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Endocrinology Vol. 145, No. 3 1050-1054
Copyright © 2004 by The Endocrine Society

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BIG-3, a Novel WD-40 Repeat Protein, Is Expressed in the Developing Growth Plate and Accelerates Chondrocyte Differentiation in Vitro

Francesca Gori and Marie B. Demay

Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Francesca Gori Ph.D., Endocrine Unit, Wellman 501, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail:gori{at}helix.mgh.harvard.edu.

Abstract

Among the local signaling pathways that regulate the sequential steps of chondrocyte differentiation is the bone morphogenetic protein (BMP) signaling pathway. We have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb) that is expressed in a BMP-regulated fashion in the prechondroblastic cell line MLB13MYC clone 17. BIG-3 is also expressed in proliferating and hypertrophic chondrocytes in the developing growth plate in vivo. We undertook studies to address whether BIG-3 played a functional role in chondrocyte differentiation, using mouse clonal chondrogenic ATDC5 cells. BIG-3 protein levels increased during ITS (insulin, transferrin, sodium selenite)-induced ATDC5 differentiation and in response to BMP-2 treatment. To determine whether stable expression of BIG-3 could alter the program of chondrocytic differentiation, ATDC5 cells were stably transfected with the full-length coding region of BIG-3 (ATDC5-BIG-3) or with the empty vector (ATDC5-EV). Accelerated matrix proteoglycan synthesis was observed in the pooled ATDC5-BIG-3 clones. Alkaline phosphatase and osteopontin mRNA levels were also increased in ATDC5-BIG-3 clones compared with ATDC5-EV clones. Stable expression of BIG-3 also accelerated mineralized matrix formation in both the presence and absence of ITS. These findings, which demonstrate that BIG-3 accelerates chondrocyte differentiation in vitro, combined with the observation that BIG-3 is expressed in the growth plate during embryonic development, suggest that this novel protein is likely to play an in vivo regulatory role in the developing growth plate.

THE SKELETON PROVIDES mechanical and protective properties and supports metabolic functions of all vertebrates. Skeletal development in vivo occurs via two major processes, intramembranous and endochondral ossification. Both intramembranous and endochondral ossification begin with condensation of mesenchymal cells that form a template for the skeleton and end with formation of calcified elements. However, although intramembranous ossification occurs by direct differentiation of mesenchymal cells into osteoblasts, endochondral bone formation occurs through a highly coordinated sequence of events beginning with chondrocyte proliferation, deposition of cartilage matrix, chondrocyte hypertrophy, mineralization of the cartilage matrix, followed by matrix degradation, apoptosis of the hypertrophic chondrocytes, vascular invasion, and formation of an ossification center containing type I collagen-expressing osteoblasts (1, 2).

Several factors are known to cooperate in regulating the onset and the sequential steps of chondrogenesis, as well as formation of the bone collar (1, 2, 3). Among the local signaling pathways that are known to play a role in these processes are the bone morphogenetic protein (BMP) signaling pathways (4, 5, 6, 7, 8). BMPs were originally identified as molecules derived from bone that were capable of inducing new bone formation ectopically when implanted sc in animal models (9). In vertebrates, BMPs are expressed in a tightly regulated spatial and temporal pattern during embryogenesis and have been shown to play a critical role in the development of many organs and tissues (6). They are also expressed at sites of new bone formation early in development, suggesting that they are key signaling molecules for limb formation and patterning. BMPs signals are transduced by two types of serine/threonine kinase receptors, type I and type II receptors that dimerize (10, 11). Several studies have demonstrated that BMP signaling plays a role in the regulation of chondrocyte proliferation, modulating Indian hedgehog expression and delaying terminal maturation of hypertrophic cells (12, 13).

Using differential display PCR, we identified a novel gene 3 kb long, named BIG-3 (BMP-2-induced gene 3 kb), which was induced in the prechondroblastic cell line, MLB13MYC clone 17, in response to BMP-2 treatment (14). BIG-3 encodes a cytoplasmic protein with a molecular mass of approximately 34 kDa. Searches based on the primary amino acid sequence of BIG-3 demonstrated that this protein is a new member of a family of structurally conserved proteins, the WD-40 repeat proteins, found in all eukaryotes (15, 16). This family of highly conserved proteins has been shown to play a role in numerous cellular functions including signal transduction, mRNA processing, gene regulation, vesicular trafficking and regulation of the cell cycle (16). These proteins contain four to 16 conserved Trp-Asp motifs (the so-called WD-40 repeats). BIG-3 is expressed in several mouse tissues including skin, muscle, liver and heart, and in marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes.

