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1 First Department of Pathology and 2 Department of Orthopedic Surgery, Wakayama Medical University, Wakayama, Japan
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Abstract |
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Introduction |
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Trps1 is mainly expressed in the joints and in the limb growth plate cartilages during late embryogenesis (Kunath et al. 2002). Mice with Trps1 deficiency die of respiratory failure soon after birth and show changes such as skeletal deformities that resemble those seen in human TRPS patients (Malik et al. 2002). These findings suggest that Trps1 acts as a transcriptional regulator during the process of cartilage formation. However, the molecules that lie upstream and downstream of Trps1 are largely unknown.
During development of the cartilage and joints, numerous cytokines and growth factors may have a role. The transforming growth factor-β (TGF-β) superfamily of proteins, especially TGF-β itself and the bone morphogenetic proteins (BMPs), play a central role in the process of cartilage formation. The BMP subfamily consists of at least 20 proteins that can be placed into distinct subgroups based on their amino acid sequences. During chondrogenesis, BMP2 and BMP4 (a subgroup of BMPs) stimulate the proliferation of chondrocytes and counteract the effects of FGFs (Yoon et al. 2006), while BMP7 promotes the differentiation of chondrocytes and the accumulation of matrix molecules (Asahina et al. 1996). Growth and differentiation factors (GDFs) 5, 6 and 7 form the other subgroup of BMPs, and play an important role in joint formation (Ducy & Karsenty 2000). Although the functions of these proteins are broadly known, the precise molecular mechanisms involved are not fully understood.
A human genetic skeletal disease caused by mutations of GDF5 (Holder-Espinasse et al. 2004; Dawson et al. 2006; Wang et al. 2006) has similar phenotypic features (like brachydactyly) to those of TRPS. In addition, brachypodism (bp) caused by a Gdf5-mutation leads to limb shortening and abnormalities of joint formation in mice (Storm et al. 1994; Storm & Kingsley 1996), in which Trps1 is strongly expressed during a development. These findings raise the possibility that GDF5 and TRPS1 could be linked in the processes of joint and cartilage formation.
ATDC5 cells are a well-characterized chondrogenic cell line derived from a mouse teratocarcinoma that mimics the multistep process of chondrocyte differentiation in culture. Several studies have demonstrated that the TGF-β superfamily of proteins induces chondrogenesis in cultured ATDC5 cells (Shukunami et al. 1998; Nakamura et al. 1999). In addition, GDF5 has been shown to promote chondrocyte differentiation and activation of p38 MAPK in these cells (Nakamura et al. 1999).
In the present study, we examined the functional interaction between Gdf5 and Trps1 using ATDC5 cells. Our results indicated that Gdf5 and Trps1 mutually regulate their expression and that Trps1 acts downstream of Gdf5 during the differentiation and apoptosis of cultured ATDC5 cells.
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Results |
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Immunohistochemical analysis showed that Trps1 was strongly expressed by chondrocytes in the phalangeal joints of E15.5 embryos (Fig. 1A,B). We found that the immunohistochemical localization of Trps1 was similar to the pattern of Gdf5 expression on E15.5 (Fig. 1C,D), as reported elsewhere (Storm & Kingsley 1996). Thus, we hypothesized that Trps1 might interact with Gdf5. To test this hypothesis, we examined possible interactions between Trps1 expression and Gdf5 signaling using the ATDC5 chondrogenic cell line.
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Since Gdf5 utilizes Alk6 as its receptor, we examined whether or not up-regulation of Trps1 by Gdf5 occurs via Alk6. When we transfected ATDC5 cells with an expression vector bearing a dominant negative form of Alk6 (dn-Alk6), the expression of Trps1 was strongly suppressed at the protein levels although the suppression of the mRNA levels was not so prominent (Fig. 3A). p38 MAPK is reported to be activated by the TGF-β superfamily of proteins, including BMPs (Moriguchi et al. 1996). To test whether p38 MAPK mediates the interaction of Gdf5 with Trps1, we examined phosphorylation of p38 MAPK by dn-Alk6 as well as expression of Trps1 after inhibition of p38 MAPK. In cells transfected with dn-Alk6, phosphorylation of p38 MAPK was suppressed, while no significant change was seen in mock-transfected control cells (Fig. 3B). In addition, when we treated ATDC5 cells with a p38 MAPK inhibitor (SB203580), Trps1 protein levels were dramatically suppressed (Fig. 3C).
