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Endocrinology, doi:10.1210/en.2006-0962
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Endocrinology Vol. 148, No. 3 1195-1205
Copyright © 2007 by The Endocrine Society

The Orphan Nuclear Estrogen Receptor-Related Receptor-{alpha} Regulates Cartilage Formation in Vitro: Implication of Sox9

E. Bonnelye, R. A. Zirngibl, P. Jurdic and J. E. Aubin

Department of Molecular and Medical Genetics (E.B., R.A.Z., J.E.A.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Laboratoire de Biologie Moleculaire de la Cellule (E.B., P.J.), Institut Fédératif de Recherche 128 Biosciences Lyon-Gerland École Normale Supérieure/Centre National de la Recherche Scientifique 5161, 69365 Lyon, France; and Mécanismes et Traitements des Métastases Osseuses des Tumeurs Solides (E.B.), Unité Institut National de la Santé et de la Recherche Médicale Unité 664, Faculté de Médecine René Théophile-Hyacinthe Laennec, 69372 Lyon, France

Address all correspondence and requests for reprints to: Jane E. Aubin, Ph.D., Department of Molecular and Medical Genetics, Faculty of Medicine, University of Toronto, Room 6230, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: jane.aubin{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report for the first time the expression of estrogen receptor-related receptor (ERR)-{alpha} in fetal and adult rat chondrocytes in growth plate and articular cartilage and the rat chondrogenic cell line C5.18 cells in vitro. ERR{alpha} mRNA and protein were expressed from proliferating chondrocyte to mature chondrocyte stages. We show that overexpressing ERR{alpha} in C5.18 cell cultures induces an increase in Sry-type high-mobility-group box transcription factor (Sox)-9 expression, a master gene in cartilage formation. In parallel, we report Sox9 promoter regulation by ERR{alpha} in C5.18 cells. To assess a functional role for ERR{alpha} in chondrogenesis, its expression was blocked by antisense oligonucleotides in C5.18 cell cultures, and this led to inhibition of cartilage formation associated with down-regulation of Sox9 and Indian hedgehog expression and maturation of proliferating chondrocytes into hypertrophic chondrocytes in vitro. Together these results implicate ERR{alpha} in the formation and maintenance of cartilage and also suggest that agonists and antagonists of ERR{alpha} may be useful as therapeutic agents in a wide variety of diseases affecting cartilage and joints.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOCHONDRAL BONE FORMATION begins with condensation of mesenchymal progenitors that undergo a series of sequential changes in proliferation and expression of genes that include the transcription factor, Sry-type high-mobility-group box transcription factor (Sox9), and extracellular matrix components (e.g. collagen type II, aggrecan, and link protein) (1). Ultimately, central cells in these cartilage primordia mature to hypertrophic chondrocytes expressing other molecules including type X collagen, the adjacent matrix then mineralizes, and is eventually replaced with bone (2). The bone grows in length through a continued and highly spatially oriented process of chondrocyte proliferation and continued differentiation to hypertrophic chondrocytes in the growth plate. This developmental program is controlled by a large number of factors, including Sox9 (early, from prechondrocytic mesenchymal condensation stage through differentiating chondrocytes), Indian hedgehog (Ihh; intermediate as prehypertrophic cells cease proliferation and become hypertrophic), and runt-related transcription factor-2 (late, hypertrophic chondrocytes) to name just a few (3, 4, 5).

A number of nuclear steroid receptors are also present in growth plate chondrocytes (6). The superfamily to which nuclear receptors belong comprises both ligand-dependent molecules such as estrogen receptors (ERs), and a large number of so-called orphan receptors for which no ligand has yet been determined (7, 8). Three orphan receptors, ER-related receptor (ERR)-{alpha}, ERRß, and ERR{gamma} (NR3B1, NR3B2, and NR3B3, respectively, according to the Nuclear Receptors Nomenclature Committee, 1999), share similarity with ER{alpha} and ERß (NR3A1 and NR3A2, respectively); however, they do not bind estrogens (9, 10). Based on its ability to recognize similar DNA sequences as the ERs and the ability of estrogens to stimulate ERR{alpha} gene expression in several tissues (uterus, heart, bone), ERR{alpha} has been proposed to modulate estrogen signaling (11, 12, 13, 14, 15). Teng and colleagues (16, 17) also showed that ERR{alpha} modulates the activating effect of estrogens on the lactoferrin promoter and suggested that ERR{alpha} may also interact with ERs through protein-protein interaction.