Because BIG-3 was regulated by BMP-2 and was expressed in chondrocytes in vitro, we undertook studies to investigate the temporal expression of BIG-3 in vivo during endochondral bone formation, and to determine whether BIG-3 has a functional role in the program of chondrocyte differentiation. The clonal chondrogenic cell line, ATDC5, was chosen for these studies because they have been shown by several investigators to be a useful in vitro model for examining the multistep differentiation of chondrocytes (17, 18, 19). Undifferentiated ATDC5 cells proliferate rapidly until they reach confluence, at which point they undergo growth arrest. When treated with insulin, transferrin, and sodium selenite (ITS), confluent ATDC5 cells reenter a proliferative phase and form cartilaginous matrix nodules. As differentiation progresses, these cells undergo a late differentiation phase, becoming hypertrophic, calcifying chondrocytes that synthesize osteopontin, a marker of terminal chondrocyte differentiation.

Materials and Methods

Immunohistochemistry
Using the Jameson-Wolf antigenicity index, an epitope in the seventh WD repeat of BIG-3 (LENDKTIKLWKSDC) was conjugated to keyhole limpet hemocyanin and used to immunize rabbits (Covance, Denver, PA). After affinity purification of serum on a peptide column, purified {alpha}-BIG-3 was shown to recognize a single, specific band of 34 kDa on Western analyses (14). Immunohistochemistry using this antibody or a nonspecific rabbit IgG (Sigma, St. Louis, MO) and the TSA Biotin System Kit (PerkinElmer, Boston, MA), was performed to examine BIG-3 expression during endochondral ossification. Tibiae from 13.5, 15.5, and 18.5 d post coitus (dpc) embryos and tibiae from newborn and 24-d-old mice were fixed in 10% buffered formalin, processed, paraffin embedded, and sectioned. Immunoreactive proteins were visualized using Streptavidin Fluorescein and Streptavidin Texas Red (PerkinElmer).

Cell culture
The mouse chondroblastic cell line, ATDC5, was cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium containing 5% FBS and 1% penicillin/streptomycin (Life Technologies, Inc./BRL, Grand Island, NY) for 7–45 d. For induction of differentiation, media were supplemented with 10 µg/ml transferrin, 3 x 10-8 M sodium selenite, and 10 µg/ml bovine insulin (ITS) (Roche Diagnostic, Mannheim, Germany). In experiments assessing mineralization, cells were incubated in 3% CO2, and medium was supplemented with ascorbic acid (50 µg/ml) (Sigma, St. Louis, MO).

Stable transfection
ATDC5 cells were plated at a density of 1.5 x 105 cells/cm2. After 24 h, they were stably transfected with pcDNA3.1 containing the full-length coding region of BIG-3 (ATDC5-BIG-3) downstream from the CMV promoter (Invitrogen Carlsbad, CA) or with the empty vector (ATDC5-EV) using 2 µg of DNA and 60 µl of Effectene transfection reagent (QIAGEN, Valencia, CA) in 10 ml of medium. After 24 h, and every 48 h thereafter for 2 wk, media were replaced with fresh media containing 300 µg/ml of G418. This dose and duration of treatment resulted in the death of all nontransfected cells within 10 d. Pools of 50 clones of ATDC5-BIG-3 and ATDC5-EV were isolated for further studies. Pools of clones between passages 2 and 5 were used for the experiments reported.

Western analysis
ATDC cells were plated at a density of 5 x 103 cells/cm2 and cultured for periods ranging from 3–7 d. Confluent ATDC5 cells were treated with ITS for 24 h before the addition of BMP-2 (1 µg/ml) for 48 h. Ten micrograms of protein were subjected to SDS-PAGE under reducing conditions. Immunodetection was performed with {alpha}-BIG-3 (14). Immunoreactive proteins were visualized using a chemiluminescence detection kit (NEN Life Science Products, Boston, MA) according to the manufacturer’s instructions.

