The ERR-1 Orphan Receptor Is a Transcriptional Activator Expressed During Bone Development
Edith Bonnelye1,
Jean Marc Vanacker1,
Tanja Dittmar,
Agnes Begue,
Xavier Desbiens,
David T. Denhardt,
Jane E. Aubin,
Vincent Laudet and
Brigitte Fournier
Endocrinos group (E.B., J.M.V., A.B., V.L.),
CNRS UMR 319 Mécanismes du développement et
de la Cancérisation Institut de Biologie de
Lille, 59021 Lille Cedex France,
Novartis Pharma AG (T.D., B.F.), K125 917,
P11002 Basel, Switzerland Department of Biological
Sciences (D.T.D.), Rutgers University,
Piscataway, New Jersey 08855, Department of
Anatomy and Cell Biology (J.E.A.), University of
Toronto, Toronto, M5S 1A8 Canada Centre
de Biologie Cellulaire du Développement (X.D.)
Université des Sciences et Techniques de Lille
Villeneuve dAscq, France
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ABSTRACT
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We studied the expression of estrogen-related
receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is
present in bones formed by both the endochondral and intramembranous
routes, and the onset of its expression coincides with bone formation.
By RT-PCR experiments, we found that ERR-1, but not the related
receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as
well as in primary osteoblastic cell populations derived from normal
human bone. By gel shift analysis we found that ERR-1 binds as a
monomer specifically to the SFRE sequence (SF-1-responsive-element;
TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif
and the TCA 5'-extension are required for efficient ERR-1 binding. In
transient transfection assays, ERR-1 acts as a potent transactivator
through the SFRE sequence. This effect is cell-specific since ERR-1
activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as
well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells.
Notably, the osteopontin (a protein expressed by osteoblasts and
released in the bone matrix) gene promoter is a target for ERR-1
transcriptional regulation. Our findings suggest a role for ERR-1 in
bone development and metabolism.
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INTRODUCTION
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Nuclear receptors are transcription factors that are
involved in various physiological regulatory processes. The superfamily
in which they are grouped comprises ligand-dependent molecules such as
the steroid hormone-, thyroid hormone-, or retinoic acid receptors, but
also an increasing number of so-called orphan receptors, for which no
ligand is known (1, 2). The various orphan receptors are likely to play
important functional roles since their sequences are highly conserved
in mammals and even between phyla (3), and they often display
restricted spatial and temporal patterns of expression. The activity of
nuclear receptors is controlled at several levels, including their
expression (and its level) in the tissues and cells in which they act.
This regulatory mechanism may be particularly important in the case of
orphan receptors that may lack ligand-associated modulation.
Conversely, determination of the expression patterns of orphan
receptors may give important clues in the search for their functions.
This was recently exemplified in the case of Steroidogenic Factor 1
(SF1), which was shown to be expressed in the hypothalamo-pituitary
axis and in the gonad and, concomitantly, demonstrated to regulate the
steroidogenic cascade (Ref. 4 and references therein).
Two orphan receptors, estrogen-related receptor-1 (ERR-1) and ERR-2
(5), were identified by low-stringency screening of cDNA libraries with
a probe encompassing the DNA-binding domain of the human estrogen
receptor (ER). Sequence alignment of ERR-1, ERR-2, and ER reveals an
high similarity (68%) in the 66 amino acids of the DNA-binding domain.
The putative ligand-binding domain, positioned between amino acids 295
and 521 of ERR-1, shows 63% identity when compared with ERR-2 and 36%
to the ER. Analysis of the tissue distribution of these two orphan
receptors in adult mice showed that ERR-2 is expressed at lower levels
and in a more restricted pattern than ERR-1. Recently, it was shown
that ERR-2 is expressed during the early stages of mouse chorion
formation (6). In contrast, ERR-1 appears to be widely distributed
although more abundant in the central nervous system (5). ERR-1 has
been identified as a regulator of the SV40 major late promoter during
the early-to-late switch of expression (7). Yang et al. (8)
also recently showed that ERR-1 modulates the activating effect of
estrogens on the lactoferrin promoter and suggested that ERR-1 may
interact with ER through protein-protein interactions.
To search for a possible physiological role for ERR-1, we
analyzed its expression during mouse embryonic development by in
situ hybridization. Among complex expression patterns that will be
published elsewhere (Bonnelye E., Vanacker J.-M., Spruyt N., Alric S.,
Fournier B., Desbiens X., and Laudet V., submitted), a striking
relationship was noted between ERR-1 and skeleton formation. In this
paper, we report our findings that ERR-1 mRNA is detected in
ossification sites of the developing mouse embryo. In parallel, we show
that ERR-1 is highly expressed in both human primary osteoblasic cells
and osteosarcoma cell lines. Because little is known about the
mechanisms of action of ERR-1, we have examined its transactivation
functions. Our data demonstrate that ERR-1 binds to the SFRE
(SF1-responsive element; TCAAGGTCA; Refs. 9 and 10) motif as a monomer
and acts through this sequence as a transcriptional activator. The
latter property is cell-type specific and occurs in osteoblastic cells
among other cell lines. Seven SFRE-like sequences are present in the
promoter of the gene encoding mouse osteopontin (OPN), a noncollagenous
protein released in the bone matrix by osteoblasts and believed to play
an important role in the formation and remodeling of bone tissues (11).