In addition to modulation of ER-mediated transcriptional activity, ERR{alpha} may regulate fatty acid oxidation. Consistent with this function, ERR{alpha} is prominently expressed in tissues with high capacity for ß-oxidation of fatty acids, such as heart and brown fat, and induces many genes involved in energy metabolism such as peroxisome proliferator-activated receptor-{alpha} and medium-chain acyl CoA dehydrogenase (18, 19, 20, 21, 22). In these tissues, ERR{alpha} is coexpressed with the transcriptional coactivator peroxisome proliferator-activated receptor-{gamma} coactivator (PGC)-1{alpha}. ERR{alpha} can be induced and activated by PGC-1{alpha}, and together they cooperate to induce mitochondrial biogenesis, suggesting that ERR{alpha} plays a role in at least some of the known PGC-1{alpha}-regulated pathways (21, 23, 24). ERR{alpha} may also be involved in bone formation, given that it is highly expressed at ossification sites and promotes osteoblast differentiation in vitro, and an ESRRA gene regulatory variant has recently been associated with bone mineral density in premenopausal women (25, 26, 27, 28). ERs, which are known to play a role in bone cells, are also expressed in cartilage in which they are thought to play roles in not only the pubertal growth spurt (29) but also cartilage damage associated with osteoarthritis and rheumatoid arthritis (30, 31).

Due to the putative roles of ERs and estrogens in cartilage, we hypothesized that ERR{alpha} may be functionally involved in chondrogenesis. To address this, we regulated ERR{alpha} expression in chondrogenic cells in vitro by transiently overexpressing ERR{alpha} via expression plasmids or underexpressing ERR{alpha} via antisense technology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
C5.18 cells, a model for chondrocyte proliferation, differentiation, and maturation/hypertrophy (32, 33, 34) were maintained in {alpha}-MEM containing 15% heat-inactivated fetal bovine serum (Flow Laboratories, McLean, VA), antibiotics comprising 100 µg/ml penicillin G (Sigma Chemical Co., St. Louis, MO), 50 µg/ml gentamicin (Sigma), 0.3 µg/ml fungizone (Flow Laboratories), and dexamethasone (10–8 M; (Merck, Sharp, and Dohme, Canada, Ltd., Kirkland, Québec, Canada). For differentiation, cells were grown in the same medium with dexamethasone and the addition of 50 µg/ml ascorbic acid and 10 mM sodium ß-glycerophosphate; medium was changed every 2 d. Cells were incubated at 37 C in a humidified atmosphere in a 95% air-5% CO2 incubator.

Northern blots
Total RNA was extracted according to the manufacturer’s directions with Trizol reagent (Invitrogen, Carlsbad, CA) from C5.18 cells at 72 h after transfection. Northern blots were prepared and hybridized with a 750-bp fragment corresponding to the rat 3' untranslated region of ERR{alpha} (provided by J. M. Vanacker, Institut National de la Santé et de la Recherche Medicale, Unité 540, Université de Montpellier 1, Montpellier, France) as described (26).

RT-PCR
Total RNA was extracted with Trizol reagent from C5.18 cells. Samples of total RNA (1.5–5 µg) were reverse transcribed and semiquantitative PCR was performed (26) (QIAGEN, Hilden, Germany) with the primers listed in Table 1Go. In preliminary experiments, we confirmed linear range of amplification for all primers and products. Real-time RT-PCR was carried out by using the LightCycler system (SYBR Green; Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Amplimers were quantified in triplicate samples in each of three independent experiments for each gene and normalized to corresponding L32 values.


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  		TABLE 1. Primers and conditions used for PCR

 
Immunolabeling
Approval of the University of Toronto Animal Care Committee was obtained for the use of rats to prepare skeletal sections for immunolabeling. Immunolabeling of cultures and sections was done as described previously (35, 36). Cells or sections were incubated for 3 h with appropriate dilutions of primary antibodies [1:50, anti-ERR{alpha}, purified rabbit polyclonal antibody raised against a peptide in the A/B domain (14–32 amino acids) characterized as described in Refs. 26 and 37 ; anti-ER{alpha} or anti-ERß (MC-20 or Y-19, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (38, 39, 40); anti-Sox9 (H-90; Santa Cruz Biotechnology)] or PBS control. For photography and printing, equal exposure times were used for specifically labeled and control cultures.

Cartilage nodule quantification
For quantification of cartilage formation, dishes or wells were fixed and stained with Alcian blue and cartilage nodules were counted on a grid (32). Results are plotted as the mean number of nodules ± SD of three wells for controls and each concentration of antisense, sense, or scrambled oligonucleotides and are representative of three independent experiments.

Cell counting
For cell growth analysis, the cell layers were rinsed in PBS, released with trypsin (Invitrogen) and collagenase (Sigma), and counted electronically. Results are plotted as the average of three counts for each of three wells for control and each concentration of oligonucleotides used and are representative of three independent experiments.

