Correspondence to: J.E. Aubin, Department of Anatomy and Cell Biology, Faculty of Medicine, University of Toronto, Rm. 6255, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel:(416) 978-4220 Fax:(416) 978-3954 E-mail:jane.aubin{at}utoronto.ca.
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Abstract |
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The orphan nuclear estrogen receptorrelated receptor (ERR
), is expressed by many cell types, but is very highly expressed by osteoblastic cells in which it transactivates at least one osteoblast-associated gene, osteopontin. To study the putative involvement of ERR
in bone, we first assessed its expression in rat calvaria (RC) in vivo and in RC cells in vitro. ERR
mRNA and protein were expressed at all developmental stages from early osteoprogenitors to bone-forming osteoblasts, but protein was most abundant in mature cuboidal osteoblasts. To assess a functional role for ERR
in osteoblast differentiation and bone formation, we blocked its expression by antisense oligonucleotides in either proliferating or differentiating RC cell cultures and found inhibition of cell growth and a proliferation-independent inhibition of differentiation. On the other hand, ERR
overexpression in RC cells increased differentiation and maturation of progenitors to mature bone-forming cells. Our findings show that ERR
is highly expressed throughout the osteoblast developmental sequence and plays a physiological role in differentiation and bone formation at both proliferation and differentiation stages. In addition, we found that manipulation of receptor levels in the absence of known ligand is a fruitful approach for functional analysis of this orphan receptor and identification of potential target genes.
Key Words: osteoblasts, bone, nuclear receptor, estrogen related, differentiation
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Introduction |
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Nuclear receptors are transcription factors involved in various physiological regulatory processes. The superfamily to which nuclear receptors belong comprises both ligand-dependent molecules such as the steroid hormone-, thyroid hormone-, retinoic acid-, and vitamin D receptors, and an increasing number of so-called orphan receptors for which no ligand has yet been determined (
Two orphan receptors, estrogen receptorrelated receptor (ERR
)1 and ERRß ([
and ERß ([
was identified by low-stringency screening of cDNA libraries with a probe encompassing the DNA-binding domain of the human estrogen receptor (ER). Recently, a third ERR, ERR3 or ERR
, was identified by yeast two-hybrid screening with the glucocorticoid receptorinteracting protein 1 (GRIP1) as bait (
and the ERs reveals a high similarity (68%) in the 66 amino acids of the DNA-binding domain and a moderate similarity (36%) in the ligand-binding E domain, which may explain the fact that ERR
does not bind estrogen. Although ligands for the ERRs have not been clearly identified, the pesticides chlordane and toxaphene have been suggested to be potential ligands for ERR
(
has been identified as a regulator of fat metabolism (
Postmenopausal osteoporosis is a condition caused primarily by the severe decrease of serum estrogen levels after cessation of ovarian function ( or ERß have relatively minor skeletal abnormalities (
may intervene in the signals induced by estrogen in bone. ERR
has a broad spectrum of expression, including fat, muscle, brain, testis, and skin (
is also highly expressed in the ossification zones of the mouse embryo (in long bones, vertebrae, ribs, and skull), and is more widely distributed in osteoblast-like cells than is ER
(
positively regulates the osteopontin (OPN) gene (
Given these observations, we sought to assess more directly whether ERR plays a functional role in osteoblast development and bone formation. Our findings show that ERR
is highly expressed and widely distributed in differentiating osteoblastic cells. They also indicate a critical role for ERR
in bone formation, with both up- and downregulation of bone nodule formation concomitant with up- and downregulation of ERR
expression in vitro. These data suggest that ERR
plays a widespread and physiologically relevant role in bone formation and predict specific ERR
target genes at different osteoblast developmental time windows.
