Abstract
The active thyroid hormone, triiodothyronine (T3), binds to thyroid hormone receptors (TR) and plays an essential role in the control of chondrocyte proliferation and differentiation. Hypo- and hyperthyroidism alter the structure of growth plate cartilage and modify chondrocyte gene expression in vivo, whilst TR mutations or deletions in mice result in altered growth plate architecture. Nevertheless, the particular roles of individual TR isoforms in mediating T3 action in chondrocytes have not been studied and are difficult to determine in vivo because of complex cellular and molecular interactions that regulate growth plate maturation. Therefore, we studied the effects of TRα and TRβ on chondrocyte growth and differentiation in primary cultures of neonatal rib chondrocytes isolated from TRα- and TRβ-deficient mice. T3 decreased proliferation but accelerated differentiation of rib chondrocytes from wild-type mice. T3 treatment resulted in similar effects in TRα-deficient chondrocytes, but in TRβ-deficient chondrocytes, all T3 responses were abrogated. Furthermore, T3 increased TRβ1 expression in wild-type and TRα-deficient chondrocytes. These data indicate that T3-stimulated differentiation of primary rib chondrocytes in vitro requires TRβ and suggest that the TRβ1 isoform mediates important T3 actions in mouse rib chondrocytes.
Introduction
Triiodothyronine (T3) plays a key role in bone growth and maturation (Bassett & Williams 2003). Hypothyroidism results in growth retardation, epiphyseal dysgenesis and delayed bone mineralization. In contrast, thyrotoxicosis accelerates growth and advances bone age, but causes short stature due to early closure of the growth plates. Longitudinal growth results from proliferation of growth plate chondrocytes, differentiation of proliferating chondrocytes and enlargement of hypertrophic chondrocytes, which increase their volume by tenfold during maturation. Hypertrophic chondrocytes also play a key role in the control of bone vascularization and thus regulate endochondral ossification (Gerber & Ferrara 2000, Goldring et al. 2006).
Both hypothyroidism and thyrotoxicosis alter the structure and organization of the growth plate in vivo (Lewinson et al. 1994, Stevens et al. 2000, Freitas et al. 2005). Hypothyroidism increases the width of the proliferative zone but reduces the size of the hypertrophic zone. In contrast, thyrotoxicosis results in a reduced width of both proliferative and hypertrophic zones. Nevertheless, the mechanisms by which altered thyroid status regulates the growth plate remain unclear. In vivo, thyroid hormones regulate expression of parathyroid hormone related peptide (PTHrP) and PTHrP receptors, which control the pace of chondrocyte proliferation during endochondral ossification (Stevens et al. 2000). In organ cultures, thyroxine (T4) is a potent stimulator of chondrocyte differentiation (Wakita et al. 1998, Miura et al. 2002), whilst in vitro T3 decreases the growth of chondrocyte colonies and inhibits cell proliferation (Bohme et al. 1992, Ohlsson et al. 1992, Robson et al. 2000, Okubo & Reddi 2003). Many investigators have also demonstrated a positive role for T3 in regulating terminal differentiation of chondrocytes in vitro in several species using different culture systems (Ohlsson et al. 1992, Quarto et al. 1992, Ballock & Reddi 1994, Bohme et al. 1995, Alini et al. 1996, Leboy et al. 1997, Ishikawa et al. 1998, Okubo & Reddi 2003). Mechanistic studies have revealed that thyroid hormone (TH) stimulates expression of p21, a cyclin D1 inhibitor, in rat epiphyseal chondrocytes in vitro (Ballock et al. 2000) and inhibits the expression of Sox-9, a transcription factor that maintains chondrocytes in an undifferentiated state, in mouse rib chondrocytes (Okubo & Reddi 2003). Furthermore, T3 stimulates fibroblast growth factor (FGF) receptor expression in ATDC5 cells and enhances FGF signalling in these cells and also in primary mouse rib chondrocytes (Barnard et al. 2005). However, precisely how these responses are involved in the T3 control of chondrocyte proliferation and differentiation has not been determined.