Antisense, sense, and scrambled oligonucleotide treatment
C5.18 cells were plated in 24-well plates at 104 cells/well. Antisense oligonucleotide inhibition of ERR{alpha} expression was accomplished with a 20-base phosphorothioate-modified oligonucleotide localized to the A/B domain (26). Control dishes were treated with the complementary sense (S) or scrambled (Sc) oligonucleotide or no oligonucleotide (Ct); no significant differences were seen between results with any of these controls. Preliminary experiments were also done to determine effective oligonucleotide concentrations that were not toxic. Then 0.5 µM to 5 µM oligonucleotides were added directly to cells during the proliferation phase (d 1–6) and 1–2 µM oligonucleotides were added during the differentiation phase (d 6–21) in differentiation medium. Medium was changed every 2 d and fresh oligonucleotides were added. Cartilage nodules were counted at d 21. mRNA was collected at d 6 for the proliferation experiments and at d 15 and 21 for the differentiation experiments.

Transfections and transactivation assay
C5.18 cells were transfected with full-length mouse ERR{alpha} (cloned into a modified pcDNA3.1-vector) (Invitrogen) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Each transfection consisted of 0.8 mg of empty vector as a control, ERR{alpha} wild-type and ERR{alpha}-{Delta}AF2 391 (helix11–12 deletion) expression plasmids, 200 ng of pGL3 mSox9 (–470 to +87) promoter vector (a 3 kb promoter fragment was the generous gift of T. M. Underhill, Department of Cellular and Physiological Sciences, University of British Columbia, Canada), and 25 ng pRL tk plasmid (Promega, Madison, WI) for internal control. Luciferase activities were measured on a Berthold microplate luminometer LB96V (EG&G Berthold GMBH & Co., Bad Wildbad, Germany) using the dual-luciferase reporter assay system (Promega). The experiments were done in triplicate and repeated at least three times.

Western blot
Total protein was extracted from C5.18 cells according to standard methods (56). Western blot analyses were performed using a semidry system. Immunoblotting was performed with the purified rabbit polyclonal antibody raised against a peptide in the A/B domain (14–32 amino acids) (26, 37); blots were incubated overnight at room temperature with the polyclonal antibody diluted at 1:60, and binding was detected using HRPO-conjugated goat antirabbit antibodies (1:3000; Bio-Rad Laboratories, Hercules, CA) and chemiluminescence.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (Tunel)
C5.18 cells were plated in 24-well plates at 104 cells/well, and 2 µM oligonucleotides (sense or antisense) were added during the differentiation phase (d 6–21). Cells were then fixed and Tunel staining performed with the Klenow FragEL DNA fragmentation detection kit (Calbiochem, San Diego, CA) according to the manufacturer’s instructions. Cells were counterstained with Methyl Green.

Statistical analysis
Results for PCR analysis, cartilage nodule number (antisense/sense/scrambled oligonucleotide experiments), and cell proliferation were expressed as mean ± SD and analyzed statistically by one-way ANOVA with treatment group as a fixed effect followed by t test post hoc tests (InStat software, version 2.01; GraphPad Software, San Diego, CA). Statistical significance was taken as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ERR{alpha} protein and mRNA are expressed in chondrocytes in vivo and in vitro
Immunohistochemistry on sections of 21 d fetal rat tibiae and metatarsals using a specific antibody (26, 37) showed that both articular chondrocyte precursors in the perichondrium (Fig. 1Go, A and C), and proliferating chondrocytes (Fig. 1Go, B and D) stain intensely for ERR{alpha}; however, hypertrophic chondrocytes are not labeled above background of controls (Fig. 1GGo). In adult bones, articular chondrocytes are intensely labeled (Fig. 1JGo). As shown previously (26), mature osteoblasts lining the bone matrix in the metaphyses of the growing bone also label intensely for ERR{alpha} (Fig. 1GGo).


Figure 1
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  		FIG. 1. Immunolabeling for ERR{alpha} in sections showing different regions of the 21-d fetal (A–D) and adult (J) rat tibia and metatarsal. For comparison, hematoxylin and eosin staining of the developing articular surfaces (E) and portion of the growth plate with hypertrophic chondrocytes (H) in 21-d fetal bones is shown. Second antibody (antirabbit antibody) control (Ct–) shows low nonspecific background (F, I, and K). Bar, 200 µm (A–K).

 
To extend the observations made in vivo to chondrocytes in vitro, semiquantitative PCR was performed over a proliferation-differentiation time course in the rat chondrogenic cell line, C5.18 (32, 33, 34). ERR{alpha} mRNA was expressed constitutively throughout proliferation (d 3), confluence (d 6), and early (d 11 and 15) and late (d 21) chondrocyte proliferation and cartilage nodule formation stages (Fig. 2Go, A and B). For comparison, mRNA levels for Sox9 and two extracellular cartilage matrix components [link protein and collagen type II (Col2a1)] are also shown (Fig. 2Go, A and B).


