR ligands with enhanced beneficial properties and reduced potential negative side effects. As an example, estrogen deficiency causes bone loss and leads to development of osteoporosis, the most prevalent skeletal disorder in postmenopausal women. Since administration of natural estrogens for the treatment of osteoporosis often associates with undesirable side effects, several synthetic estrogen receptor ligands have been developed with higher therapeutic efficacy and specificity. This review presents current progress in our understanding of the roles of various nuclear receptor-mediated signaling pathways in bone physiology and disease, and in development of advanced NR ligands for treatment of common skeletal disorders.Physiological Reviews 04/2013; 93(2):481-523. · 30.17 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.
1. Ducy P, Zhang R, Geoffroy V, Rydall AL, Karsenty G (1997) Osf2/Cbfa1, a
transcriptional regulator of osteoblast differentiation. Cell 8: 747–754.
2. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, et al. (2002) The novel
zinc finger-containing transcription factor osterix is required for osteoblast
differentiation and bone formation. Cell 108: 17–29.
3. Komori T (2006) Regulation of osteoblast differentiation by transcription
factors. J Cell Biochem 99: 1233–1239.
4. Kostenuik PJ (2005) Osteoprotegerin and RANKL regulate bone resorption,
density, geometry and strength. Curr Opin Pharmacol 5: 618–625.
5. Xing L, Schwarz EM, Boyce BF (2005) Osteoclast precursors, RANKL/RANK,
and immunology. Immunol Rev 208: 19–29.
6. Wada T, Nakashima T, Hiroshi N, Penninger JM (2006) RANKL-RANK
signaling in osteoclastogenesis and bone disease. Trends Endocrinol Metab 12:
7. Asagiri M, Takayanagi H (2006) The molecular understanding of osteoclast
differentiation. Bone 40: 251–264.
8. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, et al. (1997)
Osteoprotegerin: a novel secreted protein involved in the regulation of bone
density. Cell 89: 309–19.
9. Boyce BF, Xing L (2008) Functions of RANKL/RANK/OPG in bone modeling
and remodeling. Arch Biochem Biophys 473: 139–146.
10. Syed F, Khosla S (2004) Mechanisms of sex steroid effects on bone. Biochem
Biophys Res Commun 328: 688–696.
11. Raisz LG (2005) Pathogenesis of osteoporosis: concepts, conflicts, and prospects.
J Clin Invest 115: 3318–3325.
12. Laudet V, Gronemeyer H (2002) The nuclear receptor factbook. Academic
press: San Diego.
13. Gigue `re V (1999) Orphan nuclear receptors: from gene to function. Endocr Rev
14. Gigue `re V, Yang N, Segui P, Evans RM (1988) Identification of a new class of
steroid hormone receptors. Nature 331: 91–94.
15. Tremblay AM, Gigue `re V (2007) The NR3B subgroup: an ovERRview. Nucl
Recept Signal 5: e009.
16. Horard B, Vanacker JM (2003) Estrogen receptor-related receptors: orphan
receptors desperately seeking a ligand. J Mol Endocrinol 31: 349–357.
17. Bonnelye E, Vanacker JM, Spruyt N, Alric S, Fournier B, et al. (1997)
Expression of the estrogen related receptor 1 (ERR-1) orphan receptor during
mouse development. Mech Dev 65: 71–85.
18. Gigue `re V (2008) Transcriptional control of energy homeostasis by the estrogen
related receptors. Endocr Rev 29: 677–696.
19. Villena JA, Kralli A (2008) ERRalpha: a metabolic function for the oldest
orphan. Trends Endocrinol Metab 19: 269–76.
20. Ariazi EA, Jordan VC (2006) Estrogen-related receptors as emerging targets in
cancer and metabolic disorders. Curr Top Med Chem 6: 203–215.
21. Bianco S, Lanvin O, Tribollet V, Macari C, North S, et al. (2009) Modulating
ERRalpha activity inhibits cell proliferation. J Biol Chem 284: 23286–23292.
22. Bonnelye E, Vanacker JM, Dittmar T, Begue A, Desbiens X, et al. (1997) The
ERR-1 orphan receptor is a transcriptional activator expressed during bone
development. Mol Endocrinol 11: 905–916.
