Induction of the Sry-Related Factor SOX6 Contributes to Bone Morphogenetic Protein-2-Induced Chondroblastic Differentiation of C3H10T1/2 Cells

R. Fernández-Lloris, F. Viñals, T. López-Rovira, V. Harley, R. Bartrons, J. L. Rosa and F. Ventura

Departament de Ciències Fisiològiques II (R.F.-L., F.Vi., T.L.-R., R.B., J.L.R., F.Ve.), Campus de Bellvitge, Universitat de Barcelona, 08907 L’Hospitalet de Llobregat, Spain; and Prince Henry’s Institute (V.H.), Monash Medical Centre, Melbourne 3168, Victoria, Australia

Address all correspondence and requests for reprints to: Francesc Ventura, Unitat de Bioquímica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Spain. E-mail: fventura{at}bellvitge.bvg.ub.es.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen {alpha}1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CHONDROGENESIS IS A complex multistep process that allows the generation of mature cartilage, a tissue containing a unique set of extracellular matrix molecules, which gives its ability to withstand compression and frictional stresses. The first step in chondrogenesis is the aggregation of mesenchymal cells into prechondrogenic condensations. These condensations start to express cartilage-specific genes and further differentiate into mature chondrocytes. In endochondral ossification, bone is formed through ossification of this preexisting cartilage. Although much is known about the terminal differentiation products expressed by chondrocytes, the factors that specify the chondrocyte lineage are still mostly unknown. Only in the last few years have a number of studies identified nuclear factors that control the determinative switch for chondrocyte differentiation and which might also regulate the expression of genes involved in prechondrogenic condensation and subsequent chondrocyte differentiation (1, 2, 3). To date, some individual members of the Sry-type high-mobility group (HMG) box proteins (SOX) family have been convincingly shown to be necessary in the initiation of cartilage differentiation during embryogenesis (4). SOX proteins form a subfamily of DNA-binding proteins with a 79-amino-acid HMG domain, and have crucial functions as transcription activators in a great many developmental processes, including neurogenesis, sex determination, and skeleton formation (5, 6). Transcripts for Sox9, L-Sox5 (an alternatively spliced form of Sox5), and Sox6 are coexpressed with cartilage-specific genes and cooperate in the expression of type-II collagen {alpha}1 chain (Col2a1) and other genes of the chondrocytic program (4, 7, 8). These factors bind to the same cis element, the chondrocyte-specific enhancer, which encompasses a 48-bp region of the murine and human Col2a1 gene. This region contains four SOX protein binding sites that are required for chondrocyte- specific expression of markers such as Col2a1, Col11a2, and aggrecan (7). Several mutations in the Sox9 gene were found in patients with campomelic chondrodysplasia (9, 10, 11, 12, 13, 14), a rare congenital dwarfism syndrome characterized by hypoplastic development of endochondrally formed skeletal tissues and various nonskeletal anomalies. Overexpression of Sox9 transcripts can induce ectopic cartilage formation in vivo (15), whereas prechondrogenic mesenchyme cells with homozygous Sox9 -/- deletion are unable to differentiate as chondrocytes (16), and L-Sox-5:Sox6 double null mice fetuses die with severe, generalized chondrodysplasia (17). Thus, these and other transcription factors may be needed to specify the high-level expression of such specific chondrocytic genes.

Many molecules promote chondrogenesis in vivo and in vitro (18, 19). Among these, bone morphogenetic proteins (BMPs) are expressed in the mesenchyme in an overlapping pattern and are required in vivo for both chondroprogenitor condensation and subsequent differentiation into chondrocytes (20, 21). Studies in primary cultures of chondroblasts, as well as prechondrogenic and undifferentiated mesenchymal cell lines such as ATDC5 and C3H10T1/2 cells, show that BMPs stimulate the expression of chondrogenic markers (22, 23, 24, 25, 26). However, little is known about the relationship between this cartilage-specific transcriptional activation and the BMP transductional pathway that leads to chondrogenesis.