T3 actions are mediated by nuclear T3 receptors (TR) (Yen et al. 2006), which act as ligand-controlled transcription factors. TRα1 is encoded by the TRα gene, which also encodes TRα2, TRΔα1 and TRΔα2 isoforms that do not bind T3 and whose function is largely unknown. TRα1 and TRα2 are expressed in chondrocytes and osteoblasts. TRβ1, TRβ2 and TRβ3 are encoded by the TRβ gene. TRβ1 is expressed in chondrocytes and osteoblasts. Mice which lack TRα1 and TRα2 display abnormal growth plate architecture and impaired long bone mineralization during post-natal growth (Gauthier et al. 2001) associated with reduced FGFR1 and FGFR3 expression in osteoblasts and chondrocytes (Stevens et al. 2003, Barnard et al. 2005). Introduction of a point mutation into the TRα gene results in the expression of a dominant-negative TRα protein that inhibits the function of wild-type TRs and causes a similar, but more severe, skeletal phenotype than that observed in TRα-deficient mice (O’Shea et al. 2005). In contrast, mice with the homologous mutation in TRβ have elevated TH levels, advanced bone age and short stature with a reduced width of the growth plates affecting both the proliferative and the hypertrophic zones. These features have been interpreted to result from elevated TH levels acting via intact TRα1 in bone (O’Shea et al. 2003). Nevertheless, a specific role for TRβ in chondrocytes has also been suggested by studies of the TRβ selective agonist GC-1 (Freitas et al. 2005). Hypothyroid rats exhibited disorganized chondrocytes columns, reduced hypertrophic chondrocyte differentiation and impaired mineralization. These abnormalities were all rescued by the administration of T3, whereas the TRβ-selective agonist GC-1 also rescued the chondrocyte differentiation and bone mineralization defects but was unable to restore normal growth plate architecture.
In vivo analysis of mechanisms that account for T3 action in the growth plate is difficult because of the complex interactions between chondrocytes and other cell types and because of the interplay between T3 and other endocrine and paracrine systems, which may be affected by TR genetic modifications. In particular, local concentrations of thyroid hormone can be affected by these mutations: TRβ−/− mice have been shown not only to have elevated TH levels, but also to have altered expression of de-iodinases in some tissues, including liver (Amma et al. 2001) and cochlea (Campos-Barros et al. 2000), thus modifying the local metabolism of TH. The aim of this study was to determine the roles of Trα and TRβ in T3-stimulation of chondrocyte growth, differentiation and maturation, in an in vitro system, which allows a precise control of TH concentration. For this purpose, we isolated chondrocytes from the ribs of newborn mice. Murine rib chondrocytes cultured in a monolayer maintain a differentiated phenotype of round or polygonal cells that express type II collagen (Lefebvre et al. 1994). Cells differentiate in response to bone morphogenetic protein-2 (BMP-2) (Valcourt et al. 2003, Yagi et al. 2003), but may de-differentiate after several passages if cultured at low density (Lefebvre et al. 1994) or when maintained in the presence of TGFβ1 (Valcourt et al. 2002). To analyse the roles of TRα and TRβ, we isolated rib chondrocytes from mice, which lack either TRα (TRα0/0 mice) (Gauthier et al. 2001) or TRβ (TRβ−/− mice) (Gauthier et al. 1999). We measured the expression of TR isotypes at different times of the culture. We monitored the growth of the cultures by measuring the amount of DNA and evaluated the differentiation by measuring the activity of alkaline phosphatase (ALP) and the expression of collagen X.
Materials and Methods
Animals and cell culture
Mice were maintained with a ratio of 12 h light:12 h darkness cycle at 22–26 °C and experiments performed according to requirements of the regional committee of Ethics and the National Committee of Genetics. Homozygous wild type, TRα0/0 and TRβ−/− mice were crossed to obtain homozygous offspring. Neonatal ribs were dissected as described (Lefebvre et al. 1994). Cells were dissociated in 2 mg/ml protease (Sigma) for 20 min, washed in PBS, incubated in 2 mg/ml collagenase (Sigma) for 10 min, washed thrice in PBS, and incubated in 2 mg/ml collagenase for 5 h. The cell pellet was resuspended in DMEM/F12 medium (Gibco, Life Technology) supplemented with 10% heat-inactivated foetal bovine serum and 2 mM l-glutamine (Gibco), penicillin, streptomycin and gentamycin. Cells were seeded into 75 cm2 flasks at a density of 50 000/cm2. Confluent cells were trypsinized and seeded into 96- or 6-well plates at the same density and after 48 h the medium was replaced by a serum-free medium containing 1:500 ITS (Bio-Whittaker, Walkersville, Cambrex), 1 mg/ml BSA and combinations of 10−7 M T3 (Sigma) and 100 ng/ml BMP-2 (Peprotech, Tebu).