Evaluation of matrix proteoglycan synthesis
ATDC5-EV and ATDC5-BIG-3 pooled clones were plated at a density of 5 x 103 cells/cm2. At confluence, cells were cultured in the presence or absence of ITS for 14–45 d. Cells were fixed in 10% neutral buffered formalin for 10 min at room temperature, washed twice with PBS, and stained with 0.1% alcian blue 8GX (Sigma, St. Louis, MO) in 0.1 N HCl overnight at room temperature. Alcian blue was eluted in 6 M guanidine HCl for 6 h at room temperature and quantitated spectrophotometrically at 630 nm.

Northern analysis
Ten micrograms of total RNA were resolved on a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Biotrans ICN, Aurora, OH) by capillary blotting. Probes were radiolabeled with [{alpha}-32P] deoxy-ATP (DuPont NEN Life Science Products, Boston, MA) to a specific activity of >= 108 cpm/ng DNA (Megaprime DNA labeling systems, Amersham, Piscataway, NJ). Control hybridization with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe verified equal RNA loading.

Evaluation of mineralized matrix formation
ATDC5-EV and ATDC5-BIG-3 pooled clones were plated at a density of 5 x 103 cells/cm2 and cultured for 14–45 d. The formation of mineralized matrix nodules was assessed by alizarin red-S staining (14). In parallel experiments, calcium accumulation in the matrix was quantitated by solubilizing the deposited calcium with 0.6 N HCl overnight at room temperature. The samples and a standard curve of calcium carbonate were reacted with methylthymol blue and measured spectrophotometrically at 620 nm (20).

Statistical analyses
Student’s paired t test was used to evaluate differences between ATDC5-BIG-3 pools and ATDC5-EV pools at each time point. A P value < 0.05 was considered statistically significant.

Results

The observation that BIG-3 is expressed in a BMP-2 regulated fashion in growth plate chondrocytes in vitro (14) led us to examine the expression of BIG-3 in the developing growth plate. As shown in Fig. 1AGo, BIG-3 expression was first detected in mouse tibiae at embryonic d 13.5 pc. At this stage, BIG-3 was strongly expressed throughout the mesenchymal condensation. At 15.5, 18.5 dpc and in newborns, BIG-3 was expressed in the proliferating (P) and hypertrophic chondrocytes (H) (Fig. 1AGo and data not shown). Postnatally, BIG-3 expression was restricted to the flat proliferating (P) and hypertrophic (H) layers of the growth plate (Fig. 1AGo). No signal was detected when nonspecific rabbit IgG was used, confirming specificity of the signal (Fig. 1BGo). The observations that BIG-3 is expressed in the developing growth plate and is induced by BMP-2 in vitro suggest that BIG-3 may play a role in chondrocyte differentiation. To address this hypothesis, the expression of BIG-3 during chondrocyte differentiation was evaluated in the murine clonal chondrogenic cell line, ATDC5. We first examined whether ATDC5 cells express BIG-3 and whether endogenous levels of BIG-3 increased during their differentiation. As shown in Fig. 2AGo, BIG-3 protein levels increase during the early stage of chondrocyte differentiation similar to what is seen as MC3T3-E1 cells acquire the osteoblastic phenotype (14). Exogenous BMP-2 treatment increases BIG-3 protein levels by 2.8 ± 1.0-fold in ATDC5 cells compared with untreated cells (Fig. 2BGo).



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  		FIG. 1. Expression of BIG-3 in the developing growth plate. Immunohistochemistry on tibiae from 13.5 and 15.5 dpc embryos and 24-d-old mice was performed using an {alpha}-BIG-3. A, {alpha}-BIG-3 antibody. B, Nonspecific rabbit IgG. C, Hematoxylin and eosin staining. Horizontal lines correspond to folds in the tissues sections.

 


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  		FIG. 2. Western analyses of BIG-3 expression in ATDC5 cells. A, ATDC5 cells treated with ITS for 3 and 7 d. B, ATDC5 cells were cultured with ITS and treated or not with BMP-2 for 48 h. The experiments were carried out twice, and a representative blot is shown. Control with an {alpha}-actin antibody verified the amount of protein loaded.