By cotransfection experiments in rat osteosarcoma cells, we show that
the OPN promoter is positively regulated by ERR-1. Taken together, our
data suggest an important transcriptional regulatory role of
the orphan receptor ERR-1 in bone metabolism.
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RESULTS
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ERR-1 is Expressed in Vivo during Bone Development
Formation of bone during embryonic skeletal development is an
organized and well regulated process that involves differentiation of
uncommitted mesenchymal progenitor cells to osteoblasts. In the case of
membranous ossification, a process used in generating the flat bones of
the skull (i.e. membrane bones), as well as in adding bone
to the outer surfaces of long bones, the mesenchymal cells
differentiate directly into bone-forming osteoblasts. In contrast, in
long bone and vertebrae (i.e. cartilaginous bones)
endochondral bone formation takes place by the progressive substitution
of cartilage by bone.
With respect to bones formed by endochondral ossification, ERR-1 was
detected by in situ hybridization in forelimb bones such as
the ulna, radius, and humerus at E17.5 (u, r, and h, respectively, in
Fig. 1A
). This expression is specific
since no signal was seen with the sense probe (Fig. 1F
). As show by
picro-indigo-carmine labeling, which stains the ossification centers in
brown and the cartilage in blue, ERR-1 expression is restricted to the
bone in the midshaft and is not found in any zone of cartilage
including the hypertrophic zone (compare Fig. 1A
and Fig. 1B
). At
higher magnification of the ulna, the specificity of ERR-1 for the bone
[ossification zone (oz) in Fig. 1
, C, D, and E] but not hypertrophic
cartilage, is clear.


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Figure 1. In Situ Hybridization for ERR-1
Expression in Mouse Embryos
A, A longitudinal section of forelimb at 17.5 days post coitus (p.c.)
with ERR-1 antisense probe. Bar, 1400 µm. B,
Picro-indigo-carmine coloration of a forelimb (adjacent to A) at 17.5
days p.c showing the ossification zone (oz). Bar, 1400
µm. C, Higher magnification of the ulna bone; ERR-1 antisense probe.
Bar, 70 µm. D, Hoechst labeling of the same section,
staining the nuclei and thus the tissular organization.
Bar, 70 µm. E, Picro-indigo-carmine coloration of a
portion of the ulna showing the ossification zone. Bar,
70 µm. F, A section of an ulna bone hybridized with sense ERR-1
probe. Bar, 1000 µm. G, Section of a femur hybridized
with the antisense probe. Bar, 750 µm. H, Section of a
femur hybridized with the sense probe. Bar, 750 µm. I,
Magnification of the ossification zone (oz) of the femur hybridized by
ERR-1 probe. Bar, 200 µm. J, Picro-indigo-carmine
labeling of the femur. Bar, 750 µm. K, Humerus head
section hybridized by ERR-1 antisense probe. Bar, 750
µm. L, Picro-indigo-carmine coloration of the humeral head.
Bar, 750 µm. M, Section of a vertebral arch hybridized
with ERR-1 antisense probe. Bar, 175 µm. N,
Picro-indigo-carmine coloration of the vertebral arch.
Bar, 175 µm. O, Maxillary bone section hybridized with
ERR-1 antisense probe. Bar, 50 µm. P,
Picro-indigo-carmine coloration of a section of maxillary bone.
Bar, 175 µm. c, Cartilage; d, digit; di, diaphyse; e,
epiphyse; fh, femur head; h, humerus; hz, hypertrophic zone; ma,
maxillary bone; m, muscles; nt, neural tube; oz, ossification zone; r,
radius; u, ulna; va, vertebral arch.
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In E17.5 mice, we observed ERR-1 expression only in long bones in which
bone centers were established around E15. We thus performed in
situ hybridization on E15.5 embryos to see whether ERR-1
expression coincides with the establishment of the first ossification
centers. At E15.5, we observed the appearance of some ossification
zones in our sections of ribs and vertebrae. In these ossification
centers, ERR-1 expression began to be detected (data not shown). Taken
together, these data indicate a spatial and temporal correlation
between ERR-1 expression and the formation of the ossification zone.
Consistent with this conclusion, we observed ERR-1 expression during
ossification of the cartilage primordia of the upper-shaft region of
the humerus (oz in Fig. 1
, K and L), in the hindlimb (femur; Fig. 1
, G,
H, and I), and in the vertebrae (see va in Fig. 1
, M and N).