Figure 2
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  		FIG. 2. A and B, Semiquantitative PCR showed that ERR{alpha} is expressed over the proliferation (d 3)-confluence (d 6)-differentiation (d 11–21) time course in the rat chondrogenic cell line C5.18. ERR{alpha} signal intensity was normalized against that of the ribosomal protein gene L32. For comparison, mRNA levels for Sox 9 and two chondrocyte markers, link protein and Col2a1, are also shown and normalized against L32. C, ERR{alpha} protein is detectable by immunolabeling of C5.18 cells throughout the proliferation (d 3) (Ca), confluence (d 6) (Cb), and differentiation-cartilage nodule formation (d 11–21) (C, c and d) time course. Alcian blue positive control nodules at 21 d are also shown (Ce). ER{alpha} and ERß are detectable by immunolabeling of C5.18 cartilage nodules at d 18 (C, f and g). As expected, Sox9 is highly expressed in cartilage nodules (Cj). Antirabbit for ERR{alpha}, ER{alpha}, and Sox9 (C, h and k) and antigoat for ERß (Ci) antibody controls (Ct–) are also shown. Bar, 200 µm (C, a–k).

 
Consistent with the high levels of expression of ERR{alpha} mRNA, ERR{alpha} protein was found widely distributed in C5.18 cell cultures at all times analyzed, including proliferation (d 3–6) (Fig. 2CGo, a and b), early differentiation stages when nascent nodules were forming (d 11) (Fig. 2CcGo), and when cartilage-like nodules (containing cells with round morphology surrounded by an Alcian blue-positive matrix) were well developed (d 21) (Fig. 2CGo, d and e). Consistent with other studies documenting expression of ERs in chondrocytes (41, 42), staining for both ER{alpha} and ERß was also detected in chondrocytes in cartilage nodules (Fig. 2CGo, f and g), although ERß staining was somewhat less intense than that of ERR{alpha} or ER{alpha}. As expected, Sox9 was also found to be highly expressed in cartilage-like nodules (Fig. 2Go, Cj). Similarly to ERR{alpha}, Sox 9 staining was more intense in chondrocytes associated with cartilage-like nodules (Fig. 2CGo, d and j; white arrow) than in the surrounding fibroblastic cells (Fig. 2CGo, d and j; white asterisk).

Overexpression of ERR{alpha} stimulates Sox9 and chondrocyte marker expression in C5.18 cells
To address a putative functional role of ERR{alpha} in chondrogenesis, we first asked whether cartilage markers are altered when ERR{alpha} is overexpressed in C5.18 cells. ERR{alpha} overexpression was achieved by transient transfection of d 5 (50–60% confluent) cultures of C5.18 cells with a CMV-ERR{alpha} construct; a CMV-ßGal vector was used as control (Fig. 3Go, A and B). Using real-time PCR to assess expression of early markers of chondrogenesis, we found a significant increase in Sox9 but not aggrecan expression 72 h after transfection (Fig. 3Go, C and D). On the other hand, at d 15, when cartilage nodule formation is well progressed, link protein was also up-regulated in ERR{alpha}-overexpressing cultures (Fig. 3Go, E and F), suggesting that ERR{alpha} may be involved in chondrocyte differentiation. Concomitantly cartilage nodule number was increased by 40% in ERR{alpha} overexpressing cultures (Fig. 3GGo). To address further a putative functional role of ERR{alpha} on Sox9 expression, we performed luciferase reporter assays with a (–470 to +87 bp) Sox9 promoter fragment (pGL3sox9Luc) and WT-ERR{alpha} and found that ERR{alpha} transactivates the Sox9 promoter in C5.18 cells (Fig. 3HGo). Given that ERR{alpha} has been reported to transactivate through its activation function 2 (AF2) region located in the C-terminal end of the ligand binding domain (14), we tested the ability of ERR{alpha}-{Delta}AF2, where AF2 has been removed via deletion of helix 11and 12, to block the activation induced by WT-ERR{alpha}. As shown in Fig. 3HGo, the transactivation of the Sox9 promoter driven by WT-ERR{alpha} is abolished by ERR{alpha}-{Delta}AF2, suggesting that the activation of the Sox9 promoter by ERR{alpha} in C5.18 cells is dependent on a functional AF2 domain.


