Mouse Snail family transcription repressors regulate chondrocyte , extracellular matrix , type II collagen , and aggrecan

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Introduction
During skeletal development, condensed mesenchymal cells give rise to chondrocytes and form cartilage primordia, which serve as templates for endochondral bone formation. In cartilage primordia, chondrocytes undergo further differentiation, starting as proliferating chondrocytes, becoming prehypertrophic chondrocytes and ending as hypertrophic chondrocytes. Chondrocytes secrete abundant extracellular matrix (ECM) into the extracellular space and the composition of ECM changes during the sequential differentiation steps. Type II collagen and aggrecan are major components of the cartilage extracellular matrix produced by proliferating and prehypertrophic chondrocytes, and Type X collagen is a major collagen type produced by hypertrophic chondrocytes. The differentiation into to hypertrophic chondrocytes is accompanied with switching of collagen types from type II to type X and the disappearance of aggrecan and link protein (1,2). Dysfunction of these extracellular macromolecules in mutant mice results in striking skeletal malformation, suggesting their important roles in skeletal formation (3)(4)(5)(6)(7).
Type II collagen is a homotrimer of the alpha1 (II) chain (Col2a1). Two elements for the regulation of Col2a1 transcription are known; one is the enhancer located within the first intron that mediates cartilage-specific expression of Col2a1, and the other is in the promoter region that has both silencers and promoter elements (8)(9)(10)(11)(12). One of the E-boxes (CAGGTG) in the promoter region is involved in the negative regulation of Col2a1 but the transcription factors which bind to that E-box remain unknown (13).
Drosophila Snail was the first member identified among the Snail family transcription factors and encodes a zinc finger-type transcription factor that is necessary for gastrulation and mesoderm formation (14). Snail family members have been evolutionarily conserved among many species. They share highly conserved C2H2 type zinc finger domains, which bind to the E-box and repress the transcription of target genes (reviewed in 15). Snail-related transcription repressors were shown to play roles in a broad spectrum of biological functions such as cell differentiation, cell adhesion, cell movement, cell cycle regulation and apoptosis (16-26, reviewed in 15,27). Mouse Snail family members include Snail (Sna), Slug (Slugh) and Smuc (28). Expression patterns of mouse Snail and Slug in the early developmental stage suggest involvement of Snail and Slug in chondrogenesis (24,(28)(29)(30). Mouse Snail mRNA is expressed in condensed precartilage mesenchyme but not detected in chondrocytes at 14.5 days except in the distal phalanges of the hind limbs, which are the only sites of precartilage in the limbs at this stage (24). In the 3 day old rat, mRNA of rat Slug is detected in condensing mesenchymal cells corresponding to cartilage precursors, and at 5 days, the cartilage primordium itself was negative for the expression (29).
In this study we found an inverse correlation between the gene expression of Snail and Slug and type II HAL author manuscript inserm-00148022, version 1 collagen and aggrecan in vivo and in vitro during chondrocyte differentiation in the growth plate of mouse embryos. For the experiments in vitro, we have used well-characterized chondrogenic cell line, ATDC5, which is derived from embryonal teratocarcinoma and recapitulate the sequential steps of chondrocyte differentiation (29)(30)(31)(32). Using ATDC5 cells we found that overexpression of Snail or Slug reduced the expression level of type II collagen mRNA and aggrecan mRNA. Reporter analysis using the Col2a1 promoter revealed that Snail and Slug suppress the activity. We found that E-boxes, which are binding motifs for the Snail family, are involved in the negative regulation of Col2a1 transcription (13).
Reporter analysis showed Snail suppressed the transcriptional activity of Col2a1 through the E-boxes.
Gel shift assay demonstrated the binding of Snail to the E-box. These results suggest that Snail and Slug regulate chondrocyte differentiation by repressing the genes, the Col2a1 gene and possibly the aggrecan gene,that encode major ECM molecules in cartilage.