 
To assess whether stable expression of BIG-3 could alter the program of chondrocytic differentiation, ATDC5 cells were stably transfected with pcDNA3.1 containing the full-length coding region of BIG-3 (ATDC5-BIG-3) or with the empty vector (ATDC5-EV). Pools of 50 clones of ATDC5-BIG-3 and ATDC5-EV between passages two and five were used for these experiments.

The effect of BIG-3 on matrix proteoglycan synthesis was evaluated by examining the time course of onset and intensity of alcian blue-positive nodules in ATDC5-EV and ATDC5-BIG-3 pooled clones. Alcian blue-positive cartilaginous nodules were first seen at 14 d in ITS-treated ATDC5-BIG-3 clones (data not shown) and increased in size and number until 45 d (Fig. 3AGo). Small alcian blue-positive cartilage nodules were also seen at 35 d in ITS-treated ATDC5-EV and untreated ATDC5-BIG-3 clones; however, ATDC5-EV clones cultured without ITS failed to show specific alcian blue staining after 45 d in culture. Quantification of cartilage proteoglycan synthesis by elution of alcian blue from stained cartilage deposits demonstrated a 7.0 ± 0.7- and 4.7 ± 0.2-fold increase at 35 and 45 d, respectively, in ITS-treated ATDC5-BIG-3 clones compared with ITS-treated ATDC5-EV clones (Fig. 3BGo). These findings indicate that stable expression of BIG-3 synergizes with ITS in promoting the early differentiation of ATDC5 cells, reflected by an increase in cartilage proteoglycan synthesis.



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  		FIG. 3. Proteoglycan synthesis. Pooled clones of ATDC5-EV and ATDC5-BIG-3 cells were treated or not with ITS for up to 45 d. A, Alcian blue staining. B, Alcian blue quantification. The results shown are representative of three independent experiments ± SEM carried out in duplicate. *, P < 0.05; **, P < 0.005.

 
The effect of BIG-3 on the expression of mRNAs encoding markers of chondrocyte differentiation was examined. Alkaline phosphatase (AP) expression increases during chondrocyte differentiation. Analogous to the effect of stable expression of BIG-3 on AP activity in MC3T3E1 cells, by 21 d AP mRNA expression was 3.8 ± 0.0-fold higher in ITS-treated ATDC5-BIG-3 clones compared with ITS-treated ATDC5-EV clones (Fig. 4AGo). At this time point, untreated ATDC5-BIG-3 clones had 3.9 ± 1.0 and 3.4 ± 0.4-fold increase in AP mRNA expression compared with untreated and ITS-treated ATDC5-EV clones, respectively (Fig. 4AGo). At 21 d, untreated ATDC5-BIG-3 clones expressed 2.0 ± 0.2-fold higher osteopontin (OP) mRNA levels than untreated ATDC5-EV clones, whereas no significant difference was observed between ITS-treated ATDC5-EV and ITS-treated 5-BIG-3 clones (data not shown). By 30 d, OP mRNA expression was 2.1 ± 0.4-fold higher in ITS-treated ATDC5-BIG-3 clones compared with ITS-treated ATDC5-EV clones (Fig. 4BGo).



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  		FIG. 4. Northern analyses. Total RNA, isolated from pooled clones of ATDC5-EV and ATDC5-BIG-3 cells treated or not with ITS, was hybridized with cDNA probes for AP (panel A) and OP (panel B). Control hybridization with glyceraldehyde-3-phosphate dehydrogenase verified the amount of RNA loaded. The experiments were carried out twice, and representative blots are shown.

 
The effect of stable expression of BIG-3 on mineralized matrix formation, one of the final steps in chondrocyte differentiation, was examined. Alizarin red-positive nodules were first seen at 14 d in both untreated and ITS-treated ATDC5-BIG-3 clones, but not in the ATDC5-EV clones (data not shown). At 30 d, alizarin red-positive nodules in ITS-treated and untreated ATDC5-BIG-3 clones were larger and more numerous than those in ITS-treated and untreated ATDC5-EV clones (Fig. 5AGo). This difference persisted until 45 d. When mineralization was quantitated, at 30 d, there was a 2.0 ± 0.2-fold increase in alizarin red staining (data not shown) as well in the calcium content of the ITS-treated ATDC5-BIG-3 clones compared with ITS-treated ATDC5-EV clones (Fig. 5BGo). Calcium content in ITS-treated ATDC5-EV clones continued to increase with more prolonged culture; however, at 45 d ITS-treated ATDC5-BIG-3 clones still had a 1.9 ± 0.1-fold increase in calcium content relative to ITS-treated ATDC5-EV clones (Fig. 5BGo). At this time point, untreated ATDC5-BIG-3 clones also had a 3.1 ± 0.2 and 1.3 ± 0.1-fold increase in calcium content relative to untreated and ITS-treated ATDC5-EV clones, respectively (P < 0.05) (Fig. 5BGo).