Cell growth and ALP activity
Cells were harvested at several time points after seeding by washing twice in PBS and freeze-thawing in 50 μl Hoechst 33258 solution (5 μg/ml in 10 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.1 mM EDTA). DNA concentration was determined in a Cytofluometer2 multi-well fluorometer (excitation 360 nm/emission 460 nm) compared with a standard curve prepared from calf thymus DNA. ALP activity was determined by freeze-thawing cells in 50 μl 0.5% Triton X-100 and incubating them for 1–30 min at 37 °C with 50 μl ALP substrate (20 mM p-nitrophenyl phosphate) in alkaline buffer solution (Sigma). Absorbance was determined at 405 nm in a fluorescence microplate reader (MRX, Dynex Technologies) and ALP activity was estimated compared with a standard curve prepared from a p-nitrophenol solution (Sigma), normalized to DNA concentration and expressed as nanomoles/minute per microgram DNA.
Analysis of gene expression by real-time quantitative RT-PCR (qRT-PCR)
RNA was extracted using an RNeasy Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed with Superscript II reverse transcriptase (Invitrogen) using 1.25 μg RNA template. One microlitre of tenfold diluted cDNA was used for qRT-PCR, using specific primers (Table 1) to amplify hypoxanthine phosphoribosyl transferase (HPRT), TRβ1 (Boelen et al. 2005), TRα1 and collagen X (Cormier et al. 2003). Reactions were performed in duplicate for each cDNA using the iQ SYBR Green supermix in an i-cycler IQ thermocycler (Bio-Rad). Quantitation was performed using the Optical System Software (Bio-Rad). Ct values were determined in all amplifications and the mean Ct value for each duplicate was calculated. For each RNA extraction, the mean ΔCt between the gene of interest and the HPRT gene was calculated. For each series of analyses, the reference sample was derived from untreated wild-type cells at the earliest time point of the culture, or from tissues of untreated wild-type mice. This reference value (ΔCt0) was subtracted from the ΔCt values of all other samples to give the ΔΔCt value; thus, the ΔΔCt value of the reference sample was zero. The relative amount of cDNA in each sample was calculated using the formula RQ= 2−ΔΔCt; thus the quantity of cDNA in the reference sample was 1.
Statistical analysis
Statistical analysis was performed using unpaired two-tailed Student’s t- tests and a P value <0.05 was considered significant. These analyses were performed by comparing the data obtained from multiple wells for each experimental condition, within one primary culture. All the results presented in this manuscript have been repeated at least in three independent primary cultures for each genotype.
Results
Expression of TRα and TRβ isoforms in rib chondrocytes
Expression of TR isoforms in rib chondrocytes was determined over 21 days in culture by qRT-PCR. In the absence of T3, TRβ1 expression (Fig. 1A) was similar in wild type and TRα0/0 cells and did not vary significantly with time, other than a 50% decrease at day 12 compared with day 6 in wild-type cells. In the presence of T3, TRβ1 expression increased significantly at days 12 and 21 in the wild-type cells and at days 6 and 21 in TRα0/0 cells, as compared with the control untreated cells. TRβ1 expression in the presence of T3 did not differ between wild type and TRα0/0 cells at any time of the culture. In wild-type chondrocytes in the absence of T3, TRα1 expression (Fig. 1B) remained constant until day 21. In T3-treated cells, TRα1 expression tended to be lower at day 6 and significantly decreased at days 12 and 21 compared with the control untreated cells. In untreated TRβ−/− chondrocytes, TRα1 expression was lower than in untreated wild-type cells at all times. T3 decreased TRα1 expression at day 6 but did not have any effect at days 12 and 21.