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Materials and Methods |
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Cell Culture
Cells were enzymatically isolated from the calvaria of 21-d Wistar rat fetuses by sequential digestion with collagenase as described previously (-MEM containing 15% heat-inactivated FBS (Flow Laboratories) and antibiotics comprising 100 µg/ml penicillin G (Sigma-Aldrich), 50 µg/ml gentamycin (Sigma-Aldrich), and 0.3 µg/ml fungizone (Flow Laboratories). After 24 h incubation, attached cells were washed with PBS to remove nonviable cells and other debris, and then collected by trypsinization using 0.01% trypsin in citrate saline. Aliquots were counted with a Coulter Counter (Coulter Electronics), and the remaining cells were resuspended in the standard medium described above. The resuspended cells were plated into 100-mm tissue culture dishes at 105 cells/dish, into 35-mm tissue culture dishes at 2 x 104/dish, and in 24-well plates at 104 cells/well. After 24 h incubation, medium was changed and supplemented with 50 µg/ml ascorbic acid, 10 mM sodium ß-glycerophosphate, and with or without 10-8 M dexamethasone (Merck, Sharp, and Dohme, Canada, Ltd.). Medium was changed every 2 d. All dishes were incubated at 37°C in a humidified atmosphere in a 95% air/5% CO2 incubator.
Northern Blots
Total RNA was extracted with guanidine from rat calvaria (RC) cells at different times of the culture corresponding to different stages of proliferation, differentiation, and bone nodule formation ( (provided by J.M. Vanacker, CNRS, UMR 5665, Lyon, France) according to standard procedures (
gt11 library prepared from ROS 17/2.8 cells and rat L32 was generated from RC cell mRNA by PCR using specifics primers (
Reverse Transcription PCR
Samples of total cellular RNA (1.55µg) were reverse transcribed using oligo(dT) and the first strand synthesis kit of SuperscriptTM II (GIBCO BRL). PCR was performed with specific primers specific for ERR. Primers were as follows: ERR
upstream (3'UTR), CAG GAA AGT GAA TGC CCA GG; ERR
downstream (3'UTR), CTT TGC AGC AAA TAT ACA TT; L32 upstream, CAT GGC TGC CCT TCG GCC TC; and L32 downstream, CAT TCT CTT CGC TGC GTA GCC.
The PCR reaction mixture contained cDNA (1 µl), 1 µl dNTP mix (10 mM), 10x PCR buffer, Q solution, 25 pmol primers, and 5 U of Taq polymerase from Quiagen. PCR was done for 25 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final elongation step of 7 min at 72°C) for ERR and L32. Amplimers were sequenced for verification.
Osteoblast associated and other markers were also amplified by PCR using specific primers for rat OCN, OPN, ALP, bone sialoprotein (BSP), Cbfa1, collagen type I chain (COLLI), C-fos, cyclin D1, Bax, and Bcl-2. PCR was done for 25 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final elongation step of 7 min at 72°C) for OCN, OPN, ALP, BSP, L32, Bax, and 30 cycles for Bcl-2, 32 cycles for c-Fos, 35 cycles for cyclin D1 and Cbfa1 (with annealing temperatures of 58°C and 62°C, respectively), and 23 cycles for COLLI with annealing temperature of 59°C.
Primers were OCN upstream: AGG ACC CTC TCT CTG CTC AC; OC downstream: AAC GGT GGT GCC ATA GAT GC; BSP upstream: CGC CTA CTT TTA TCC TCC TCT G; BSP downstream: CTG ACC CTC GTA GCC TTC ATA G; ALP upstream: CCC GCA TCC TTA AGG GCC AG; ALP downstream: TAG GCG ATG TCC TTG CAG C; OPN upstream: GCC ACT TGG CTG AAG CCT G; OPN downstream: GAA ACT CCT GGA CTT TGA CC; Cbfa1 upstream: CTT CAT TCG CCT CAC AAA C; Cbfa1 downstream: CAC GTC GCT CAT CTT GCC GG; cyclin D1 upstream: TCC CGC CAG CAG CAA GAC AC; cyclin D1 downstream: TGA GCT TGT TCA CCA GAA GC; c-Fos upstream: ATA GAG CCG GCG GAG CCG CG; c-Fos downstream: AAG CCC CGG TCG ACG GGG TG; Bax upstream: CCT TGG AGC AGC CGC CCC AG; Bax downstream: ATG TGG GCG TCC CGA AGT AGG; Bcl-2 upstream: GGG GAA ACA CCA GAA TCA AG; Bcl-2 downstream: AGA GAA GTC ATC CCC AGC CC; COLLI upstream: GGA GAG AGT GCC AAC TCC AG; COLLI downstream: CCA CCC CAG GGA TAA AAA CT.