In the present study, we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2 in the pluripotent mesenchymal cell line C3H10T1/2. Our findings show that BMP-2 transcriptional activation of pro{alpha}1(II) collagen gene expression takes place through its chondrocyte-specific enhancer located in the first intron. Our data also suggest that increased expression of SOX6 transcription factor acts as a downstream mediator of these BMP-2-mediated transcriptional effects.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BMP-2 Induces Chondrogenic Differentiation in C3H10T1/2 Cells
The pluripotent embryonic cell line C3H10T1/2 has been shown to undergo chondrogenic differentiation in the presence of BMP-2 (34, 35). We first analyzed this induction using two cartilage matrix markers: sulfated glycosaminglycans, evaluated by Alcian-blue staining, and type-II collagen expression. In BMP-2-treated cultures supplemented with 1% fetal bovine serum (FBS), Alcian blue-positive material appeared at d 1, whereas in control cultures it did not appear at d 1 (Fig. 1AGo). The appearance of Alcian-blue-positive material at d 7 showed BMP-2 dose dependence (Fig. 1BGo).



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Figure 1. BMP-2 Induces Chondrogenic Differentiation in C3H10T1/2 Cells

A, Time-course assay of sulfated glycosaminglycans staining by Alcian blue. Cells were cultured in DMEM supplemented with 1%, 50 µg/ml L-ascorbic acid and 10 mM ß-glycerophosphate and treated with 3 nM BMP-2. Results are the mean ± SEM of three separate experiments. B, C3H10T1/2 cells were incubated with the indicated doses of BMP-2 for 7 d in the same media as above. Results are the mean ± SEM of three separate experiments. C, Cells were cultured in DMEM supplemented with 1% FBS, 50 µg/ml L-ascorbic acid, and 10 mM ß-glycerophosphate and incubated with 3 nM BMP-2 for different times. Total RNA was extracted, analyzed by Northern blot analysis with Col2a1 or glyceraldehyde-3-phosphate dehydrogenase probes. D, Cells were cultured in the presence or absence of 3 nM BMP-2 and/or 10 µg/ml cycloheximide (CHX) as indicated. Northern blot analysis were performed as above.

 
The regulation of Col2a1 mRNA expression by BMP-2 was studied in the same C2H10T1/2 cell line. RNAs from C3H10T1/2 cells treated with 3 nM BMP-2 were analyzed by Northern blot analysis. Induction of Col2a1 mRNA expression begins 8–12 h after BMP-2 addition and increases in a time-dependent manner up to 24 h (Fig. 1CGo). We also analyzed whether induction of Col2a1 gene expression required new protein synthesis. As shown in Fig. 1DGo, addition of cycloheximide completely blocked the induction of type-II collagen mRNA either at 6 or 12 h after BMP-2 addition. Altogether, these data suggest that the major component of the Col2a1 induction by BMP-2 requires synthesis of additional proteins.

The Chondrocyte-Specific Enhancer Is the BMP-2-Responsive Element in the Col2a1 Gene
To delineate more precisely how BMP-2 controls the expression of cartilage-specific genes, we used a reporter-construct, PI1P1L, containing the promoter and the first intron of Col2a1 gene. The PI1P1L reporter transfected in C3H10T1/2 cells showed a 2.4-fold induction when cells were incubated with 3 nM BMP-2 for 24 h (Fig. 2AGo). Furthermore, cotransfection of constitutively active forms of BMP receptor (BMPR)-I (36, 37, 38, 39) induced luciferase activity up to 5-fold whereas BMPR-II plus constitutively activated BMPR-IB induced luciferase activity 25-fold even in the absence of BMP-2 (Fig. 2BGo). Similarly, overexpression of SMAD1 or coexpression of SMAD1 and BMPR-IB increased reporter activity up to 35-fold. These data indicate that BMP-2 is able to regulate type-II collagen expression at the transcriptional level.