Restriction of ERR-1 transcripts to areas of bone is clear when the
in situ hybridization is compared with the
picro-indigo-carmine histochemistry (oz in Fig. 1
, G-J). ERR-1
hybridization is exclusively localized to bone in the midshaft
(diaphyses) and is not present in the cartilaginous epiphyses. Notably,
ERR-1 transcripts are also detected in the ossification centers of the
ribs at the same stage (not shown). To confirm that ERR-1 is not
expressed in cartilage cells, E11 limb bud cells were cultured for 24
or 96 h and then analyzed by in situ hybridization.
ERR-1 expression was not observed at either time point, although
cartilage nodules were clearly present by 96 h.
Notably, ERR-1 was also expressed in bones formed by intramembraneous
ossification, e.g. strong signal was seen in the maxillary
bones of the face (see ma in Fig. 1
, O and P). Thus, ERR-1 mRNA levels
are high in all ossification centers during both intramembranous and
endochondral bone formation.
ERR-1 Is Expressed in Human Osteoblastic Cells
The expression of ERR-1 during in vivo bone development
led us to investigate the presence of its mRNA in cultured bone-derived
cells. Since the process of ossification involves many cell populations
that cannot be easily distinguished by in situ
hybridization, such an experiment may also provide a first
discrimination of the cell type displaying ERR-1 expression. We
performed RT-PCR experiments with primers designed to amplify
separately the three ER-related mRNAs: ERR-1, ERR-2, and ER itself
(Fig. 2A
). Figure 2B
shows that ER is expressed in
osteoblastic cell lines SaOs, TE85, and primary human osteoblastic
populations (NHB), but not in DAMI platelet cells, as expected. To
address further the specificity of ERR-1 for osteoblastic cells, we
screened other nonosteoblastic cells using TFIID as a positive control.
Consistent with our observation that ERR-1 is expressed in the
developing mammary gland (Bonnelye et al., submitted), T47D
(a mammary carcinoma cell line) cells contain ERR-1 and a weak amount
of ER (Fig. 2C
). On the contrary, HeLa- and FLG 29.1 cells [a human
leukemic line that exhibits osteoclastic differentiation markers upon
TPA treatment (12)]) are devoid of ERR-1 mRNA.

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Figure 2. Expression of ER, ERR-1, and ERR-2
A, Scheme of the RT-PCR strategy. The domain organization of nuclear
receptors is shown. Numbers indicate the nucleotide
position of the ends of each primer used. B, C, and D, RT-PCR
experiments. mRNA from the cell lines indicated (B and C) and ES cells
(D) were reverse transcribed and amplified with primers specific for
ER, ERR-1, ERR-2, or TFIID as stated. Reactions were run on a native
gel. Values on the left indicate the expected size of
the amplification products of ERR-1 (482 bp), ER (339 bp), ERR-2 (281
bp), and TFIID (260 bp). M, Marker lane. Slash bars
indicate PCR reactions performed in the absence of RNA as negative
control.
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As expected from the earlier observations of a restricted pattern of
expression in adult (5, 6), ERR-2 was not detected in any of the cells
tested except in the positive control embryonic stem (ES) cells (Fig. 2D
). In agreement with the data published by Petersson et
al. (6), we found that these cells express ERR-2. ERR-1 is also
present in ES cells, suggesting an early onset of its expression in the
developing embryo. Taken together, the detection of ERR-1 mRNA in
cultured cells is consistent with its expression in vivo, in
particular in bone-derived populations where it appears to be
restricted to osteoblastic cells.
ERR-1 binds to DNA as a Monomer on the SFRE Element
As a first step toward understanding the molecular effects of the
ERR-1 gene, we determined the DNA target of its product. ERR-1, like
all members of the nuclear receptor superfamily, can be expected to
bind to specific DNA sequences that contain the general core responsive
element (AGGTCA). Electrophoretic mobility shift analyses (EMSAs) were
performed using in vitro translated ERR-1 product and
various synthetic oligonucleotides containing the core element as
probes. These included palindromic sequences (HREpal, see Fig. 3A
, and estrogen responsive element), direct repeats
motifs [response elements to the thyroid hormone-, the vitamin
D3- or the retinoic acid receptor; see Glass (13) for a
review], or monomeric half- sites (response elements to Rev-erb, SF-1,
or NGF1B) in the presence or absence of unlabeled oligonucleotides as
competitors (Fig. 3
and data not shown). We found that ERR-1
specifically binds to the SFRE probe (SF1/FTZ-F1 responsive element;
TCAAGGTCA; Refs. 9 and 10), as illustrated in Fig. 3B
. ERR-1-programmed
reticulocyte lysate (lane 2) but not unprogrammed lysate (lane 1) forms
a single retarded complex with a SFRE probe. The recognition was
specific since complex formation was inhibited by an excess of
unlabeled homologous- (SFRE; lanes 3 and 4), but not of related but
distinct oligonucleotides (lanes 5 to 10; sequences in Fig. 3A
). Other
receptors have been described to bind to a single element
(e.g. SF-1/FTZ-F1, NGF1B, Rev-erb ... ; see Refs. 13 and
14 and references therein). The sequence immediately 5' of the core
element is, in this case, assumed to mediate the specificity of binding
of each type of receptor (9, 13). We therefore assayed the requirement
of ERR-1 toward the nucleotides present upstream of the core element
(Fig. 3b
). Addition of excesses of NGRE (NGF1B responsive element; Ref.