Figure 3
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  		FIG. 3. C5.18 cells were transfected with either a pcDNA3 empty plasmid or pcDNA3-ERR{alpha} (0.5 µg DNA per transfection). Successful overexpression of ERR{alpha} after transfection was judged by extracting total RNA (pools from three 35-mm dishes for each condition) 72 h after transfection and Northern blotting with ribosomal protein L32 as control (A and B). Total RNA was extracted from parallel dishes 72 h after transfection and at d 15, and real-time PCR was performed (RNA pooled from three 35-mm dishes) for chondrocyte markers Sox9, aggrecan, and link protein (C–F). The amount of PCR product for each marker was normalized to ribosomal protein L32 PCR product. Sox9 and link protein were significantly increased in cultures overexpressing ERR{alpha} (ANOVA; P < 0.001, P < 0.05; Student’s t posttests, *, P < 0.05, ***, P < 0.001 empty vector vs. ERR{alpha} transfected vector). The experiments were done in triplicate and are representative of three independent experiments. C–F, Overexpressing ERR{alpha} resulted in a slight increase in the number of cartilage nodules (ANOVA; P < 0.05; Student’s t posttests, *, P < 0.05, empty vector vs. ERR{alpha} transfected vector). The experiments were done on four independent dishes and are representative of two independent experiments. G, C5.18 cells were cotransfected with a luciferase reporter construct containing a (–470 to +87 bp) fragment of the mouse Sox 9 (pGL3 mSox9) promoter with 0.8 mg of either empty pcDNA3 or pcDNA3-ERR{alpha} or pcDNA3-ERR{alpha}{Delta} AF-2. Reporter activity was measured 48 h after transfection and normalized to internal control vector. The experiments were done in triplicate and repeated a minimum of three times. Student’s t posttests, **, P < 0.01; *, P < 0.05 (H).

 
Inhibition of ERR{alpha} expression down-regulates Sox9 and Ihh expression and alters cartilage formation in vitro
To further examine the role of ERR{alpha} during chondrogenesis, we blocked ERR{alpha} protein production by using antisense oligonucleotides designed to be specific for ERR{alpha} and previously used in osteoblast studies (12, 26). Preliminary experiments were done to determine oligonucleotide concentrations that were effective but not toxic (0.5–5 µM; see also Ref. 26); ERR{alpha} was markedly reduced in C5.18 cultures treated with antisense (AS) oligonucleotides (Fig. 4Go, A and B). To dissect the possible involvement of ERR{alpha} in chondrocyte differentiation and cartilage formation, C5.18 cells were treated with AS or control oligonucleotides from d 6 to 13 (beginning of cartilage nodule formation in control cultures). A significant dose-dependent increase in the number of alcian blue-positive cartilage nodules formed was seen in AS-treated C5.18 cells (25% at 1 µM and 30% at 2 µM at d 21; Fig. 4Go, C and D). Notably, however, the cartilage-like nodules that formed in AS-treated cultures appeared very flat and stained relatively faintly, compared with the markedly three-dimensional dark blue nodules observed in untreated, Sc- and S-treated cells (Fig. 4Go, C and E). In parallel, the number of mature dark blue three-dimensional nodules was significantly reduced in AS-treated vs. Sc- and S-treated cells (Fig. 4FGo), even though the total (light and dark blue) number of alcian blue-positive colonies was higher in AS-treated cells (Fig. 4Go, C and D). These data suggest that inhibition of ERR{alpha} levels has two opposing effects: induction of commitment or differentiation of some progenitors but inhibition of chondrocyte differentiation at an early stage such that progression to robust matrix deposition and maturation is reduced. To determine whether this is due to Sox9 regulation, we assessed Sox9 expression by semiquantitative PCR (Fig. 5Go) and real-time PCR (data not shown) at two times: midway through the differentiation process (d 15) and at late terminal differentiation/hypertrophic stages (d 21). Consistent with what we found in ERR{alpha}-overexpressing cultures, Sox9 was dramatically down-regulated at d 15 after inhibition of ERR{alpha} in C5.18. Concomitantly, we found Col2a1, aggrecan, and link protein also decreased in cells treated with AS, compared with control oligonucleotides (Fig. 5Go, A and B).


Figure 4
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  		FIG. 4. Inhibition of ERR{alpha} expression was accomplished with AS oligonucleotide. Control C5.18 cells were treated with either the complementary S oligonucleotide or no oligonucleotide (Ct). The efficacy of the AS treatment is shown by decreased intensity of label for ERR{alpha} protein after treatment with 2 µM of AS but not 2 µM S or no (Ct) oligonucleotide (A and B; B is a quantification of A). C5.18 cells were treated with AS, Sc, or S oligonucleotides at 1 and 2 µM or no (Ct) oligonucleotide from d 6 (confluence) to d 13 (cartilage nodules beginning to form); cultures were fixed on d 21 and stained with alcian blue (C and E). Inhibition of ERR{alpha} protein synthesis resulted in a slight increase in the number of alcian blue-positive colonies (ANOVA; P < 0.0001; Student’s t posttests, **, P < 0.01, AS vs. S) (D) but a decrease in the number of three-dimensional dark blue cartilage nodules (ANOVA; P < 0.05; Student’s t posttests, *, P < 0.01, AS vs. S) (F). Data are expressed as the mean number of nodules ± SD of triplicate wells and are representative of three independent experiments; cartilage nodules were identified by alcian blue staining at d 21 (C and E). E, Original magnifications, x10 (a and b) and x30 (c and d). Bar, 200 µm (a–d).