Poly(A) PCR Library Selection
19 poly(A) PCR libraries representative of five transitional stages in osteoblast lineage progression were selected from >100 available amplified colonies (I collagen or ALP, both early markers of osteoprogenitor cells. Stage B and C colonies are progressively more mature, i.e., expressing type I
I collagen or both type I
I collagen and ALP, respectively. Stage D colonies represent multilayered cells and contain histologically identifiable cuboidal osteoblasts. Stage E colonies comprised terminal differentiation stages, with multilayered cells and mineralized bone matrix.
Total RNA was extracted using a mini-guanidine thiocyanate method as described previously ( was standardized against total cDNA.
Western Blots
Total protein was extracted from confluent HeLa and RC cells according to standard methods (
Immunocytochemistry
Immunolabeling of cultures was done essentially as described previously ( and in 3% BSA in PBS (denaturated) for OCN, ALP, OPN, and BSP. After rinsing, cells or sections were incubated for 3 h with appropriate dilutions of purified rabbit polyclonal antibody described previously (1/50, anti-ERR
). The antirat OCN antiserum was provided by D. Modrowski (INSERM U349, Hospital Lariboisiere, Paris, France) and used at 1/100 dilution. The anti-OPN (MPIIIB10) and anti-BSP (WWVIDI) antibodies were purchased from the Hybridoma Bank and used at a 1/300 and 1/100 dilution, respectively. The production and characterization of monoclonal antibody RBM 211.13 directed to rat bone/liver/kidney ALP have been described elsewhere (
, OC, and ALP or secondary antibody antimouse (1/300 final dilution; Jackson ImmunoResearch Laboratories) for BSP and OPN. After rinsing, samples were mounted in Moviol (Hoechst Ltd.) and observed by epifluorescence microscopy on a ZEISS Photomicroscope III (ZEISS). For photography and printing, equal exposure times were used for specifically labeled and control cultures.
Nodule Quantification
For quantification of nodule formation, dishes or wells were fixed and stained by the Von Kossa technique and bone nodules were counted on a grid ( of five independent experiments.
Cell Counting
For cell growth analysis, the cell layers were rinsed in PBS, released with trypsin and collagenase (1:1, vol/vol, of solutions described above), and the harvested cells were counted electronically. Results are plotted as the average of three counts for each of three dishes for control and pcDNA3-ERR or three wells for each concentration of antisense or sense primers used.
ALP Histochemistry
The histochemical stain for ALP is a modification of
Transient Transfections
Primary RC cells were grown in 35-mm tissue culture dishes at 2 x 104/dish in -MEM containing 10% heat-inactivated FBS (Flow Laboratories) and supplemented with 50 µg/ml ascorbic acid, 10 mM sodium ß-glycerophosphate, and 10-8 M dexamethasone. Cells were transfected at 50% of confluence according to the Effectene transfection protocol (QIAGEN) using a pcDNA3 empty plasmid as a control and pcDNA3-ERR
(mouse cDNA full-length from 12,211 bp; in the EcoRI cloning site) at 0.5 µg of total DNA per transfection. As control of transfection efficiency, we used a CMV-ßGal vector. Nodules were counted at day 15. mRNA was extracted at 72 h, day 10 (beginning of nodules formation), and day 13 (formed nodules) after transfection.