9; lanes 5 and 6) or of the RevRE (Rev-erb responsive element; Refs. 15
and 16 and data not shown) to the binding reaction did not affect the
fixation of ERR-1 on the SFRE. Mutations inside the core element (as
present in competitor SFREm; lanes 7 and 8) impaired the ability to
compete. Finally, the palindromic hormone responsive element (HREpal;
lanes 9 and 10) did not interfere with ERR-1 binding. The formation of
the ERR-1-SFRE complex is thus strikingly dependent upon the integrity
of the TCA sequence located 5' of the core element.

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Figure 3. Binding Specificity of ERR-1
A, Sequences of the products used in binding experiments. The
upper panel describes the oligonucleotides. Core
responsive element is underlined by an
arrow. The consensus site in each oligonucleotide is in
capital letters. Mutated nucleotides in SFREm are in
boldface. The lower panel schematizes the
ERR-1 derivatives used in Fig. 3C . B, Competition EMSA. ERR-1 protein
was in vitro synthesized and allowed to bind on a SFRE
probe. Where stated, unlabeled oligonucleotides were added at the
indicated molar excesses. Lane 1: unprogrammed (empty pSG5 vector)
reticulocyte. Unbound probe is indicated ("free probe"). C, Monomer
binding of ERR-1. Indicated proteins were in vitro
synthesized and allowed to bind to a SFRE probe as stated. Lane 1:
unprogrammed (empty pSG5 vector) reticulocyte lysate. Unbound probe is
indicated ("free probe").
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Monomer vs. dimer binding is a key question in the
mechanism of action of orphan receptors. To address this problem in the
case of ERR-1, deletion of 96 amino acids of its A/B domain (which is
dispensable for DNA binding) was performed by PCR (schematized on Fig. 3A
). The resulting construct (
96ERR-1) was (after sequencing)
in vitro translated and used in EMSA (Fig. 3C
).
96ERR-1
forms a complex with SFRE that migrates faster than wild type (wt)
ERR-1-SFRE. Combination of wt ERR-1 and
96ERR-1 in the binding
reaction produces two distinct complexes and no intermediary one, which
would be expected in the case of dimer binding. ERR-1 is thus a new
example of monomer binding orphan receptor.
Transcriptional Activity of ERR-1
As the ERR-1 gene product binds to a specific DNA sequence, we
have tested its ability to regulate transcription. To this end, SFRE
oligonucleotides were cloned as pentamers in front of the
HSVtk promoter and a chloramphenicol acetyltransferase (CAT)
reporter gene. After sequencing, resulting constructs were
cotransfected in HeLa cells with varying amounts of pSGERR-1 plasmid
(Fig. 4A
). Reporter gene expression from pSFREtk was
enhanced in a dose-dependent manner by cotransfection of ERR-1-encoding
plasmid. As a positive control, the FTZ-F1 group member xFF1r acts
through the same sequence. It should be noted that a high (10-fold)
reporter-effector ratio was required to reach a maximal effect.
Stimulation by ERR-1 requires the presence of SFRE sites in the
plasmid, since no transactivation was observed with the minimal
promoter supplemented with the nonbinding HREpal sequence (plasmid
pHREptk). We also tested the ability of several other cell lines to
support ERR-1-induced transactivation. As shown in Fig. 4B
, ERR-1
exerts a pleiotropic activity, ranging from high (HeLa cells) to
low or no (simian COS1, human HepG2 cell) stimulation capacity. Human
NB-E and rat FREJ4 cell lines supported ERR-1 transactivation to an
intermediate level. This phenomenon cannot be interpreted in terms of
differences of expression of the transfected ERR-1 in the various cell
lines as verified by EMSA and examplified in Fig. 4C
. Transfection of
ERR-1 in HeLa cells (lane 2) resulted in a SFRE-protein complex that
was undetectable in mock-transfected cells (lane 1). This complex arose
in comparable amounts when NB-E (lane 4) or COS cells (lane 6) were
transfected even though the latter cells already express endogenous
ERR-1 (see lane 5). We therefore conclude that the transcription
activation potential of ERR-1 exhibits strong cell type
specificity.