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  		FIG. 5. Mineral deposition. Pooled clones of ATDC5-EV and ATDC5-BIG-3 cells were cultured for periods ranging from 14–45 d in the presence or absence of ITS. A, Alizarin red staining. B, Calcium accumulation in the matrix. The results shown are representative of three independent experiments ± SEM carried out in triplicate. *, P < 0.005.

 
Discussion

These studies demonstrate that BIG-3 accelerates chondrocyte differentiation and maturation in vitro. In addition, the temporal and spatial expression of BIG-3 in proliferative and hypertrophic chondrocytes in the embryonic growth plate is consistent with a potential role of BIG-3 as a regulator of chondrocyte differentiation and maturation. These observations, combined with the findings that BIG-3 is expressed in osteoblasts and accelerates their differentiation, suggest that this novel protein is likely to be involved in endochondral bone formation in vivo. Although BIG-3 accelerates chondrocytes in vitro, further investigation will be required to determine whether BIG-3 expression is essential for this process.

The molecular mechanism(s) by which this novel protein regulates the program of chondrocyte differentiation is not yet certain. The observation that BIG-3 is expressed in several tissues suggests that BIG-3 is not a specific regulator of chondrocyte differentiation. However, BIG-3 may regulate the expression and the activity of key chondrocyte genes, resulting in an acceleration of chondrocyte differentiation and maturation. Analogous to other members of the WD-40 family of proteins, it is likely that the ß-propeller structure of BIG-3 forms a scaffold, recruiting novel or known factors involved in chondrocyte differentiation. The resultant protein-protein interactions and potential posttranslational modifications of these key chondrogenic factors may be the mechanism by which BIG-3 regulates chondrogenic differentiation. Characterization of factors recruited to the ß- propeller of BIG-3 will provide insight into the molecular mechanisms by which BIG-3 accelerates chondrocyte differentiation and perhaps synergizes with or converges upon other pathways involved in this process.

In the absence of exogenous differentiating agents such as ITS, expression of BIG-3 is capable of promoting differentiation of ATDC5 cells as reflected by acquisition of markers of late chondrocyte differentiation. However, ITS treatment increases the effect of BIG-3, suggesting that BIG-3 synergizes with other differentiating factors in inducing chondrogenesis. Insulin induces cellular condensation and formation of cartilage nodules in ATDC5 cells acting through both insulin and IGF signaling pathways. It has been shown that insulin/IGF stimulation of chondrogenesis and myoblast differentiation is induced by activation of the phosphatidylinositol-3-kinase pathway that in turn activates the serine/threonine kinase Akt (21). The downstream actions of BIG-3 may, therefore, converge on the IGF signaling pathways, or alternatively BIG-3 may mediate its effects on cellular differentiation by an independent, parallel pathway. Characterization of the interactions of BIG-3 with other signaling molecules will clarify the mechanisms by which seemingly unrelated signaling pathways converge to promote chondrocyte differentiation. Although in these studies we demonstrated that BIG-3 accelerates chondrocyte differentiation, we did not examine whether the expression of BIG-3 is required for chondrocyte differentiation.

These data, which demonstrate that BIG-3 accelerates chondrocyte differentiation in vitro, combined with the observation that BIG-3 is developmentally expressed in the embryonic growth plate, suggest that this novel protein plays a developmental role in endochondral bone in vivo.

Footnotes

This work was supported by Grants 1 F32 DE-05754 (to F.G.) and DK36597 (to M.D.) from the NIH.

Abbreviations: AP, Alkaline phosphatase; BIG-3, BMP-2-induced gene 3 kb; BMP, bone morphogenetic protein; dpc, days post coitus; EV, empty vector; ITS, insulin, transferrin, sodium selenite; OP, osteopontin.

Received October 1, 2003.

Accepted for publication November 25, 2003.

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