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

Endocrin’os 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 9–17, 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 d’Ascq, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). This expression is specific since no signal was seen with the sense probe (Fig. 1FGo). 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. 1AGo and Fig. 1BGo). At higher magnification of the ulna, the specificity of ERR-1 for the bone [ossification zone (oz) in Fig. 1Go, 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.

 
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. 1Go, K and L), in the hindlimb (femur; Fig. 1Go, G, H, and I), and in the vertebrae (see va in Fig. 1Go, 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. 1Go, 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. 1Go, 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. 2AGo). Figure 2BGo 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. 2CGo). 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.

 
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. 2DGo). 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. 3AGo, 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. 3Go 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. 3BGo. 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. 3AGo). 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. 3bGo). 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. 3CGo. 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").

 
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. 3AGo). The resulting construct ({Delta}96ERR-1) was (after sequencing) in vitro translated and used in EMSA (Fig. 3CGo). {Delta}96ERR-1 forms a complex with SFRE that migrates faster than wild type (wt) ERR-1-SFRE. Combination of wt ERR-1 and {Delta}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. 4AGo). 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. 4BGo, 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. 4CGo. 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. 3AGo), 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.

 
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. 5AGo). 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.

 
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 5BGo 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (200–1000 molecules per cell; Refs. 21–23) relative to reproductive tissues (10000–100000 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. 2Go) 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. 6Go) that allow to define a TCA consensus. In contrast, these T and A boxes are divergent from those of NGF1B or Rev-erb{alpha}. This suggested that SF-1/FTZ-F1 and ERR-1 could recognize common sequences, distinct from the ones bound by NGF1B and Rev-erb{alpha}. 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{alpha} (which binds to a highly divergent sequence). +, Positively charged a.a.

 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 3AGo) 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 {Delta}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 [{gamma}-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 Back

Received for publication July 8, 1996. Revision received February 28, 1997. Accepted for publication March 19, 1997.


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