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Figure 4. Transactivation by ERR-1
Cells were cotransfected with 0.5 µg reporter plasmid, together with
the indicated molar excess of effector plasmid. CAT activites were
determined 48 h after transfection and are plotted relative to the
effector-free sample. Graphs represent the average of at
least three independent experiments, with error bars indicating the
internal variation. A, Target specificity of ERR-1 response. HeLa cells
were cotransfected with pSFREtk or pHREptk (containing pentamers of
SFRE and HREpal sites, respectively; see sequences on Fig. 3A ),
together with ERR-1 or FTZ-F1(xFF1r)-encoding plasmid, as indicated. B,
Cell specificity of ERR-1 response. Indicated cells were cotransfected
with pSFREtk together with ERR-1-encoding plasmid. C, Control of ERR-1
expression. Indicated cells were transfected with 5 µg plasmid pSG5
ERR-1 (even lanes) or wt pSG5 (odd
lanes). Forty eight hours after transfection, whole cell extracts were
performed. Equal amounts of proteins were allowed to bind on a SFRE
probe. Arrow indicates SFRE-protein complex, appearing
in ERR-1-transfected cells.
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Activities of ERR-1 in Osteoblastic Cells
Because ERR-1 is expressed in vivo during bone
formation and regulates transcription in a cell-specific manner, we
addressed the question of its activity in bone cells. Interestingly, we
found that ERR-1 is a functional transcription factor in the rat
osteosarcoma cell line Ros 17.2/8. Increasing the expression of ERR-1
in these bone cells led to an enhanced (up to 12-fold) expression of
the CAT gene cloned in the pSFREtk plasmid (Fig. 5A
).
Again, no effect of ERR-1 was observed on the pHREtk vector (data not
shown).

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Figure 5. Transcriptional Activity of ERR-1 in ROS 17.2/8
Cells
Cells were cotransfected with 0.5 µg reporter plasmid, together with
the indicated molar excess of effector plasmid. Reporter activities
were determined 48 h after transfection and are plotted relative
to the effector-free sample. Graphs represent the
average of at least three independent experiments, with error bars
indicating the internal variation. A, Activity of ERR-1 on
SFRE-supplemented minimal promoter. CAT activity is used as a reporter.
B, Activity of ERR-1 on osteopontin gene promoter. The region spanning
nucleotides -882 to +79 (relative to the transcription start site and
described in Ref. 20), cloned in front of the Luc reporter gene was
used in this study.
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Bone cells express a number of differentiation markers (see Refs.
17 and 18 for a review), the expression of some being temporally
regulated during the process of ossification. In search of a potential
molecular target for the transcription activator ERR-1, we screened the
published promoters of bone-associated genes for the presence of SFRE
sites. Computer-assisted analysis of the mouse osteopontin (OPN)
promoter (19, 20) revealed the presence of seven SFRE-like motifs
(bearing one or two nucleotide differences with the consensus SFRE).
OPN is a noncollagenous protein expressed by osteoblastic cells and
released in the bone matrix. To determine the effect of ERR-1 on this
promoter, cotransfections were performed in rat osteosarcoma Ros 17.2/8
cells. Figure 5B
shows that ERR-1 positively regulates the OPN promoter
in a dose-dependent manner, thus suggesting a physiological relevance
of ERR-1 expression in bone cells.
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DISCUSSION
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ERR-1 and Bone Formation
In situ hybridization of mouse embryo sections reveals
a temporal and spatial correlation between the presence of ERR-1 mRNA
and the process of ossification. Bone formation and patterning require
several cell types, including bone-forming osteoblasts and
bone-degrading osteoclasts. Consistent with the in situ
hybridization data, the RT-PCR results presented here clearly show that
ERR-1 is expressed by osteoblastic cell lines and cell populations
derived from normal human bone that contains a high percentage of
osteoblasts. Furthermore, the presence of ERR-1 mRNA could not be
detected in the osteoclast-like FLG 29.1 cell line, suggesting that
ERR-1 is, among bone-derived cells, osteoblast-specific. Although not
restricted to bone (Ref. 5; Bonnelye et al., submitted; and
our results presented here), the pattern of ERR-1 expression suggests
that this receptor may play a role in the formation of both
endochondral and intramembranous bones. Study of the osteoblast lineage
has been hampered by a relative lack of markers as summarized by Aubin
and colleagues (17, 18). The potential of ERR-1 as a regulator and as a
new marker of the osteoblast lineage and its use together with already
defined osteoblastic markers such as as alkaline phosphatase, collagen
type I, bone sialoprotein, and osteocalcin may contribute to a better
understanding of the stage of differentiation of the osteoblastic cells
during both intramembranous and endochondral ossification.
Bone formation and turnover are known to be under the control of
many hormones and cytokines, among which estrogens play a critical
role, as exemplified by the accelerated bone loss leading to
osteoporosis at menopause. The low concentration of ER in osteoblasts
(2001000 molecules per cell; Refs. 2123) relative to reproductive
tissues (10000100000 molecules per target cell) is consistent with
the hormone having a more limited number of direct actions on the
skeleton. It is rather commonly believed that most of the known effects
of estrogens in bone may be mediated by the action of specific growth
factors and cytokines (24, 25). It is also possible that ERR-1 plays a
role in this complex series of events by regulating estrogen response.