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To investigate the role of Gdf5–Trps1 signaling in chondrogenesis, we examined the expression of chondrogenic markers (type II and type X collagen) and Trps1, as well as Alcian Blue staining. Cartilage-like nodules stained by Alcian Blue were larger and grew faster in cultures of Trps1-overexpressing and Gdf5-treated cells compared with cultures of control cells (Fig. 4A). Conversely, in cultures of cells transfected with dn-Alk6, the cartilage-like nodules were fewer and grew more slowly compared with cultures of control cells (Fig. 4A). In addition, the expression of chondrogenic marker proteins (type II and type X collagen) was up-regulated earlier in Trps1-overexpressing cells compared with control cells, whereas their expression was delayed in dn-Alk6-transfected cells compared with control cells (Fig. 4B). Treatment with Gdf5 also up-regulated the expression of chondrogenic marker proteins in control ATDC5 cells, but not in dn-Alk6-transfected cells (Fig. 4C). Trps1 mRNA levels were not significantly enhanced by Gdf5 compared with Trps1 protein levels (Figs 2B and 4C). These results suggested that chondrogenic differentiation of ATDC5 cells was accelerated by Gdf5–Trps1 signaling.
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In the growth plates, hypertrophic chondrocytes die of apoptosis during the final phase of cartilage development (Gibson et al. 1995). Based on the fact that ATDC5 cells undergo apoptotic death after chondrogenic differentiation (Mushtaq et al. 2002), we examined the expression of proteins associated with apoptosis in Trps1-overexpressing and dn-Alk6-transfected ATDC5 cells. Among these proteins, expression of Bcl-2 mRNA was significantly decreased by the over-expression of Trps1, whereas transfection with dn-Alk6 resulted in elevated Bcl-2 expression (Fig. 5A).
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Discussion |
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The regulation of Gdf5 expression by Trps1 is poorly understood. At least four GATA motifs have been reported to exist in the 3.5 kb 5'-flanking region of the human GDF5 gene (Sugiura et al. 1999). In the mouse Gdf5 gene, multiple GATA motifs also exist in 5'-flanking region (Mouse genome informatics: 95688). Given that Trps1 is a GATA-type transcriptional repressor, it is possible that it binds directly to these GATA motifs in the promoter region of Gdf5 and thus regulates its expression.
Noggin is an antagonist of BMPs including Gdf5 that may influence Gdf5–Trps1 signaling. It has been reported that Noggin is expressed during joint formation and contributes to the regulation of Gdf5 expression (Brunet et al. 1998). Although it antagonizes Gdf5 itself, Noggin induces Gdf5 expression by inhibiting Bmp7 during joint formation (Merino et al. 1999). Like Trps1-deficient mice or bp mice, Noggin-deficient mice lack Gdf5 expression and display fusions between the phalanges and carpal bones (Brunet et al. 1998; Suemoto & Muragaki, unpublished data). These findings suggest that Trps1–Gdf5 signaling is regulated by Noggin.
Although it has been reported that Trps1 interacts with LC8a and RNF4, and localizes in the nucleus (Kaiser et al. 2003a,b), we demonstrated in this study that Trps1 underwent nuclear translocation in ATDC5 cells after treatment with Gdf5. Since Gdf5 and Trps1 are co-expressed at virtually the same time in ATDC5 cells, Gdf5 signaling occurs in these cells and Trps1 is always detected in the nuclei by immunohistochemistry. To exclude the effect of endogenous Gdf5, we used ATDC5 cells after 1 day of insulin treatment for the nuclear translocation assay. Nuclear translocation of Trps1 was completely blocked by treatment with SB203580 (data not shown). Thus, we assume that the mechanism of nuclear translocation could involve the phosphorylation of certain amino acid residues of Trps1.