Antisense and Sense Oligonucleotide Treatment
The resuspended RC cells were plated in 24-well plates at 104 cells/well. Antisense oligonucleotide inhibition of ERR expression was accomplished with a 20-base phosphorothioate-modified oligonucleotide, localized to the A/B domain, close to the start methionine. This domain is known not to be highly conserved among members of the nuclear receptor family. The ERR
antisense oligonucleotide sequence was: 5'-TCACCGGGGGTTCAGTCTCA-3'. This sequence was rigorously analyzed by Blast search and no homologies were found; this included no homology with either ER
or ERß, ERRß or ERR
, or any other currently known nuclear receptors. The corresponding sense and a scrambled oligonucleotide (5'-TCGGCTCACACGGGTTAGCT-3') were used as negative controls. Other control dishes were treated with no oligonucleotide. Preliminary experiments were done to determine effective oligonucleotide concentrations that were not toxic. 0.1 to 5 µM oligonucleotides were added directly to cells either during the proliferation phase (days 1 to 6) and 0.5 to 2 µM oligonucleotides were added during the differentiation phase (day 5 [end of proliferation] to 11) in standard medium as above supplemented with 50 µg/ml ascorbic acid, 10 mM sodium ß-glycerophosphate, and 10-8 M dexamethasone. Medium was changed every 2 d and fresh oligonucleotides were added. mRNA was collected at day 6 for the proliferation experiments and at day 15 for the differentiation experiments. Nodules were counted at 15 d.
Statistical Analysis
Results for PCR analysis, bone nodule number (antisense/sense experiments), and cell proliferation were expressed as mean ± SD, and analyzed statistically by one-way analysis of variance (ANOVA) with treatment group as variance and Bonferroni and Tukey post tests with InStat software (v2.01; GraphPad Software). Results for bone nodule number (overexpression experiment) were expressed as mean ± SD and analyzed statistically by the Mann-Whitney test and Welch test (InStat). Statistical significance was taken as P < 0.05.
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Results |
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ERR mRNA Is Expressed at All Developmental Stages of Osteoblast Differentiation and Maturation in RC Cells In Vitro
ERR mRNA expression levels assessed over a proliferation-differentiation time course by Northern blotting of primary RC cell populations indicated that ERR
mRNA was expressed at all times analyzed, including proliferation (day 6), early nodule formation (day 10), and nodule mineralization (day 15) (not shown). However, because RC cell cultures comprise a heterogeneous mixture of cell types and osteoblasts at different differentiation stages, we sought to confirm that ERR
is expressed by osteoblast lineage cells and clarify its expression pattern over the proliferation-differentiation sequence of the osteoprogenitors. To do this, we used globally amplified (poly[A] PCR) cDNA pools prepared previously from single isolated osteoblast colonies at different stages of differentiation (
was amplified in each cDNA pool with specific primers for sequences in the 3'UTR of ERR
and normalized to the relative amounts of total cDNA. ERR
mRNA was found to be expressed at all developmental times assessed including in colonies containing primitive progenitors, expressing only OPN (Fig 1 A), in progressively more mature colonies expressing COLLI and OPN (Fig 1 B) or COLLI, ALP, and OPN (Fig 1 C), in multilayered colonies containing identifiable cuboidal osteoblasts also expressing OCN (Fig 1 D) and, finally, in mineralized bone nodules (Fig 1 E).
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ERR Protein Is Expressed in Osteoblastic RC Cells In Vitro and in Fetal RC In Vivo
To determine whether ERR protein is expressed in RC cell cultures, we performed immunocytochemistry. First, however, a Western blot of HeLa cell extracts was used to confirm the specificity of the ERR
antibody. As expected based on previously published data (
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As predicted based on the relatively wide tissue distribution of ERR mRNA in developing mouse embryos (
protein was found to be widely distributed in most, if not all, cells in RC cultures at all times analyzed, including early proliferation stages, confluence (data not shown), when nascent bone nodules were forming (Fig 2 B, a), and when nodules were mineralizing (Fig 2 B, b). However, staining for ERR
was more intense in cuboidal osteoblasts associated with both early and late bone nodules than in the surrounding fibroblastic cells (Fig 2 B, a and b). For comparison, protein expression of two well-established osteoblast markers, OCN (Fig 2 B, d) and OPN (Fig 2 B, e), both of which are coexpressed at high levels in the same cuboidal osteoblastic cells expressing ERR
, is also shown. As expected, ERR
was also highly expressed in cells expressing ALP and BSP (data not shown).