Interestingly, the level of mRNA expression of ERR-1 is much higher
than of ER, as determined both by RT-PCR (Fig. 2
) and in
situ hybridization experiments (not shown). Whether this reflects
the amount of protein available inside the cells remains to be
established. A detailed study of the level of ER and ERR-1 mRNA and
proteins during bone differentiation is therefore of particular
importance in this context. Although highly related to the ER, ERR-1 is
unlikely to bind estrogen, since its transcriptional activity seems to
be ligand-independent (see below). In this respect, an indirect
regulatory pathway seems likely, consistent with the in
vitro demonstration of a physical interaction between ER and ERR-1
and the suggestion that, in given promoter contexts, ERR-1 may enhance
the transcriptional effects of ER (8). The two factors could thus
regulate overlapping gene networks. Whether such an effect takes place
in osteoblastic cells constitutes an interesting possibility that
remains to be addressed. This awaits the identification of common
target genes of ER and ERR-1, expressed during bone formation
(e.g. in osteoblasts).
Osteoblasts are very active cells that synthesize many different
products as type I collagen (26), osteocalcin (27), osteopontin (28)
and bone sialoprotein (29, 30). Any of these genes could be candidates
for ERR-1 target genes. Indeed, we found that the osteopontin promoter,
which contains seven SFRE-like elements, positively responds to ERR-1
in transient cotransfection experiments, suggesting an in
vivo regulation of osteopontin by ERR-1. In support of this
hypothesis, there is a temporal correlation between the onset of
osteopontin expression (E16; 31 and ERR-1 (E15.5). Taken together
with our expression data, these results suggest that ERR-1 may play a
key role in bone development. This could be achieved through direct
promoter regulation and/or by interference with the estrogen-controlled
pathway, through interaction with ER.
Relations of ERR-1 with Other Nuclear Receptors
The SFRE motif was first described as the response element of the
members of the SF1/FTZ-F1 subfamily of nuclear receptors. These orphan
receptors bind to DNA as monomers, and it has been demonstrated that,
when they do so, the conserved T and A boxes (which lie downstream of
the Zn-fingers) play an important role in the recognition of the
5'-extension of the core motif (9). It is interesting to note that in
these critical regions, ERR-1 and SF1 harbor a high level of sequence
identity (adapted from Ref. 9 in our Fig. 6
) that allow
to define a TCA consensus. In contrast, these T and A boxes are
divergent from those of NGF1B or Rev-erb
. This suggested that
SF-1/FTZ-F1 and ERR-1 could recognize common sequences, distinct from
the ones bound by NGF1B and Rev-erb
. In agreement with this, we
indeed found that ERR-1 binds specifically to the SFRE motif, an
interaction that is not competed by NBRE or RevRE sequences. Analysis
of the T and A box sequences in the work mentioned above (9) also
predicted monomer binding for ERR-1, a feature confirmed in our
experiments. However, the behavior of ERR-1 in the presence of repeats
of SFRE has not been investigated, and it remains possible that, as
with Rev-erb on the RevDR2 elements (32), ERR-1 could homodimerize on
specific direct repeats or heterodimerize with RXR as is the case for
several other nuclear receptors.

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Figure 6. Sequence Analysis of Monomer Binding Receptors
Schematic domain organization of nuclear receptors is given, with a
magnification of the T- and A-box. Sequences are given in the
one-letter-code amino acids (a.a.). The comparison of
ERR-1 and SF-1 allows the definition of a TCA consensus, where
identical a.a. are shown, and periods represent variable ones. This can
be compared with the same regions of AAA-binding receptors (AAA
consensus , as defined in Ref. 9) and of Rev-erb (which binds to a
highly divergent sequence). +, Positively charged a.a.
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The transient cotransfection experiments presented here indicate
that the SFRE is sufficient to render a minimal promoter responsive to
ERR-1. Transactivation by ERR-1 occurs without addition of any factor
to the culture and even when cells are cultured in charcoal-stripped
medium (i.e. steroid-devoid; data not shown). Nevertheless,
this does not rule out the possibility of a ligand-activated function
of ERR-1 since a ligand could be synthesized and be active inside the
cell. Such an intracrine mode of action is, for example, thought to
occur for the PPAR gene products that may be activated by specific
ligands synthesized inside the cell from inactive precursors (33, 34).
The fact that ERR-1 is able to positively regulate transcription is in
agreement with the results published by Lydon et al. (35),
who found that a PR-ERR-1 fusion protein is able to activate
transcription without addition of any exogenous factor. The level of
transcription stimulation achieved by ERR-1 is dependent on the cell
type examined. This is reminiscent of the behavior of SF1/FTZ-F1 that
exert low (36) or high (10, 37) transactivation upon its cognate
sequence, depending on the cell system used. This could suggest that
cells in which no ERR-1-induced transactivation is observed
(e.g. HepG2) lack a cofactor that is present to a large
amount in HeLa cells. Cells supporting an intermediate ERR-1
stimulation (such as osteoblastic Ros cells) would, accordingly,
contain intermediate levels of this factor. It may also explain the
discrepancy of our work with reports of a repressive effect of ERR-1 on
the SV40 major late promoter (MLP; 7 . On the other hand, in the
absence of any SFRE (as in the case of the SV40 MLP), other sequences
might mediate a negative effect.