 

Figure 5
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  		FIG. 5. Total RNA was extracted from parallel wells at d 15 (A and B) and d 21 (C and D) and semiquantitative PCR was performed (RNA pooled from three 24-mm dishes) by using primers specific for chondrocyte markers (Sox9, Ihh, aggrecan, link protein, Col2a1), hypertrophic chondrocyte markers (Col10a1, PPR), and apoptosis markers (Bcl2 and Bax). The amount of PCR product for each marker was normalized to ribosomal protein L32 PCR product. Band intensity was quantified in triplicate samples. Results are representative of three independent experiments. A–D, All marker PCR products were normalized to L32 PCR product. Sox9, Ihh, aggrecan, link protein, and Col2a1 were reduced significantly by AS treatment (ANOVA, P < 0.0001 at d 15 and 21; Student’s t posttests, *, P < 0.05, **, P < 0.01, ***, P < 0.001 AS vs. S), whereas Col10a1 was increased significantly (ANOVA, P < 0.001 at d 15 and P < 0.001 at d 21; Student’s t posttests, *, P < 0.05, **, P < 0.01, ***, P < 0.001, AS vs. S) at d 15 (A and B) and 21 (C and D) and Bcl2 decreased (ANOVA, P < 0.001 ***, P < 0.001, AS vs. S) day 21 (C an and D). C5.18 cells were treated with AS or S oligonucleotides at 2 µM or no (Ct) oligonucleotide from d 6 (confluence) to d 13 (cartilage nodules beginning to form), fixed on d 21 and Tunel labeled; a marked increase in the number of brown apoptotic bodies is clear in AS- vs. S-treated and untreated (Ct) cells (E). Bar, 200 µm.

 
Because terminal differentiation of chondrocytes to the hypertrophic phenotype is accelerated in Sox9–/– mice (3, 4), we next examined expression of the prehypertrophic marker Ihh and the hypertrophic chondrocyte marker collagen type X (Col10a1) (43). Consistent with the down-regulation of Sox9 expression, we found a decrease in Ihh expression and a small increase in Col10a1 in AS-treated cells at d 15, suggesting increased maturation to a hypertrophic state (Fig. 5Go, A and B). At terminal differentiation stages (d 21), the expression of Sox9, Ihh, aggrecan, link protein, and Col2a1 were still strikingly decreased, whereas Col10a1 was strongly increased at this stage along with a very small increase in PTH/PTHrP receptor expression (PPR; a marker of prehypertrophic and hypertrophic chondrocytes) in AS-treated compared with control cultures (Fig. 5Go, C and D), although the increase in PPR did not reach statistical significance. Because hypertrophic chondrocytes are well known to go through apoptosis, we also assessed Bcl2, which was decreased, and the proapoptotic Bax, which was unchanged, in AS-treated cells (Fig. 5Go, C and D), suggesting an increase in the maturation of chondrocytes into hypertrophic chondrocytes with induction of apoptosis (Fig. 5Go, C and D). The increase in apoptosis was confirmed with Tunel staining, which revealed a marked increase in labeled apoptotic bodies in AS- vs. S-treated or Ct cells (Fig. 5EGo).

Sox9 expression is also regulated by ERR{alpha} during proliferation stage in C5.18 cultures
Because Sox9 is one of earliest markers expressed in mesenchymal cells undergoing prechondrogenic condensation, we also treated C5.18 cells during the proliferation stage (d 1–6). AS treatment caused a significant and specific dose-dependent decrease (~30%) in cell number at d 6 in dishes treated with AS compared with S, Sc, or untreated controls (Fig. 6AGo). Concomitantly, Sox9, Ihh, and cyclin D1 (cell cycle regulator and proliferation marker) were reduced significantly (30, 20, and 35%, respectively; Fig. 6Go, B and C), whereas FGFR-3, Col2a1, aggrecan, Bax, and Bcl2 were not significantly affected at that early stage of the culture (Fig. 6Go, B and C).