T3 inhibits growth of wild-type and TRα-deficient, but not TRβ-deficient rib chondrocytes in the presence of BMP-2
When wild-type rib chondrocytes were grown in 10% foetal bovine serum, cell numbers increased during the first 7 days of culture but remained stable until day 21 (Fig. 2A). In serum-free medium, cell numbers declined between day 7 and day 14, but remained stable until day 21. T3 treatment had no effect on cell growth in serum-free medium. In contrast, treatment with BMP-2 increased cell number by 40–100% at days 7, 14 and 21 (four separate cultures from four different litters, eight wells per treatment for each culture). T3 significantly decreased the number of cells measured in the presence of BMP-2 at days 7, 14 and 21 by 20–40% in four separate experiments. Thus, T3 inhibits BMP-2-induced growth of wild-type mouse rib chondrocytes. Chondrocytes from TRα0/0 mice cultured in 10% serum or in the absence of T3 displayed a similar pattern of growth as wild-type cells (Fig. 2B). Treatment with T3 alone had no significant effect on cell growth. BMP-2 stimulated cell growth by 40–120% (three separate experiments, eight wells per treatment for each culture), and T3 reduced the cell number obtained in the presence of BMP-2 by 20–30% (three separate experiments). In 10% serum and in the absence of T3, chondrocytes from TRβ−/− mice had a similar growth pattern to that observed in wild-type cells (Fig. 2C). T3 did not modify the growth of TRβ−/− chondrocytes. BMP-2 stimulated chondrocyte growth by 25–100%, but T3 did not significantly affect this stimulation in three separate experiments (eight wells per treatment for each culture).
T3 stimulates differentiation of wild type and TRα-deficient, but not TRβ-deficient rib chondrocytes
Chondrocyte differentiation was monitored by measuring ALP activity at days 14 and 21 (Fig. 3), and collagen X expression at days 6, 12 and 21 (Fig. 4). When wild-type chondrocytes were cultured in the absence of T3, ALP activity increased by fourfold between day 14 and day 21 (Fig. 3A). ALP activity was increased five- and threefold at days 14 and 21 respectively, following T3 treatment. A two- to fivefold stimulation of ALP activity by T3 was observed at day 21 in five separate experiments. Treatment with BMP-2 elicited a nine- and a fivefold increase in ALP activity at days 14 and 21 respectively, compared with untreated cells. A further 2- to 2.5-fold activation was observed when cells were treated with both BMP-2 and T3. In TRα0/0 chondrocytes, basal ALP activity was stable between days 14 and 21. T3 increased ALP activity by 30% at day 14 and by 2.5-fold at day 21 (Fig. 3B). In four separate experiments, ALP activity was stimulated by T3 by two- to fivefold in TRα0/0 chondrocytes. BMP-2 stimulated ALP activity at days 14 and 21 by two- and fourfold respectively. T3 further increased this activity by 40% at day 14 and twofold at day 21. Notably, ALP activity was lower in TRα0/0 chondrocytes than wild-type cells in all cultures. In untreated TRβ−/− chondrocytes, ALP activity displayed a similar pattern as in wild-type cells, with an increase between days 14 and 21. However, T3 failed to stimulate ALP activity in TRβ−/− chondrocytes (Fig. 3C). In contrast, BMP-2 stimulated ALP activity 22-fold at day 14 and eightfold at day 21, indicating that TRβ−/− chondrocytes retain their differentiation capacity. T3 had no additional effect on ALP activity in the presence of BMP-2.
Expression of collagen X, another specific marker of chondrocyte differentiation, was also determined (Fig. 4). Basal expression of collagen X in the absence of T3 was similar in wild type, TRα0/0 and TRβ−/− chondrocytes throughout the 21-day period of culture. Collagen X expression was increased by T3 at all times in wild type and TRα0/0 chondrocytes, but this stimulation was less in TRα0/0 cells at day 6 compared with wild type. In contrast, T3 failed to stimulate collagen X expression in TRβ−/− chondrocytes.