The behavior of ERR-1 is different from that described for
its close relative ERR-2, which binds as a homodimer to the estrogen
responsive element and appears to be transcriptionally inactive on this
element (6). Although the result obtained for transactivation may be
also explained by a cell-specific action of ERR-2, the difference in
DNA binding specificities of these two strongly related factors appears
provocative. ERR-1 and ERR-2 display high sequence identity in the C
domain as well as the T box, whereas differences exist in the A box
that might be responsible for preference toward their target DNA site.
On the other hand, it is not excluded that, though binding to the ERE,
ERR-2 might also recognize another sequence, such as the SFRE.
Altogether, both ERRs may interconnect with ER-regulatory pathways,
through DNA binding on ERE (i.e. ERR-2) or through protein
interactions (i.e. ERR-1).
The action of ERR-1 through the SFRE element emphasizes the
promiscuous behavior of many members of the nuclear receptor
superfamily. Indeed, our study demonstrates that several unrelated
groups of orphan receptors are able to bind and regulate transcription
through the SFRE site. Until now, the SFRE site has been demonstrated
to be a target only for members of the SF1 group. These orphan
receptors are expressed in steroidogenic tissues as well as in gonads
and appear to be important for the regulation of the endocrine cascade
that controls gonad differentiation and sex determination. Our study
extends the number of orphan receptors and tissue types for which the
SFRE element is a key determinant. The expression territories of SF1
and ERR-1 are partly overlapping, but no competition or synergy in
transcriptional regulation by both proteins could be observed
(discussed in Bonnelye et al., submitted). Nevertheless, we
cannot exclude that in particular tissues or physiological situations,
a cross-talk between SF1 and ERR-1-mediated transcriptional regulation
may occur. Interestingly, the GCNF orphan receptor binds as a dimer to
a DR0 element (38). This site harbors the TCAAGGTCA motif, and here
again we cannot exclude a possible cross-talk between GCNF, ERR-1, and
SF1 on given response elements.
Our results give the first hints as to the possible in vivo
functions of the ERR-1 orphan receptor. Expression studies as well as
in vitro experiments suggest that ERR-1 may play an
important role in the formation of the skeleton. ERR-1 could act
independently and/or intervene in regulation pathways controlled by
other members of the nuclear receptor superfamily. These
interconnections may be necessary for the differential regulation of a
given set of target genes.
 |
MATERIALS AND METHODS
|
---|
Cells
Cells other than those of the osteoblastic lineage were
grown in DMEM with 10% FCS. The clonal human osteoblast-like cell line
SaOS-2 was obtained from Dr. S. Rodan (Merck Sharp and Dohme Research
Laboratories, West Point, PA). The human osteosarcoma cell TE-85 (ATCC,
CRL 1543) was obtained from the American Type Cell Culture Collection
(Rockville, MD). The ROS 17/2.8 rat osteosarcoma cell line was kindly
provided by Dr. J. Fisher (University of Zurich). FLG-29.1 cells were
obtained from Dr. M. L. Brandi (University of Firenze, Italy). The
cells were maintained in Hams F12/DMEM supplemented with 10% FCS.
Normal human bone was obtained from orthopedic surgery and comprised
cancellous bone from the tibial head of a 27-yr-old woman and
subsequently cultured. Briefly, the trabecular bone was minced, and
incubated with 250 U/ml collagenase (type IV) in medium lacking serum,
and the resultant bone chips were cultured in low calcium medium
(CaCl2-free DMEM) containing 10% heat inactivated FBS, 2
mM glutamine, 100 U/ml penicillin-streptomycin, and 50
µg/ml ascorbate.
RT-PCR
Total RNA was extracted from confluent cells using the Quiagen
kit for total RNA preparation from cell cultures (Quiagen, Chatsworth,
CA). Samples of total cellular RNA (5 µg) were reverse-transcribed
using the first strand synthesis kit of Pharmacia Biotech (Uppsala,
Sweden).
PCR was performed to generate amplified fragments of ERR-1, ERR-2, and
ER. The primers used, located in different exons, were as follow:
ER upstream: AGT ATG GCT ATG GAA TCT GCC A
ER downstream: TTT CAA CAT TCT CCC TCC TCT
ERR-1 upstream: TGG TCC AGC TCC CAC TCG CT
ERR-1 downstream: TGA GAC ACC AGT GCA TTC ACT G
ERR-2 upstream: TCA AGT GCG AGT ACA TGC TT
ERR-2 downstream: GAA ATC TGT AAG CTC AGG TA
TFIID upstream: ACA GGA GCC AAG AGT GAA GAA
TFIID downstream: CCA GAA ACA AAA ATA AGG AGA
The PCR reaction mixture contained cDNA (10 µl), 2 µl dNTP mix (10
mM), 1x PCR buffer with Mg++, 20 pmol primers
and 5 U of Taq polymerase from Boehringer (Mannheim,
Germany). Reactions were carried out in a PCR apparatus Master cycle
5330 plus from Eppendorf. One PCR cycle consisted of denaturation for
30 sec (94 C), annealing for 30 sec (60 C), and extension for 30 sec
(72 C). Each PCR reaction consisted of 30 cycles.