Figure 6
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  		FIG. 6. C5.18 cells were treated with AS, Sc, or S oligonucleotides at 1, 2, and 5 µM or no (Ct) oligonucleotide during the proliferation stage (d 1–6). Inhibition of ERR{alpha} decreased cell proliferation as evident from decreased cell number at d 6 (ANOVA, P < 0.0001; Student’s t post tests, ***, P < 0.001 AS vs. S, AS vs. Sc). Data are expressed as the cell number mean ± SD of triplicate wells and are representative of three independent experiments (A). Total RNA was extracted from parallel wells and semiquantitative PCR performed on triplicate samples by using primers specific for early markers of chondrocyte differentiation (Sox 9, Ihh, FGFR-3, Col2a1, aggrecan), proliferation (cyclin D1), and apoptosis (Bcl2, Bax) at d 6 (B). The amount of PCR product for each marker was normalized to ribosomal protein L32 PCR product (C). Sox 9, Ihh, and cyclin D1 were significantly reduced in AS-treated cultures (ANOVA; P < 0.05, P < 0.001, P < 0.0001, respectively; Student’s t post tests, *, P < 0.05; **, P < 0.01, AS vs. S). Results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report that ERR{alpha} mRNA and protein are expressed throughout proliferation and differentiation stages in the C5.18 cell model in vitro and in fetal and adult cartilage in vivo. Manipulation of ERR{alpha} levels by transient transfection (overexpression) and ERR{alpha} AS treatment (inhibition) induces an up- and down-regulation, respectively, of Sox9 and the cartilage extracellular matrix components, Col2a1, aggrecan, and link protein. Our results suggest that ERR{alpha} plays a widespread functional role in proliferation and maturation of proliferating chondrocytes into hypertrophic chondrocytes. The fact that ER{alpha} and ERß (albeit at lower levels than ER{alpha}) are also expressed in C5.18 cells at all times analyzed and that both receptors are present in adult articular chondrocytes (44), suggest that ERR{alpha} and ER{alpha} and/or ERß are coexpressed in chondrogenic cells, in which they may act alone or together to regulate the expression of target genes in cartilage.

By modulating ERR{alpha} expression in a chondrogenic cell line, we found marked regulation of Sox9, a transcription factor required in successive steps of chondrogenesis, as indicated by the severe chondrodysplasia that occurs when it is inactivated in limb buds (3). In mouse embryos, Sox9 is expressed in chondroprogenitors and chondrocytes but not hypertrophic chondrocytes, paralleling ERR{alpha} expression (Fig. 7AGo) and consistent with the down-regulation of Sox9 expression in ERR{alpha} AS-treated cells and with the activation by ERR{alpha} of Sox9 promoter activity in C5.18 cells. In Sox9–/– mice, most cells were arrested and did not undergo overt differentiation into chondrocytes resulting in a decrease in cartilage extracellular matrix proteins, Col2a1, and aggrecan and also in Ihh-PTHrP signaling pathways (3). In keeping with Sox9 function in chondrogenesis, its down-regulation after ERR{alpha} AS treatment of differentiating C5.18 cells leads to a decrease in expression of Ihh and cartilage extracellular matrix proteins, Col2a1, aggrecan, and link protein. In addition to a marked decrease in chondrocyte proliferation, Sox9–/– mice also show an increase in the fraction of hypertrophic chondrocytes, again consistent with the up-regulation of Col10a1 and Bcl2 and the small increase in PPR expression we observed after down-regulation of Sox9 expression by ERR{alpha} AS treatment in C5.18 (Fig. 7BGo). Taken together, our data suggest that inhibition of ERR{alpha} in chondrocytic cells accelerates maturation of proliferating chondrocytes to hypertrophy and increases apoptosis, possibly due to the direct or indirect effect of ERR{alpha} on regulation of Sox9.


Figure 7
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  		FIG. 7. Schematic representation of a potential ERR{alpha} function in chondrogenesis. ERR{alpha} and Sox9 coexpression patterns in the growth plate (A) and in vitro regulation of Sox9 and other markers expression in chondrocytes (B) are shown.

 
It must be acknowledged that no cartilage/bone phenotype was described in ERR{alpha}–/– mice, although it seems likely that compensatory mechanisms involving ERRß and/or ERR{gamma} may be operative (20). In any case, our findings suggest a critical role for ERR{alpha} in cartilage formation and the transition from proliferating chondrocytes into hypertrophic chondrocytes. In this regard, Ihh–/– mice also have pronounced abnormalities in bone growth with a marked decrease in chondrocyte proliferation and an increase in the fraction of hypertrophic chondrocyte present (45, 46, 47). Whether ERR{alpha} function in cartilage formation is due to its ability to directly regulate Sox9 upstream of other cartilage-associated markers and/or the Ihh-PTHrP axis, which is a key regulator of chondrocyte hypertrophy (4, 47, 48), remains to be determined. In addition to the role of ERR{alpha} in regulating maturation to the hypertrophic state, it will also be of interest to uncover the mechanisms underlying the increase in Alcian blue colony starts when ERR{alpha} is diminished because it implies an inhibitory role for the receptor in commitment or very early differentiation events. To address further the role of ERR{alpha} in cartilage, we are in the process of preparing transgenic mice with the Col2a1 promoter driving ERR{alpha} expression specifically in chondrocytes.