Discussion
Chondrocytes are sensitive to thyroid hormones in vivo and in vitro (Nilsson et al. 2005). In vitro, T3 inhibits cell proliferation but accelerates hypertrophic chondrocyte differentiation; however, the mechanisms responsible for these effects are poorly understood. In order to understand the mechanisms of T3 action in chondrocytes, it is important to define which TR mediates its biological effects, and thus we investigated chondrocyte proliferation and differentiation in primary cells obtained from TRα and TRβ knockout mice. Inhibition of chondrocyte proliferation by T3 has been demonstrated in several cell culture systems, including epiphyseal chondrocyte monolayer (Ishikawa et al. 1998) and three-dimensional cultures (Ballock et al. 2000, Robson et al. 2000). When mouse rib chondrocytes were cultured in serum-free medium, cell proliferation declined within the first 48 h (data not shown) before reaching a plateau. Addition of T3 had no effect on cell growth in serum-free medium, which is not surprising because T3 has not been reported to exert effects on quiescent chondrocyte cultures, although it is has been shown to inhibit expansion of proliferating chondrocyte cultures (Ballock et al. 2000, Robson et al. 2000). In contrast and consistent with studies showing that BMP-2 stimulates mouse growth plate chondrocyte proliferation in vivo (Minina et al. 2001), BMP-2 not only prevented cell loss but also stimulated growth of primary rib chondrocytes. Importantly, T3 prevented the BMP-2-induced increase in cell number, indicating that T3 inhibits BMP-2-stimulated growth of mouse rib chondro-cytes. We also demonstrated that growth of TRα- and TRβ-deficient chondrocytes in serum-free medium and in the presence of BMP-2 was similar to the growth of wild-type cells, suggesting that TRs do not play a role in T3-independent chondrocyte growth. As observed in wild-type cells, T3 had no effect on the growth of TRα0/0 or TRβ−/− cells in basal medium. Addition of T3 to BMP-2-treated cultures impaired growth of TRα0/0 chondrocytes, as observed with wild-type cells, but had no effect on BMP-2-treated TRβ−/− chondrocytes. These data suggest that TRβ1 mediates the inhibitory action of T3 on the BMP-2-stimulated proliferation of mouse rib chondrocytes.
T3 has been shown to stimulate terminal differentiation of chondrocytes in a variety of systems (Alini et al. 1996, Leboy et al. 1997, Ishikawa et al. 1998, Rosenthal & Henry 1999, Robson et al. 2000, Stevens et al. 2000, Okubo & Reddi 2003). In particular, T3 stimulates ALP activity and increases the expression of collagen X in these models. When mouse rib chondrocytes were cultured for 21 days in serum-free medium in the absence of T3, ALP activity and collagen X expression were similar to the levels at the onset of culture, indicating a lack of hypertrophic differentiation. In contrast, in the presence of T3, ALP activity increased rapidly and collagen X expression was stimulated at all time points indicating that T3 stimulates differentiation of primary mouse rib chondrocytes in monolayer cultures.
ALP activity in TRα0/0 chondrocytes was consistently lower than in wild-type cells in all culture conditions; however, the amplitude of responses to T3, BMP-2 and both agents was similar in wild type and TRα-deficient cells. Collagen X expression was also stimulated by T3 to a similar degree in both TRα0/0 and wild-type chondrocytes. In contrast, T3-stimulation of ALP and collagen X was abolished in TRβ−/− chondrocytes. Explanations for this discrepancy include an absence or low proportion of chondrocyte precursors within the ribs of TRβ−/− mice, or an impaired intrinsic inability of TRβ−/− cells to differentiate into mature chondrocytes. However, BMP-2 stimulated the differentiation of TRβ−/− cells to the same extent as in wild type and TRα-deficient cells, indicating that TRβ-deficient cells have the intrinsic capacity to differentiate into mature chondrocytes. Furthermore, whereas T3 could enhance the ALP response of wild type and TRα0/0 chondrocytes to BMP-2, it had no effect in BMP-2-treated TRβ−/− cells. All these data suggest that the T3-dependent differentiation pathway is selectively impaired in TRβ-deficient cells.