Although Gdf5 was previously shown to activate p38 MAPK in ATDC5 cells (Nakamura et al. 1999), the molecular events downstream of p38 MAPK were not understood. In this study, we demonstrated for the first time that Trps1 expression is regulated via Alk6 and p38 MAPK in ATDC5 cells. When cells were transfected with a dominant negative form of Alk6, phosphorylation of p38 MAPK and Trps1 protein levels were dramatically suppressed. In addition, treatment of ATDC5 cells with SB203580 (an inhibitor of p38 MAPK) led to a substantial reduction of Trps1 protein levels. These results indicate that Trps1 expression in ATDC5 cells is induced via Alk6 and p38 MAPK.
It is interesting to note that Trps1 protein but not mRNA levels are strongly enhanced by Gdf5 treatment and are strongly affected by dnALK6 over-expression. For some reason, the fluctuation quantity of Trps1 mRNA levels was little. This would support the notion that p38 MAPK stabilizes proteins by protecting from proteasomal degradation (Zimmermann et al. 2001; Brook et al. 2006). p38 MAPK, which is up-regulated by Gdf5 and is down-regulated by dnAlk6, would be essential for maintaining Trps1 protein levels in ATDC5 cells.
Increased phosphorylation of Smad1/5 and ERK1/2 was also observed after treatment with Gdf5 (data not shown). Since addition of SB203580 resulted in a dramatic reduction of Trps1 protein levels, p38 MAPK is considered to be involved in the main pathway for Gdf5–Trps1 signaling.
Previous studies have shown that Gdf5 stimulates chondrogenic differentiation in ATDC5 cells (Nakamura et al. 1999). In this study, we also found that over-expression of Trps1 promoted chondrogenesis of ATDC5 cells. This is in good agreement with the concept that Trps1 acts downstream of Gdf5, but whether Gdf5 and Trps1 act synergistically or antagonistically in chondrogenesis of ATDC5 cells still remains unclear. In contrast, when ATDC5 cells were transfected with dn-Alk6, fewer cartilage-like nodules were formed compared with cultures of control cells. This could be explained by the fact that Trps1 expression is reduced by dn-Alk6. Taken together, it is conceivable that chondrogenic differentiation of ATDC5 cells is promoted in proportion to the level of Trps1 expression.
Since Trps1 is a transcriptional repressor (Malik et al. 2001), it is difficult to consider that it regulates the expression of types II and X collagen. One possibility is that Trps1 might negatively regulate a transcriptional repressor of the type II and type X collagen genes. It has been reported that the other transcription factor downstream of Gdf5, Barx2, binds to the intronic regulatory region of the Col2a1 gene during chondrogenesis (Meech et al. 2005). We found that Trps1 binds to two GATA binding sites in the promoter of the Stat3 gene and represses the expression of Stat3 (Suemoto et al. 2007). It is not yet clear how this effect on Stat3 influences chondrogenesis. Further study is needed to explain how Trps1 regulates the expression of cartilage-specific collagens.
We also demonstrated that the other effect of Trps1 on ATDC5 cells is the promotion of apoptosis and that Trps1 suppressed the expression of anti-apoptotic Bcl-2. During growth plate development, hypertrophic chondrocytes die by apoptosis and are replaced by osteoblasts. Given that Trps1 is reportedly expressed by prehypertrophic chondrocytes (Kunath et al. 2002), it could promote apoptosis of hypertrophic chondrocytes by suppressing Bcl-2 expression.
Gdf5 is considered to be involved in joint formation, but the precise molecular mechanisms remain unclear. Previous studies have shown that Gdf5 induces apoptosis of interzone cells during phalangeal joint development (Storm & Kingsley 1996; Merino et al. 1999). In the present study, we showed that Trps1 promotes the apoptosis of ATDC5 cells. In parallel with these results, we also found that the phalangeal joints of Trps1-deficient newborn mice were not completely formed because interzone cells did not die of apoptosis (Suemoto & Muragaki, unpublished data). Some of the interzone cells, which are expected to disappear completely due to apoptosis, persisted in the joint spaces of the newborn mice. These findings suggest that apoptosis of interzone cells could be induced by Gdf5–Trps1 signaling.