Experimental Procedures
Cell Culture. ATDC5 cells were maintained in DMEM/F-12 medium (LifeTechnology) supplemented with 5% FBS and antibiotics in plastic dishes at 37 °C under 5% CO 2 . For the induction of chondrocyte differentiation in three-dimension culture, the cells were trypsinized and embedded in alginate beads in a differentiation medium consisting of DMEM/F-12, 10% FBS, 10ug/ml human insulin, 10ug/ml human transferrin, 10ng/ml sodium selenite (Roche) and antibiotics. To embed the cells into beads, trypsinized cells were centrifuged for 5 min at 1,500 rpm, washed once with a 150mM NaCl solution and suspended in a alginate solution (1.2% alginate, 150 mM NaCl, 1mM CaCl 2 , and 20mM HEPES) at a cell density of 1.0 x 10 6 cells/ml. This mixture was poured into a plastic syringe and dropped through a 22-gauge needle into 40ml of a 50mM CaCl 2 solution. The CaCl 2 solution was decanted and the beads were washed once with DMEM/F-12 medium. Beads were placed at 37°C in 100mm dishes with 20ml of the differentiation medium. For the cell recovery, the culture medium was discarded and the beads were washed once with PBS and dissociated in 150mM NaCl. The cells were pelleted from the suspension.
Transfection Assay. ATDC5 cells were transiently transfected using FuGENE 6 (Roche). First, 250,000 cells were seeded per 100mm dish. Then after 16h culture, they were transfected with 12mg of plasmids mixture. The medium was renewed 24 hr after the transfection. For Northern blotting, the cells were harvested 48 hr after transfection. Stable transfectants of ATDC5 expressing exogenous Snail or Slug were established following transfection with Lipofectamine 2000 (Life Technologies). At 16 hr after the seeding of 80,000 cells per well onto 24-well plates, cells were transfected with 0.9mg of plasmid.
Stable transfectants were selected in 400 mg/ml of G418 for 10 days. pcDNA3 mSna was kindly provided HAL author manuscript inserm-00148022, version 1 by A. Cano (18) and pcDTet-mtmSna by J. Cross. PCR3 Mslug S was previously described (26). For tetracycline-induced exogenous Snail expression, ATDC5 cells transfected with pcDTet-mtmSna were cultured in beads for 48 hours with 3 ng/ml of doxycyclin in the differentiation medium. At this concentration, doxycyclin was not toxic to cells undergoing chondrogenic differentiation in the three dimension culture or in monolayer culture.
Slides were dipped into NTB-2 (Eastman Kodak Co.) and stored at 4°C for three to four days to visualize signals. After development, sections were counterstained with hematoxylin and eosin and mounted.
Northern Blot Analysis. Total RNA was extracted from ATDC5 cells using an RNeasy Mini Kit (Qiagen). The 8mg RNA was applied to an agarose gel for electrophoresis, then transferred onto a Hybond N membrane (Amersham). cDNAs were labeled with [32P] dCTP using a Random Primed DNA Labeling Kit (Promega) and used for probes. The membranes were hybridized with the labeled probe at 68°C, washed first at room temperature in 2X SSC and 0.1% sodium dodecyl sulfate (SDS) and then at 65°C in 0.2X SSC and 0.1% SDS, and analyzed with a Cyclone Phosphorimager (Packard). Probes for type II collagen, type X collagen, and the PTH/PTHrP receptor were prepared from plasmids described previously (33). Probes for Snail and Slug were prepared from fragments of the plasmids used for in situ hybridization. Sox9 probes were made from mouse cDNA (1070-1324).
Reporter Assays. The plasmid constructs pCII-312, pCII 977, pCII-312E, and pCII-977E have been described previously (34). Briefly, these constructs contain the 5'-flanking sequence from the rat Col2a1 gene upstream of the CAT coding sequence. pCII-312E and pCII-977 also contains a 1,500-bp region from the first intron that has enhancer activity. For CAT assays, ATDC5 cells were transfected in 100mm dishes using Lipofectamine 2000 (Life Technologies) or FuGENE 6 (Roche) according to the manufacturer's instructions. The cells were cotransfected with Snail or Slug expression plasmid, 2.5mg of reporter plasmid (pCII-312, pCII-977, pCII-312E, or pCII-977E), 2.5mg of Beta-galactosidase expression plasmid (CMV beta-gal) and pcDNA3.1. For Lipofectamine 2000, the total amount of DNA was kept at 20mg with pcDNA3.1 and cells were exposed to the DNA solution for 2h and then cultured in fresh DMEM/F-12 medium supplemented with 5% FBS. For FuGENE6, the total amount of DNA was kept 12mg with pcDNA3.1 and the DNA solution was added to the culture medium and left for 24 hr. The medium was then renewed. For three-dimension culture, the cells were embedded in alginate beads 24 hours after transfection and cultured in the differentiation medium for 48 hr. For monolayer culturem, the 6 cells were cultured in the maintaining medium for 48 hours after transfection. Nuclear extracts were prepared for the analysis of CAT activity (35). Equivalent amounts of cellular proteins were incubated in the reaction buffer (2.5M Tris-HCl, pH 7.8, 1mM acetyl-CoA (Roche), and 2mCi/ml [14C]chloramphenicol (NEN)) for 2 hr at 37°C. The level of acetylation was examined by TLC followed by quantitation using a Cyclone Phosphorimager (Packard). CAT activity was normalized to beta-galactosidase activity with a Galacto-Light Plus system (Tropix). For luciferase assays, ATDC5 cells were transfected in six-well plates using FuGENE6 (Roche) according to the manufacturer's instructions.