To extend the observations made in vitro to bones in vivo, we also performed immunocytochemistry on sections of 21-d fetal RC, the same bones used for preparation of cell cultures. Consistent with the in vitro results, ERR was found in all detectable cohorts of osteoblasts from those associated with nascent bone at the osteogenic front (Fig 2 B, f) to those in the more mature growing bone trabecula (Fig 2 B, g) and remodeling bone (Fig 2 B, h). Preincubation of the ERR
antibody with the peptide used as immunogen abolished all specific labeling (Fig 2 B, i). Consistent with our reverse transcription (RT)-PCR results on single bone nodules (see for example Fig 1), ERR
was also detectable in sutural cells (arrows, Fig 2 B, f), preosteoblasts, and osteocytes (Fig 2 B, g and h). ERR
was also high in osteocytes present in postnatal (4 wk) RC (data not shown), suggesting that ERR
may be involved not only in the formation but also in the maintenance of bone.
Inhibition of ERR Expression Blocks the Proliferation of RC Cells and Their Differentiation to Mature Bone-forming Osteoblasts
Given its expression in both proliferating osteoprogenitor cells and more mature osteoblasts and osteocytes, we next asked whether ERR is a critical factor in osteoblast proliferation and/or differentiation. Antisense oligonucleotides form DNARNA duplexes with specific mRNA species, thereby blocking binding of the mRNA to the 40S ribosomal subunit and preventing translation (
and, by Blast searching, found no evidence for homology with either ERs or any other nuclear receptors; controls comprised both a scrambled and a sense oligonucleotide. Preliminary experiments were done to determine effective oligonucleotide concentrations that were not toxic (not shown) and the specificity of the antisense was confirmed by Western blot on RC cell extracts. After 24 h of treatment or not with sense or antisense oligonucleotides on RC cells at day 12, ERR
is detectable as expected in extracts of untreated cultures and those treated with 2 µM sense oligonucleotides (Fig 3 B) but is almost undetectable in cultures treated with 2 µM antisense after quantification with actin used as control (Fig 3 B). The specificity of the antisense was also confirmed by immunocytochemistry for ERR
on bone nodules (data not shown).
To dissect the possible involvement of ERR in osteoblast differentiation and bone formation, we treated RC cells at different developmental times from early proliferation stages until mineralized nodule formation (summary, Fig 3 A). Treatment of RC cells between days 16, the proliferation stage, caused a significant and specific dose-dependent decrease, i.e., 13% at 0.5 µM, 30% at 1 µM, and 40% at 2 µM, in cell number at day 6 in dishes treated with antisense compared with sense, scrambled, or untreated controls (Fig 4 A). These results suggest that ERR
may play a role in the proliferation or very early differentiation phases of RC cells. To analyze the underlying mechanism of ERR
action during the proliferation phase, we assessed expression of early markers of osteoblast differentiation (ALP, BSP, OPN, Cbfa1, COLLI), proliferation (cyclin D1, c-Fos [data not shown]), and apoptosis (Bcl2, Bax) at day 6 (Fig 4 B). BSP and Cbfa1 were reduced significantly by 35% (Fig 4B and Fig C); cyclin D1 was also reduced. On the other hand, ALP, OPN, COLLI, c-Fos, Bax, and Bcl2 were not affected (Fig 4B and Fig C, and data not shown). To determine whether treatment during the proliferation time window caused a sustained alteration in differentiation, we assessed terminal differentiation/bone nodule formation at day 15 in cultures treated between days 16 with antisense and then switched to normal differentiation medium. A significant decrease in mineralized bone nodule number, i.e., 29% at 1 µM and 45% at 2 µM antisense oligonucleotides compared with sense or untreated controls (Fig 5 A), was seen. Concomitantly, we found that ALP and BSP expression were lower than levels seen in control or sense-treated cultures, whereas OPN, OCN, and COLLI were not significantly altered (Fig 5B and Fig C).