It was reported that Pthrp inhibits apoptosis in cartilage (Yamanaka et al. 2003) and in lung tumors (Hastings et al. 2004). Because the pattern of Pthrp expression is similar to that of Gdf5 (Kobayashi et al. 2005) and because Trps1 is a transcriptional repressor, it is conceivable that Gdf5–Trps1 signaling could suppress Pthrp expression and thus induce apoptosis. In fact, we found that Trps1 suppressed the transcription of Pthrp by binding to two GATA binding sites in the promoter (Nishioka & Muragaki, unpublished data).
In summary, we identified Trps1 as a regulator of chondrogenesis and apoptosis in ATDC5 cells, which is a downstream target of the Gdf5 signaling pathway. Further studies are needed to identify target(s) of Trps1 and the details of Gdf5–Trps1 signaling during chondrogenesis and joint formation.
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Experimental procedures |
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ATDC5 cells were obtained from the RIKEN cell bank (Tsukuba, Japan). Cells were maintained in D-MEM/F12 medium (Gibco BRL, Grand Island, NY) supplemented with 5% FBS (MBL, Nagoya, Japan), 10 µg/mL transferrin (Sigma, St Louis, MO) and 3 x 10–8 M sodium selenite (Sigma). The culture medium was renewed every second day. Cells were plated into plastic dishes or 24-well plates at a density of 1 x 104/cm2. After reaching semiconfluence, the cells were treated with insulin (Sigma) at a concentration of 10 µg/mL and cultured for the indicated periods.
Construction of expression vectors
DNA fragments for the entire coding region of Trps1 and Alk6 were amplified by PCR using the following primer sets: 5'-gag ggtgttcctgacgatta-3' and 5'-tggataaggcaggctctatg-3' for Trps1 and 5'-catgctcttacgaagctctg-3' and 5'-gtccacaagtatctgacgtc-3' for Alk 6. PCR products were cloned into the pCR II vector (Invitrogen, Carlsbad, CA) and sequenced. It is known that the K231R mutation of Alk6 has a dominant negative effect (Fujii et al. 1999). To introduce this mutation into Alk6 cDNA, a QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used. The full-length Trps1 and mutated Alk6 cDNAs were subcloned into a mammalian expression vector, pcDNA3.1(+) (Invitrogen) and transfected into ATDC5 cells by using a Gene Pulser (BioRad, Hercules, CA).
Production of anti-Trps1 antibodies
cDNA for the carboxy-terminal region of Trps1 was amplified by PCR using a forward primer (5'-ggaagccccttagagag-3') and a reverse primer tagged with the PstI site (5'-gtgctaactgcagaggttttactctttagg-3'). The PCR product was cloned into the pQE41 vector (Qiagen, Venlo, the Netherlands) for protein expression. The peptides thus produced were then purified and used to immunize rabbits. After the fourth dose, antiserum was prepared and purified by using a HiTrap (Amersham, Uppsala, Sweden) conjugated with the antigen.
Immunohistochemistry
Forelimbs removed from E15.5 and E16.5 embryos were fixed overnight in phosphate-buffered saline (PBS) with 4% paraformaldehyde and then embedded in paraffin. Sections were cut at 5 mm. Immunostaining was performed using the dextran polymer conjugate two-step visualization system (Dako Envision System; DAKO, Carpinteria, CA) and the specimens were observed by light microscopy (Nikon BW40).
In situ hybridization
In situ hybridization was performed using digoxigenin-labeled riboprobes (Roche, Basel, Switzerland). The mouse Gdf5 probe was generated from a 493-bp RT-PCR product (bases 1872–2160 of the mouse Gdf5 mRNA, accession no.: NM_008109) and was cloned into pCRII-TOPO (Invitrogen). Hybridization was performed overnight at 50 °C, and washed were performed at 42 °C. Sections were then incubated for 1 h with alkaline phosphatase-labeled anti-digoxigenin antibody (1 : 1000, Roche) at RT followed by color reactions with BM purple substrate (Roche). Sections were finally counterstained with methyl green and mounted with Permount.