The EBx1 Luc construct was generated by inserting a PCR-amplified 283 bp promoter region into pE1bluc (previously described by A. Hata) and was verified by sequencing. mutEBx1 Luc was generated by PCR using a mismatch primer. 977-luc reporter was generated by inserting a PCR-amplified 977 bp sequence from pCII-977 into pE1b-luc, and the E-box mutant series was made by introducing mutations in each E-box using a mismatch primer to CATGCG. All plasmids were verified by sequencing.
Luciferase assays were carried out with a Luciferase Assay System (Promega) and Berthold luminometer.
A beta-galactosidase expression vector was co-transfected for normalization and assayed with a Galacto-Light Plus kit (Tropix). pCR3 Mslug S and antisense Slug plasmid were previously described (26) Electromobility Shift Assay (EMSA). The expression plasmid for HA-tagged Snail (17)   In three-dimension culture systems, once dedifferentiated primary chondrocytes cultured in monolayers recover the chondrocyte phenotype. Therefore three-dimension culture is thought to mimic the situation in vivo better than monolayer culture. We used an alginate beads culture system (38)(39)(40), in which the ATDC5 cells are embedded in alginate beads and cultured with insulin. After two days in the three-dimension culture, ATDC5 cells started to express type II collagen mRNA which is a hallmark of chondrocyte differentiation ( Fig. 2A). In monolayer cultures of ATDC5 cells, chondrocytes form few condensations and only a minor expression of type II collagen mRNA was detected four days after confluence (32,37). Therefore, this three-dimension culture system using ATDC5 cells is a useful in vitro system with which to study chondrocyte differentiation.

Expression of Snail
We examined the expression of Snail family transcription repressor mRNAs by Northern hybridization during the differentiation of ATDC5 cells. In subconfluent monolayer cultures, mRNAs of chondrocyte differentiation markers; type II collagen, PTH/PTHrP receptor and Sox9 were undetectable (36,37,41). Two days after initiating the three-dimension culture, chondrocyte differentiation markers were detected ( Fig. 2A). After four days of culture, the level of type II collagen and PTH/PTHrP receptor mRNA were enforced, whereas the levels of Snail and Slug mRNA were reduced to 60% and 50%, respectively when quantified with a phosphorimager. These results show inverse correlation of expression levels between Snail/Slug mRNA and type II collagen mRNA during chondrocyte differentiation in vitro.
Smuc mRNA was not detected in ATDC5 cells by Northern bloting.  2B). We also looked for this reduction in type II collagen mRNA using a tetracycline-inducible Snail expression plasmid (Fig. 2C). Interestingly, mRNA of Sox9, which is a positive regulator of Col2a1 gene, was expressed normally despite that level of type II collagen mRNA was reduced. In our system, the level of Sox9 mRNA increased upon differentiation and the timing coincided well with the onset of type II collagen mRNA expression (Fig. 2A). These results suggested that when overexpressed Snail and Slug can overcome the positive regulatory effect of Sox9 and can regulate chondrocyte differentiation by counteracting Sox9.

Overexpression of Snail
Next we analyzed ATDC5 cells stably transfected with Snail or Slug expression plasmids. After selection for drug resistance, ten clones were picked and expanded. The expression level of Snail or Slug mRNA in each clone cultured in monolayers was tested by Northern blotting. All the clones showed higher level of expression of Snail or Slug mRNA than the mock transfectant (data not shown). For further analysis, we chose clones expressing two to three times more Snail or Slug mRNA than parental cells (Fig. 2E, quantified by phosphorimaging). In the stable transformants, the expression of both Snail and Slug was increased in the undifferentiated state, indicating some cross-regulatory pathway between the two genes.
In the undifferentiated state, the morphology of Slug transformants was distinct from that of the untransfected or Snail-transfected clones. The Slug transfected cells displayed longer cellular protrusions and a thiner cell body (Fig. 2D). Stable clones were induced to differentiate in three-dimension culture for two days and the expression of the chondrocyte markers was analyzed by Northern Blotting (Fig. 2F).