AS oligonucleotide-induced down-regulation of ERR{alpha} in early stage cultures inhibits proliferation in the C5.18 cell model without significant changes in expression levels of several proliferation and apoptosis/survival-associated genes including FGFR3, Bcl-2, and Bax. However, cyclin D1, a regulator of G1 phase progression, is significantly inhibited, consistent with our previous suggestion that cyclin D1 is a target gene of ERR{alpha} in osteoblasts (26). Ihh binds to Patched-1, which leads to activation of Smoothened, a membrane protein required for the cellular action of Ihh (2, 4). Interestingly, in chondrocytes lacking either Ihh or Smoothened, cyclin D1 expression is markedly decreased, suggesting that Ihh regulation of chondrocyte proliferation requires this cell cycle regulator. The fact that Ihh is down-regulated in the Sox9 null mice leads to the interesting hypothesis that ERR{alpha} regulates chondrocyte proliferation via its ability to regulate Sox9 upstream of Ihh and cyclin D1. On the other hand, we cannot exclude the possibility that ERR{alpha} directly regulates Ihh and cyclin D1 expression independently of Sox9.

Very recently Sox9 activity has been shown to be regulated by the transcriptional coactivator PGC-1{alpha} (49). PGC-1{alpha} exhibits differential expression during chondrocyte differentiation and directly interacts with Sox9 to activate Sox9-dependant Col2a1 expression (49). Coexpression of Sox9 and PGC-1{alpha} also activates expression of other chondrogenic genes (e.g. aggrecan and link protein), suggesting that PGC-1{alpha} not only directly regulates Col2a1 expression but also may play a more widespread role in chondrogenesis. PGC-1{alpha} is known to play a role in energy metabolism (50, 51) and has been implicated in mitochondrial biogenesis and fatty acid oxidation with ERR{alpha}, which is coexpressed with, induced, and activated by PGC-1{alpha} in heart and brown fat (21, 23, 24). Our findings on the expression of ERR{alpha} in chondrocytes in which it regulates Sox9 expression in vitro together with the finding that PGC-1{alpha} participates in Sox9 activation and chondrogenesis in vivo provide support for the hypothesis that ERR{alpha} and PGC-1{alpha} may function together in chondrogenesis and cartilage formation.

Finally, the presence of both ER{alpha} and ERß in human articular chondrocytes (44) and the capacity of estrogen to stimulate matrix protein turnover in articular chondrocyte cultures (52) provide further evidence that chondrocytes are sensitive to estrogen. In this regard, it is notable that estrogen stimulates the expression of ERR{alpha} in bone (12). ER{alpha} has also been shown to bind the ERR{alpha} promoter and activate its expression, indicating that the ERR{alpha} gene is a downstream target of ER{alpha} (13). There are as yet no known direct estrogen target genes in chondrocytes to explain the hormone’s anabolic effects on cartilage. However, our data showing the coexpression of ERR{alpha} and ERs in articular chondrocytes, together with its functional role in chondrocytes and putative capacity to regulate such master genes as Sox9, provide a potential mechanism by which estrogen may regulate cartilage in adult organisms.

In conclusion, ERR{alpha} is expressed in chondrocytic cells, regulates the expression of the cartilage master gene Sox9 and plays a role in cartilage formation in a cell culture model. Further understanding of the mechanisms by which ERR{alpha} either alone or through convergence on the ER or PGC-1 pathways modulates cartilage formation in normal development and disease is required. In the meantime, our data suggest that regulation of ERR{alpha} activity may be useful as a therapeutic strategy in a wide variety of metabolic and other diseases of bone and cartilage.


   Acknowledgments
 
We thank Usha Bhargava for technical help and Dr. T. Michael Underhill for the Sox9 promoter.


   Footnotes
 
This work was supported by the Institute of Musculoskeletal Health and Arthritis/Canadian Institutes of Health Research, the Arthritis Society of Canada (TAS), and the Canadian Arthritis Network and Milestone Medica (to J.E.A.) and fellowship support from the Association Jacques Cartier, the Association pour la Recherche sur la Polyarthrite (France), the Canadian Arthritis Network (to R.A.Z.) and TAS and Centre National de la Recherche Scientifique (to E.B.).

First Published Online December 14, 2006

Abbreviations: AF2, Activation function 2; AS, antisense; Col2a1, collagen type II; Col10a1, collagen type X; Ct, no oligonucleotide; ER, estrogen receptor; ERR, ER-related receptor; Ihh, Indian hedgehog; PGC, peroxisome proliferator-activated receptor-{gamma} coactivator; PPR, PTH/PTHrP receptor; S, sense; Sc, scrambled; Sox9, Sry-type high-mobility-group box transcription factor; Tunel, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received July 20, 2006.

Accepted for publication December 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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