In Situ Hybridization
E11, E15.5, and E17.5 mouse embryos were fixed at 4 C for
16 h in 4% paraformaldehyde in PBS containing 5 mM
MgCl2, dehydrated, and embedded in paraffin.
Five-micrometer-thick sections were transferred to
3-aminopropyltriethoxysilane (TESPA; Aldrich, Milwaukee, WI) coated
slides and dried at 42 C for 2 days. In situ hybridization
was performed as described (39) using 35S-labeled antisense
RNA probe synthesized from a full-length mouse ERR-1 cDNA cloned in
Bluescript II KS (Stratagene, La Jolla, CA). To avoid any
cross-hybridization with ERR-2 mRNA, hybridization was performed with
stringent conditions (60 C). As a negative control, adjacent sections
were hybridized with a 35S-labeled sense RNA probe
synthesized from the same template. At the end of the in
situ hybridization protocol, nuclei were stained with bisbenzimide
and appeared blue under fluorescent light.
Histological Staining
The sections were treated with toluene, hydrated, and stained
with Nuclear Red for 20 min. They were rinsed, stained with
picro-indigo-carmine for 15 min, and rinsed with 0.3% acetic acid.
Finally, the sections were dehydrated with ethanol and treated with
toluene before mounting under a coverslip.
Transfections
Transient transfections were performed according to the calcium
phosphate precipitation method (40), using plasmid pSG5 as a carrier up
to 6 µg of total DNA per sample. Cells were collected 48 h after
transfection. CAT activities were determined and normalized to the
total amount of protein present in the cell lysates.
ROS17/2.8 cells were plated at 50% confluency in Hams F12/Dulbecco MEM
in 100-mm plates. At 80% confluence DNA-mediated gene transfer was
performed using lipofectamine from GIBCO-BRL (Basel, Switzerland).
Transfection time was 4 h and total DNA amount of 8 µg consisted
of the reporter plasmid (pSFREtk) with respectively 4- and
10-fold ERR-1 expression vector. One microgram of pCMV ß-gal
expression vector was used as an internal control for transfection
efficiency. The cells were then cultured for another 48 h and
harvested, and CAT activity was analyzed in cell extracts (prepared by
three freeze-thaw cycles) using a procedure described previously
(Gorman et al., 1982). Samples were run for 1 h on TLC
plates (Merck, Darmstadt, Germany), and radioactivity was measured by a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). ß-Galactosidase
was determined in cell extracts using the Promega kit based on a
technique described by Rosenthal (42). CAT enzyme results are
normalized by ß-galactosidase activity.
Plasmid Constructions
Mouse ERR-1 cDNA (a generous gift of Vincent Giguère) was
subcloned in the EcoRI site of plasmid pSG5. To clone HRE
sequences upstream from the minimal tk promoter, synthetic
oligonucleotides containing SFRE or HREpal sites (see sequences on Fig. 3A
) and flanked by BamHI and BglII sites were
phosphorylated and ligated. Pentamers were isolated in 5%
polyacrylamide gel and inserted into the BamHI site of
plasmid pBL Cat5 (43). For plasmid
96ERR-1, PCR was performed with
specific oligonucleotides. The resulting fragment was sequenced and
then reinserted in pSG5 plasmid.
Electrophoretic Mobility Shift Assays
Probes were labeled with T4 polynucleotide kinase in the
presence of [
-32P] ATP. In vitro
translations were performed using the TNT kit (Promega, Madison, WI)
and pSG5 either wild type (unprogrammed) or directing ERR-1 derivatives
expression. Binding reactions were performed as described (44), using 2
µl of translation reaction.
 |
ACKNOWLEDGMENTS
|
---|
Acknowledgements
The authors are indebted to Katja Sucker for technical help.
pOPN-Luc plasmid was constructed by Xiaojia Guo. We thank Dominique
Stéhelin for support, Vincent Giguere for ERR-1 expression vector
and sharing unpublished results, and Christine Dreyer for xFF1r
expression vector. Nicole Devassine and Marie-Christine Bouchez
performed excellent secretarial assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Vincent Laudet, Oncologie Moleculaire, Institut Pasteur de Lille, Endocrin Group CNRS URA 1160, 1 Rue Calmette, Lille Cedex, France 92521.
This work was supported by Association pour la Re-cherche sur le
Cancer (ARC), Centre Nationale Recherche Scientifique, and Institut
Pasteur de Lille. E.B. is supported by a grant from ARC. J.E.A. is
funded by MRC of Canada (Grant MT12380).
1 Equal first authors 
Received for publication July 8, 1996.
Revision received February 28, 1997.
Accepted for publication March 19, 1997.
 |
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