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To determine whether ERR also plays a role in osteoblast differentiation independently of an effect on proliferation, we next treated RC cells with the antisense or control oligonucleotides from day 5 (after cells had reached confluence and proliferation was decreased) to day 11. Although cell number was decreased by day 15 (19% at 1 µM and 35% at 2 µM; data not shown) in antisense-treated cultures, a much larger dose-dependent decrease in mineralized bone nodule formation was seen, i.e., 30% at 0.5 µM, 60% at 1 µM, and 100% decrease at 2 µM (Fig 6, AC); the sense and scrambled oligonucleotides had a nonspecific nondose-dependent effect on nodule numbers (Fig 6, AC). A similar inhibition of bone nodule formation was also observed when we treated the osteoblastic cell line MC3T3-E1 with the antisense oligonucleotides over a comparable time period (data not shown). In antisense-treated cultures, ALP-positive colonies were present and large in diameter but flat, suggesting that inhibition of ERR
blocked differentiation at an early stage such that progression to matrix deposition and maturation (Fig 6 B) was reduced. Consistent with this interpretation, Cbfa1, BSP, and OCN were all decreased in antisense-treated cultures whereas OPN, COLLI, and ALP were not affected (Fig 7A and Fig B). Immunocytochemistry confirmed the decrease in OCN- and BSP-expressing cells, but the maintenance of ALP expression in incipient bone nodules (Fig 7 C).
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Overexpression of ERR Increases Differentiation and Bone Nodule Formation in RC Cells
To address further the putative functional role of ERR in osteoblast differentiation suggested by the antisense oligonucleotide treatment, we next asked whether osteoblast differentiation and bone formation were altered when ERR
is overexpressed in RC cells. ERR
overexpression was achieved by transient transfection of day 5 (5060% confluent) cultures of RC cells with a CMV-ERR
construct. By using a CMV-ßGal control vector, we estimated that we reproducibly obtained a maximal efficiency of transfection of 1015%, which resulted in a 30% increase in ERR
levels observed on Northern blots (Fig 8 A). Consistent with the relatively low transfection efficiency, a small but statistically significant increase (15%) in the number of mineralized bone nodules formed by day 15 was observed (Fig 8 B). In parallel, we assessed expression levels of osteoblast markers (ALP, OPN, OCN) 72 h after transfection, at day 10 when bone nodules were beginning to form and at day 13 when nodules were well developed. We observed an increase in ALP and OPN at 72 h (Fig 8 C) and day 10 (data not shown) in cultures overexpressing ERR
, consistent with previous data describing OPN as a target gene of ERR
in reporter assays (
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Discussion |
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Our findings show that ERR is expressed in fetal and adult RC in vivo and in RC cell cultures in vitro throughout all osteoblast differentiation stages, from early osteoprogenitors to mature osteoblasts. Inhibition of ERR
expression during the proliferation phase of RC cell cultures with phosphorothioate-modified antisense oligonucleotides decreased proliferation, as seen by a decrease in cell number and proliferation markers, and reduced differentiation, as seen by a decrease in both mineralized bone nodule formation and expression levels of osteoblast-associated markers. Inhibition of ERR
early during the differentiation phase also markedly decreased differentiation and bone nodule formation. Overexpression of ERR
by transient transfection in differentiating RC cell cultures caused a small (15%) but significant increase in mineralized bone nodule formation and markers of osteoblast differentiation, consistent with the
1015% transfection efficiency and increased ERR
mRNA expression seen in the primary cell cultures. Although the antisense experiments are not sufficient to formally link functionality of the receptor with biological activity during osteoblast proliferation and differentiation, taken together with the results of ERR
overexpression, the data suggest that ERR
may play a role in bone formation and turnover, with specific effects on proliferation, progression of differentiation, and mineralization activity of osteoprogenitors and more mature osteoblasts.