The predominant role of TRβ1 in the mediation of T3 action in mouse rib chondrocytes is consistent with the pattern of expression of TR isoforms during differentiation of wild type and TR-deficient cells. In wild-type cells, T3 increased expression of TRβ1 and decreased expression of TRα1. This differential regulation of TR-isotype expression by T3 suggests TRβ1 as the main mediator of T3 action in these cells. The observations that, following treatment with T3, increased TRβ1 expression is preserved in TRα-deficient cells, whereas the decrease in TRα1 expression is attenuated in TRβ-deficient cells also support a key role for TRβ1. Our observations do not exclude the possibility that in wild-type chondrocytes, both TRs can mediate T3 action and that TRβ1 can substitute for TRα1 in TRα0/0 cells, whereas TRα1 cannot substitute for TRβ1 in TRβ−/−cells. Indeed, in the absence of T3, the level of expression of TRα1 was lower in TRβ−/−than in wild-type cells. One explanation for this low amount of TRα1 in TRβ-deficient cells is that TRβ1 as an aporeceptor positively regulates the expression of TRα1. The absence of TRβ would therefore decrease the expression of TRα1 and diminish the response to T3. Consequently, it is possible that TRα1 is active in wild-type cells but, because less abundant, unable to mediate T3 action in TRβ−/−cells. In TRα-deficient cells, the response to T3 may be mediated by TRβ1, however, no compensatory over-expression of this isotype was observed in TRα0/0 cells. Although it is not possible to exclude that TRα1 is involved in T3 action in wild-type chondrocytes, upregulation of TRβ1 and the downregulation of TRα1 by T3 in these cells are more consistent with a major role of TRβ1. Such a key role of TRβ1 in the modulation of chondrocyte differentiation by T3 is supported by in vivo studies using the TRβ-selective agonist GC-1 (Freitas et al. 2005). In these studies, young hypothyroid rats displayed altered organization of chondrocyte columns within the growth plate with impaired hypertrophic differentiation, as assessed by the decreased size of the hypertrophic zone and reduced expression of collagen X. GC-1, like T3, restored normal morphometric parameters of the growth plate and normal expression of collagen X, strongly suggesting that TRβ1 mediates the effect of T3 on chondrocyte differentiation. These findings are consistent with an important role for TRβ1 in the control of chondrocyte differentiation and, thus, the role of TRα1 in chondrocyte biology remains to be elucidated. In hypothyroid rats, T3, but not the TRβ-selective agonist GC-1, restored normal organization of the growth plate, suggesting that TRα1 mediates the effects of T3 on the spatial organization of chondrocytes (Freitas et al. 2005). However, our findings using rib chondrocytes in monolayer cultures did not demonstrate a major role for TRα in the regulation of cell proliferation and differentiation. It is possible that, in vivo or in a three-dimensional environment, TRα1 is the key mediator of T3 action, whereas in monolayer cultures, TRβ1 becomes predominant. Nonetheless, in TRα0/0-chondrocytes, ALP activity was blunted compared with wild-type cells, raising the possibility that TRα may be a permissive factor that facilitates chondrocyte differentiation. The importance of TRα is further suggested by the growth plate phenotype of mice lacking the TRα gene (Fraichard et al. 1997, Gauthier et al. 2001) or expressing a dominant negative TRα1 isoform (O’Shea et al. 2005). These mice have disorganized chondrocyte columns and reduced width of the hypertrophic zone. However, they also have mild hypothyroxinemia (Macchia et al. 2001, Weiss et al. 2002), raising the possibility that the observed phenotype results from reduced local TH availability.
Altogether, our data are consistent with the observations from TR-knockout and knock-in mice, and suggest that TRβ1 mediates important T3 effects on hypertrophic chondrocyte terminal differentiation, whereas TRα1 is likely to be involved in the control of cellular and molecular interactions that are important for the three-dimensional organization of growth plate cartilage and the initiation of chondrocyte differentiation. These findings may have important applications for the potential use of T3 analogues as therapeutic agents for the treatment of bone and articular cartilage lesions.