23. Laflamme N, Giroux S, Loredo-Osti JC, Elfassihi L, Dodin S, et al. (2005) A
frequent regulatory variant of the estrogen-related receptor alpha gene
associated with BMD in French-Canadian premenopausal women. J Bone
Miner Res 20: 938–944.
24. Reinholt FP, Hultenby K, Oldberg A, Heingard D (1990) Osteopontin, a
possible anchor of osteoblasts to bone. Proc Natl Acad Sci USA 87: 4473–4475.
25. Ross FP, Chappel J, Alvarez JI, Sander D, Buttler WT, et al. (1993) Interactions
between the bone matrix proteins osteopontin and bone sialoprotein and the
osteoclast integrin avb3 potentiate bone resorption. J Biol Chem 268:
26. Ishijima M, Rittling SR, Yamashite T, Tsuji K, Kurosawa H, et al. (2001)
Enhancement of osteoclastic bone resorption and suppression of osteoblastic
bone formation in response to reduced mechanical stress do not occur in the
absence of osteopontin. J Exp Med 193: 399–404.
27. Noda M, Denhardt DT (2002) Osteopontin. In: Bilezikian JP, Raisz LG,
Rodan GA, eds. Principle of bone biology, 2nded. San Diego: Academic Press,
vol1, pp 239–250.
28. Boskey AL, Spevak L, Paschalis E, Doty SD, McKee MD (2002) Osteopontin
deficiency increases mineral content and mineral crystallinity in mouse bone.
Calcif Tissue Int 71: 145–154.
29. Shapses SA, Cifuentes M, Spevak L, Chowdury H, Brittingham J, et al. (2003)
Osteopontin facilitates bone resorption, decreasing bone mineral crystallinity
and content during calcium deficiency. Calcif Tissue Int 73: 86–92.
30. Kavukcuoglu NB, Denhardt DT, Guzelsu N, Mann AB (2007) Osteopontin
deficiency and aging on nanomechanics of mouse bone. J Biomed Mater Res A
31. Vanacker JM, Delmarre C, Guo X, Laudet V (1998) Activation of the
osteopontin promoter by the orphan nuclear receptor estrogen receptor related
alpha. Cell Growth Differ 9: 1007–1014.
32. Vanacker JM, Pettersson K, Gustafsson JA, Laudet V (1999) Transcriptional
targets shared by estrogen receptor- related receptors (ERRs) and estrogen
receptor (ER) alpha, but not by ERbeta. EMBO J 18: 4270–9.
33. Zirngibl RA, Chan JS, Aubin JE (2008) Estrogen receptor-related receptor alpha
(ERRalpha) regulates osteopontin expression through a non-canonical ERRal-
pha response element in a cell context-dependent manner. J Mol Endocrinol 40:
34. Bonnelye B, Merdad L, Kung V, Aubin JE (2001) The orphan nuclear estrogen
receptor-related receptor a (ERRa) is expressed throughout osteoblast
differentiation and regulates bone formation in vitro. J Cell Biol 153: 971–983.
35. Delhon I, Gutzwiller S, Morvan F, Rangwala S, Wyder L, et al. (2009) Absence
of estrogen receptor related alpha increases osteoblastic differentiation and
cancellous bone mineral density. Endocrinology 150: 4463–4472.
36. Jeong BC, Lee YS, Park YY, Bae ICH, Kim DK, et al. (2009) The orphan
nuclear receptor Estrogen-receptor-Related Receptor c negatively regulates
BMP2-induced osteoblast differentiation and bone formation. J Biol Chem 284:
37. Yoshitake H, Rittling SR, Denhardt DT, Noda M (1999) Osteopontin-deficient
mice are resistant to ovariectomy-induced bone resorption. Proc Natl Acad Sci
USA 96: 8156–8160.
38. Luo J, Sladek R, Carrier J, Bader JA, Richard D, et al. (2003) Reduced fat mass
in mice lacking orphan nuclear receptor estrogen-related receptor a. Mol Cell
Biol 23: 7947–7956.
39. Schreiber SN, Emter R, Hock MB, Knutti D, Cardenas J, et al. (2004) The
estrogen-related receptor a (ERRa) functions in PPARc coactivator 1a (PGC-
1a)-induced mitochondrial biogenesis. Proc Natl Acad Sci USA 101:
ERRa and Bone Loss
PLoS ONE | www.plosone.org6 November 2009 | Volume 4 | Issue 11 | e7942