ERR Is Expressed in Osteoblast Lineage Cells throughout Their Developmental Lifetime
We have found that ERR is expressed at all detectable stages of osteoblast development, suggesting that ERR
may have a function in osteoblasts throughout their developmental lifetime (see also below). In addition to its expression in fetal calvaria, ERR
is also highly expressed in adult calvaria (and other fetal and adult bones, data not shown) and is expressed throughout osteogenesis in adult rat bone marrow stromal cell cultures (Bonnelye and Aubin, in preparation), suggesting that it may function throughout the lifetime of the organism and may be required not only for bone formation but also for maintenance. In adult quiescent bone, labeling appears highest in osteocytes, which are thought to be mechanosensors that send strain-related signals to lining cells located at the bone surface through the canicular syncytium (
to transactivate the OPN promoter in osteoblasts suggests a possible mechanism by which ERR
may contribute to mechanical stress responses. OPN is expressed in osteocytes and has been postulated previously to play a bone role in response to mechanical stress; for example, the enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of OPN (
in osteocytes suggest that it may regulate OPN in this cell type and that disregulation of ERR
expression may alter OPN expression and concomitantly modify the response to mechanical stress.
ERR and Proliferation
Consistent with its expression in proliferating osteoblastic populations, we found that antisense oligonucleotide-induced downregulation of ERR inhibits proliferation of RC cell populations, an inhibition that appears to have consequences on bone nodule formation at later times (see below). The decrease in proliferation of RC cells was somewhat unexpected, given our previous observation that ERR
expression appears to correlate with exit from proliferation and the onset of the differentiation process in at least certain other cell types, including the nervous system, the epidermis, and muscles in the developing mouse (
may play cell typespecific functions and is in keeping with its detection from the onset of osteogenesis in vivo (
The molecular basis for the ERR effect on proliferation is of interest. As OPN has been described as a target gene of ERR
in vitro promoter-reporter assays (
later during the differentiation phase (see below). The mechanisms underlying this apparently differentiation stage-specific regulation of OPN and perhaps other target genes of ERR
is not yet clear; however, differentiation stage-specific expression of other coactivators or repressors including ERR
binding partners is one interesting hypothesis. We also found no significant changes in the antisense-treated RC populations in expression levels of a variety of proliferation and apoptosis/survival-associated genes expressed in osteoblasts including c-fos, Bcl-2, and Bax, although we cannot exclude possible changes in activity. However, we did observe a significant decrease in cyclin D1, a regulator of G1 phase progression. Interestingly, estrogen induces cell proliferation by stimulating progression through the G1 phase of the cell cycle (
has been described as a modulator of the ERmediated response of the human lactoferrin gene promoter (
ERR, Osteoblast Differentiation, and Matrix Mineralization
Our findings suggest a critical role for ERR in bone formation, with both up- and downregulation of bone nodule formation concomitant with up- and downregulation of ERR
expression in vitro. Even in a background of high endogenous ERR
expression, further upregulation of ERR
by transfection of RC cells with a full-length ERR
expression vector late in the proliferation time window increased bone nodule formation by an amount approximately equivalent to the transfection efficiency of the population. Concomitantly, bone markers were also upregulated. Whether the increase in osteoprogenitor differentiation and bone nodule formation is a consequence of upregulation of any of these bone markers, or results from regulation of another currently unknown ERR
target gene, remains to be explicitly tested. Further insight into mechanisms may also be gained from experiments in which forced expression of ERR
is carried out with systematic time course and dose response studies.