DNA sequences of primers used in real-time PCR
| Upstream primer | Downstream primer | |
|---|---|---|
| Gene products | ||
| HPRT | 5′ GCAGTACAGCCCCAAAATGG 3′ | 5′ AACAAAGTCTGGCCTGTATCCAA 3′ |
| TRα1 | 5′ CATCTTTGAACTGGGCAAGT 3′ | 5′ CTGAGGCTTTAGACTTCCTGATC 3′ |
| TRβ1 | 5′ CACCTGGATCCTGACGATGT 3′ | 5′ ACAGGTGATGCAGCGATAGT 3′ |
| CollagenX | 5′ CAAACGGCCTCTACTCCTCTGA 3′ | 5′ CGATGGAATTGGGTGGAAAG 3′ |
Expression of TR isoforms in rib chondrocytes. (A) TRβ1 mRNA concentrations normalized to HPRT, in primary wild-type and TRα0/0 mouse rib chondrocytes cultured for 6, 12 and 21 days in the absence or presence of 10−7 M T3. Results are shown as mean±s.e m. n≥3 for all conditions except for T3-treated TRα 0/0 cells at day 21 (n=2). (B) Expression of TRα1-mRNA normalized to HPRT in TRβ−/− chondrocytes cultured for 6, 12 and 21 days in the absence or presence of 10−7 M T3. Results are shown as mean±s.e.m. n≥2 for all conditions. Data were analysed by two-tailed Student’s t-test. NS, not significant; *P<0.001; †P<0.05; ‡P<0.01.
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06838
Effect of T3 and BMP-2 on growth of primary rib chondrocytes. Results are expressed as micrograms DNA per well. Cells were seeded on day 0 (50 000 cells/cm2) and maintained in the absence or presence of T3 (100 nM)±BMP-2 (100 ng/ml) for 21 days. All experiments were performed four times (four independent cultures). For each culture, eight wells per condition were seeded. The graphs show a single typical experiment for each genotype. The bars indicate mean±s.e.m. Results were analysed by two-tailed Student’s t-test. NS, not significant; *P<0.001; †P<0.01. (A) Wild-type chondrocytes. (B) TRα0/0 chondrocytes. (C) TRβ−/− chondrocytes. Hatched bars, 10% FCS; white bars, control, serum-free medium; black bars, serum-free medium with T3 (10−7 M); vertically hatched bars, serum-free medium with BMP-2 (100 ng/ml), horizontally hatched bars, serum-free medium with T3 (10−7 M) and BMP-2 (100 ng/ml).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06838
Effect of T3 on alkaline phosphatase activity in primary rib chondrocytes. Chondrocytes were cultured for 21 days in the absence or presence of 10−7 M T3±100 ng/ml BMP-2. Alkaline phosphatase activity is expressed as nanomoles of substrate hydrolysed per minute per micrograms of DNA±s.e.m. (n=4 experiments, eight wells per condition). Results were analysed by two-tailed Student’s t-test. NS, not significant. *P<0.001; †P<0.01. (A) Wild-type chondrocytes. (B) TRα0/0 chondrocytes. (C) TRβ−/− chondrocytes. Hatched bars, 10% FCS; white bars, control, serum-free medium; −7 M); vertically hatched black bars, serum-free medium with T3 (10 bars, serum-free medium with BMP-2 (100 ng/ml), horizontally −7 M) and BMP-2 hatched bars, serum-free medium with T3 (10 (100 ng/ml).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06838
Effect of T3 on collagen X mRNA expression in rib chondrocytes. Wild type, TRα0/0 and TRβ−/− chondrocytes were grown for 6, 12 and 21 days in the absence (white bars) or presence (black bars) of 10−7 M T3-ColX mRNA was normalized to the concentration of HPRT mRNA. Data are expressed as mean±s.e.m. (n=2 each condition). Results were analysed by two-tailed Student’s t-test. NS, not significant. *P<0.05; †P<0.01.
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06838
This work was supported by Centre National de la Recherche Scientifique (CNRS), by Institut National de la Santé et de la Recherche Médicale (INSERM), and by grant ACI no. 0220601 of the French Ministry of Research. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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