Detection of nuclear translocation
Confluent cells at 1 day after insulin treatment were incubated with or without Gdf5 (100 ng/mL) for 1 h. Then the cells were fixed with cold acetone and reacted with an anti-Trps1 antibody overnight at 4 °C, followed by reaction with a Cy3-labeled anti-rabbit IgG secondary antibody. Nuclei were stained with DAPI.
Immunoblotting
Cells were washed with PBS and lysed in the sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol and 5% β-mercaptoethanol). Then the cell lysate was subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies for β-actin (Santa Cruz, Santa Cruz, CA), cleaved caspase 3 (Cell Signaling, Danvers, MA) and phosphorylated p38 MAPK (Cell Signaling).
Northern blot analysis
Total RNA was extracted from cells using TRIzol (Invitrogen), separated on 1% formaldehyde-agarose gel, transferred to Hybond N+ membranes (Amersham) and hybridized with a radiolabeled cDNA probe for nucleotides 2925–3854 of Trps1 cDNA. Then the membranes were washed in 0.1x SSC/0.1%SDS, and exposed to an X-ray film.
Reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative PCR
Using 1 µg of total RNA, cDNAs were synthesized by the SuperScript III system (Invitrogen). Amplification was performed with a GeneAmp PCR system 9600 (Perkin Elmer Life Science, Boston, MA) using AmpliTaq Gold DNA polymerase (Applied Biosystems. Foster City, CA). Specific primers were designed from the sequences available in GenBank: 5'-ccatgtttgtgatgggtgtg-3' and 5'-taggccatgaggtccaccac-3' for Gapdh; 5'-cttgccaagacctgaaactc-3' and 5'-caccaaattcctgttcagcc-3' for collagen 
1(II); 5'-acttcctgt caagctcatcc-3' and 5'-tcctgcatgtttcctagatg-3' for collagen 1(X); 5'-caaatctcaggcctgagtga-3' and 5'-gtgaagagctgatatcctgcag-3' for Trps1; and 5'-aatgccagggccaagggaag-3' and 5'-gacagcttcagttgggcaac-3' for Gdf5. The PCR products were separated by electrophoresis on 1%–2% agarose gel and the gels were stained with ethidium bromide. The PCR product of Gapdh cDNA was used to ascertain that an equivalent amount of cDNA was obtained from each sample.
Quantitative real time-PCR was performed with SYBR GREEN PCR Master Mix (Applied Biosystems) and the products were measured by using a 7500 Real Time PCR System (Applied Biosystems). The following primers were designed with PrimerExpress Software (Applied Biosystems): 5'-tgagcaagagaggccctatc-3' and 5'-aggcccctcctgttattatg-3' for Gapdh, and 5'-cgccgctgcctttttg-3' and 5'-gagaatgtcaatccgtaggaatcc-3' for Bcl-2. Each sample was measured in triplicate, and the level of expression of the target gene was normalized for a Gapdh standard curve run in duplicate on the same plate.
Analysis of chondrogenesis
The extent of chondrogenesis was evaluated by staining with Alcian Blue. Cells were washed with PBS, fixed with 4% paraformaldehyde, stained with 1% Alcian Blue 8 GX (Wako Pure Chemicals, Tokyo, Japan) in 3% acetic acid and rinsed with distilled water. For quantitative analysis, cells stained with Alcian Blue were extracted with 6 M guanidine–HCl and the optical density was measured at 630 nm by a spectrophotometer.
TUNNEL staining
For examination of apoptosis induced by an agonistic anti-Fas antibody, we used the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method with an ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA). Cells were cultured in differentiation medium for 1 week and then treated with an agonistic anti-Fas antibody (0.1 µg/mL) and cycloheximide (5 µM) for 10 h. Next, the cells were washed twice with PBS, fixed with 1% paraformaldehyde and stained. Subsequently, the number of TUNEL-positive cells was counted in randomly chosen fields under a fluorescence microscope.
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: ymuragak{at}wakayama-med.ac.jp
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References |
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Received: 25 September 2007
Accepted: 31 December 2007
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