Downregulation of ERR either during proliferation phase or earlier or later in the differentiation sequence also had marked inhibitory effects on differentiation and bone nodule formation. Although one can speculate that the decrease seen when cells are treated only transiently during the proliferation phase reflects the downregulation of cyclin D1 and decreased proliferation of osteoprogenitors among other cells, the decrease could also reflect the concomitant downregulation of the bone "master" gene Cbfa1 (
are coexpressed in very early osteoprogenitors (
after proliferation has largely ceased (antisense treatment from day 511) results in complete inhibition of mineralized bone nodule formation and concomitant downregulation of Cbfa1, BSP, and OCN. These observations, together with the data on increased bone formation when ERR
is upregulated early, suggest that at least part of the effect of ERR
on osteoblast differentiation and bone formation occurs early during the differentiation sequence, such that differentiation may not progress beyond a certain point when ERR
levels are low. In keeping with this hypothesis, large flat but ALP-positive colonies are present in antisense-treated cultures, and a few cells express diminished levels of other osteoblast markers (Fig 6).
Manipulation of ERR levels in this study has served as a powerful approach to elucidating ERR
function in osteoblast development and bone formation in the absence of a known ligand. ERR
and the ERs share only moderate similarity (36%) in the ligand-binding E domain, which may explain the fact that ERR
does not bind estrogen. Although the pesticides chlordane and toxaphene, two organochlorine compounds with estrogen-like activity, have been reported to be antagonists of ERR
(
may act as a ligand-dependent repressor, no other ligands/agonists have yet been reported. On the other hand, the possibility of constitutive transactivation remains of interest. Constitutive activation of transcription of the lactoferrin gene by ERR
has been reported (
may indeed function as a constitutive transactivator of at least some genes, although the experiments do not exclude the possibility of an endogenous ligand in particular cell types. Whether osteoblastic cells may be capable of producing an endogenous ligand or whether ERR
is functioning as a constitutive transactivator of at least some genes (e.g., BSP, Cbfa1, OCN) in this cell lineage remains to be determined. In the meantime, modulation of levels of this orphan receptor offers an alternative strategy for functional analysis and identification of potential target genes. Such modulation of receptor itself may also contribute to understanding of bone diseases characterized by decreased osteoblast numbers and reduced bone mass such as osteoporosis and arthritic conditions and to therapeutic strategies to deal with them.
In conclusion, ERR is expressed in osteoblastic cells in vitro and in vivo and appears to have a function in proliferation, differentiation, bone nodule formation, and mineralization in RC cells. ERR
is expressed in fetal and adult calvaria and long bone, suggesting that ERR
may be involved not only in the formation but also in the maintenance and turnover of bone throughout the lifetime of the organism. These results indicate that ERR
may have an important function in the formation and turnover of the skeleton but the mechanism by which it does so, i.e., through convergence on the ER pathways or by other mechanisms, remains to be established. They also suggest that agonists and antagonists of ERR
may be useful as therapeutic agents in a wide variety of bone metabolic and other diseases affecting bone.
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Footnotes |
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1 Abbreviations used in this paper: ALP, alkaline phosphatase; ANOVA, analysis of variance; COLLI, collagen type I chain; ER, estrogen receptor; ERR
, estrogen receptorrelated receptor
; OCN, osteocalcin; OPN, osteopontin; RC, rat calvaria; RT, reverse transcription; UTR, untranslated region.
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Acknowledgements |
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We thank Usha Bhargava for her technical help, Jean-Marc Vanacker and Vincent Laudet for the sequence of the 3'UTR of ERR, and Dominic Falconi and Afshin Raouf for discussions on the manuscript.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MT-12390) and the Canadian Arthritis Network (CAN) to J.E. Aubin, and by fellowship support from the Association pour la Recherche sur le Cancer (ARC; France) and the Arthritis Society of Canada to E. Bonnelye, and by a CAN Graduate Student Training Award to V. Kung.
Note added in proof: After this manuscript was submitted, a paper appeared reporting that diethylstilbestrol is a ligand for ERR alpha (Tremblay, G.B., T. Kunath, D. Bergeron, L. Lapointe, C. Champigny, J.-A. Bader, J. Rossant, and V. Giguere. 2001. Genes Dev. 15:833838).
Submitted: 10 January 2001
Revised: 11 April 2001
Accepted: 19 April 2001
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References |
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