In transformants, the expression levels of collagen type II mRNA were dramatically reduced in . Three E-boxes were found in the human gene, and four E-boxes in the rat and mouse genes ( Fig. 4A and 4B). Rat E-box2 and 3 (CAGGTG) were conserved among the species both in sequence and in relative location ( Fig. 3A and   3B). Then we took advantage of well-characterized reporter constructs containing promoter and enhancer regions of rat col2a1 (34). pCII-977E and pCII-312E are CAT reporter constructs containing -977 to +110 and -312 to +110 of the genomic sequence respectively and a 1500-bp first intron sequence from the rat Col2a1 gene (Fig. 3C). pCII-977E has four E-boxes and pCII-312E has one. It has been reported that the 977-bp genomic fragment contains not only promoter activity but also silencing activity, which is observed in chicken embryonic fibroblast and HeLa cells (38).
We transiently transfected ATDC5 cells with the reporter constructs, and Snail or Slug expression plasmid. CAT activity was repressed when Snail or Slug was co-transfected, but not when the empty vector was co-transfected. (Fig. 3C). These results suggest that Snail and Slug suppress the transcription of Col2a1 through the promoter and/or the enhancer. The promoter region is more likely to include the responsive elements, because pCII-977E had weaker activity than pCII-312E (Fig. 3C), indicating that the length of the promoter affected the suppressive effect of Snail/Slug. pCII-977E has three more E-boxes (E-box 1-3 in Fig. 3A ) than pCII-312E (E-box 4 in Fig. 3A) in the promoter region, therefore, we speculated that these E-boxes may have the additive suppressive effect.
The enhancer sequence was dispensable for suppressive activity of Snail/Slug. Next, we tested the possibility that Snail/Slug can counteract the positive regulation mediated by the enhancer in the reporter constructs. The enhancer contains the binding sites for Sox9 (42), which is a chondrocytespecific positive regulator of the Col2a1 gene in chondrocytes, and the enhancer also contains E-boxes.
We compared the activities of reporter constructs with or without the enhancer: pCII-977E had the enhancer and pCII-977 lacked it. The activities of pCII-977 and pCII-977E were suppressed by Snail and Slug almost equally in both undifferentiated and differentiated ATDC5 cells ( Fig. 3D and data not shown). These results suggest that Snail and Slug suppress the activity through the promoter and not through the enhancer. Because of the significant similarity among the Snail family transcription repressors in the DNA binding domains, they are thought to regulate the same target genes (20). The identity of the last four zinc fingers between Snail and Slug is 82%. In this study, overexpression of Snail and Slug (also Smuc, data not shown) gave similar results using the reporter. Therefore, we used only the effect of Snail in further analysis.

Suppressive effect of Snail was mediated by E-box. To test whether each E-box is involved in
the regulation, a mutation was introduced into each of the E-boxes in the rat 977-bp promoter sequence and the sequence was subcloned into luciferase vectors. Reporter assay revealed that a mutation in E-box1, 2 and 4 increased the activity whereas a mutation in E-box 3 had no effect on transcription (Fig. 4).
The sequence and the location of rat E-box 3 were not conserved well among the three species (Fig. 3A and 3B). These data suggested that rat E-box 3 was not involved in the regulation of Col2a1. The strong desuppressive effect of disrupton of E-box 2 and 4 indicated the importance of these two conserved sites.
It has been reported that mutation in E-box 4 desuppressed the activity of the reporter construct containing -307 to +110 of Col2a1 in chicken limb mesenchyme cells and transregulatory factors which bind to this site remained unknown (13). Therefore to study the mechanism, we focused on E-box 4 located at -167 to -162.
To test whether Snail is a candidate transcription factor, we made two luciferase constructs, EBx1-Luc and mutEBx1-Luc, both containing the -173 to +110 stretch of the promoter sequence of rat Col2a1 and mutEBx1-Luc having a mutation in the E-box 4 (CAGGTG to CATGCG, Fig. 4B) that abrogates the binding of Snail and Slug. We compared the activity of the two constructs and found the mutation in the E-box abolished the repressor activity in ATDC5 cells (Fig. 4C).
Next we examined the effect of the chimeric protein VP16-Snail, in which the N-terminal half of Snail was removed and the DNA binding domain of Snail was fused with the transcription activator domain of VP16. This chimeric protein can work as a competitor for endogenous Snail-related repressors and can counteract their suppressive activity (23). Mutation in the E-box abolished this effect of VP16 (Fig. 4D). These results suggested that VP16-Snail competed for binding to the E-box with endogenous factors in a dose-dependent manner and desuppressed the activity through the E-box in ATDC5 cells.

Major suppression of Col2a1 transcription was mediated by the Snail family.
To further define the negative regulator of the E-box, we examined the effect of antisense Slug mRNA expression, which has been reported to inhibit the functions of endogenous Slug (26). It was reported that injection of antisense Slug mRNA into Xenopus embryo reduced not only XSlug but also Xsnail expression. This is thought to be due to the high homology between XSnail and Xslug (43,44). Therefore, we checked the effect of antisense Slug on Snail protein expression by Western bloting. HA-tagged Snail protein expression was reduced when the Snail expression plasmid was co-transfected with the antisense Slug expression plasmid (Fig. 5A) in a dose-dependent manner. This result suggested that the gene product of mouse Snail can be reduced by the introduction of Slug antisense mRNA. This result was similar to the phenomenon observed in the experiment on Xenopus embryos. Next we examined if mouse Snail protein can reverse the desuppressive effect of antisense Slug mRNA. Fig. 5B shows that antisense Slug mRNA had a desuppressive effect on the 977-luc reporter (Fig. 5B, lane2) and cotransfection with HA-Snail reversed the suppressive effect in dose dependent manner (Fig. 5B, lane 3 to 5), indicating the change in the amount of Snail family protein affected the expression of the Col2a1 gene. We also tested 12 the effect of antisense Slug mRNA on EBx1 and mutEBx1 reporters. Antisense mRNA treatment resulted in increased activity of the EBx1 reporter, but had no effect on the mutEBx1 reporter (data not shown).
These results suggest that the suppression of Col2a1 transcription was mainly dependent on the effect of the Snail family through E-boxes.
Snail binds to the E-box in the Col2a1 promoter. We tested whether Snail protein interacts directly with the E-box using electrophoretic mobility-shift assays. The 24-bp oligonucleotides corresponding to the -174 to -151 sequence containing the E-box 4 formed two retarded bands when incubated with the nuclear extract prepared from ATDC5 cells transfected with HA-tagged Snail expression plasmid (Fig. 6). A supershifted band was observed when anti-HA antibody was added to the reaction mixture but not when the control antibody was added. The shifted band disappeared only when cold competitor was added to the reaction and not when mutated oligonucleotide was added. Thus the lower band (Fig. 6, arrow) contained HA-tagged Snail in the complex whereas the upper band (Fig. 6, dot) was non-specific. These results suggest that Snail protein binds to the E-box of Col2a1 in a sequence-specific manner. Sox9 is one of the major activators of the Col2a1 gene and its specific binding site locates in the enhancer in the first intron (4,8,10,41). Northern blot analysis showed that, although Sox9 mRNA was expressed, type II collagen mRNA was only weakly expressed in Snail or Slug-transfected ATDC5 cells when they were induced to differentiate in a three-dimension culture. Reporter analysis showed the suppressive activity of Snail both in undifferentiated and in differentiated ATDC5 cells. These results suggested a dominant suppressive effect of Snail over Sox9 in differentiating ATDC5 cells and also showed that the enhancer sequence (8) was unnecessary for the suppressive activity of Snail.

Snail-related transcription factors are repressors of the
We studied the mechanism involved by focusing on the E-box in the promoter region of the Col2a1 gene, which is highly conserved among species. The E-box has been reported to negatively regulate Col2a1 gene transcription (13). In addition, it is a binding site of Snail-related repressors. We found that It will be of special interest to investigate whether members of the Snail family also regulate the expression of the aggrecan gene or not. This gene is evolutionary well conserved, but the regulatory regions have not been described yet.

Snail-related transcription factors modulate ECM.
We found that Snail and Slug can modulate the expression of collagen type II and aggrecan during chondrocyte differentiation. The transition of these components of ECM is an essential step for chondrocyte differentiation. Other than chondrocytes, Snail/Slug is expressed in premigratory neural crests and plays important roles during their migration.
Aggrecan is present along their migratory pathway and guides their migration (46). Collagen type II is transiently present at the interface between epithelial cells and mesenchymal cells during the development of neurocranium (46). These expression patterns suggested that Col2a1 and Aggrecan are regulated by Snail/Slug in neural crest cells. In addition, Snail is expressed at the invasive front of cancer cell lines and primary tumors induced in mice (11). Therefore, we propose that regulating the ECM is another fundamental role of Snail family. Such a mechanism in addition to the cell-cell adhesion and cytoskeleton modulations induced by Snail factors (reviewed in 41) could be involved in cancer development and metastasis. .