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Figure 2. Mapping of the BMP-Responsive Element on the Col2a1 Gene

A, C3H10T1/2 cells were transfected with 1 µg of the PI1P1L reporter. One day later, they were split and incubated with 3 nM BMP-2 for 24 h in differentiation media. Luciferase activity was determined as described in Materials and Methods. Results are expressed as mean ± SEM of five separate transfections. B, Cells were transfected with the indicated receptors and/or SMAD expression vectors. Luciferase activity was analyzed 48 h later. C, C3H10T1/2 cells were transfected with 1 µg of the different constructs and treated with 3 nM BMP-2 for 24 h in differentiation media. Results are expressed as mean ± SEM of four separate transfections.

 
To define the BMP-2-responsive elements, we analyzed four different constructs containing the 886-bp promoter, a 1448-bp region (from +1877 to +3325) and a 465-bp region (from +1877 to +2342) of intron I, and a 48-bp sequence of the intron I, previously described as a potent activator of the Col2aI (40). Whereas deletion of the first intron completely abolished BMP-2 responsiveness, deletion constructs that maintained the 48-bp chondrocyte-specific enhancer of intron I retained the 2.4-fold induction of PI1P1L (Fig. 2CGo). Moreover, a construct containing this 48-bp enhancer alone is sufficient to drive BMP-2 transcriptional induction; and point mutations in the three HMG sequences within the 48-bp enhancer suppressed BMP-2 responsiveness. These results confirmed that transcription factors that bind to HMG sites of the chondrocyte-specific enhancer mediate the BMP transcriptional response in the Col2a1 gene.

BMP-2 Does Not Induce SOX9 Expression
There is a lot of evidence that SOX9, a HMG-containing transcription factor, plays an important role in chondrocyte differentiation and cartilage formation (4, 5). Moreover, the SOX9 transcription factor enhances specific chondrocytic gene expression through its binding to a HMG-like site (4, 7, 12). These previous studies suggested that induction of SOX9 could mediate activation of Col2a1 expression by BMP-2. To test this hypothesis, we first analyzed the abilities of full-length SOX9 and two mutant SOX9 proteins lacking one or both transactivation domains (Fig. 3AGo). Transient overexpression of SOX9 protein increased transcriptional activity, enhancing more than 20-fold the activity of the reporter containing the chondrocyte-specific enhancer. However, expression of the two truncated SOX9 proteins lacking one or both transactivation domains resulted in partial (mutant 1–410) or complete suppression of reporter activation (Fig. 3AGo). Thus, it was verified that SOX9 activates Col2a1 expression and that not only the HMG-binding domain but also the two transactivation domains are required.



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Figure 3. BMP-2 Does Not Induce SOX9 Expression at Early Steps of Chondrocytic Differentiation in C3H10T1/2

A, Schematic comparison of SOX9 and their deletion mutants. HMG for DNA binding, PQS (proline-, glutamine-, and serine-rich domain), and PQA (proline-, glutamine-, and alanine-rich domain) are boxed. Cells were transfected with the Col2a1-enhancer reporter and the indicated constructs. Luciferase activity was determined 48 h later. B, RNA extracts were obtained from C3H10T1/2 cells treated with 3 nM BMP-2 in differentiation media for the indicated times. RT-PCR was performed as described in Materials and Methods. C, C3H10T1/2 cells were transfected with a reporter construct containing 6.8 kb of Sox9 promoter. One day later, they were split and incubated with 3 nM BMP-2 and/or 1 µM RA for 24 h in differentiation media. Results are shown as mean ± SEM of three independent experiments. D, C3H10T1/2 cells were cotransfected with the Col2a1-enhancer reporter and 1 µg of Sox9 expression vector or empty vector as indicated. One day later, they were split and incubated with 3 nM BMP-2 for 24 h in differentiation media. Results are shown as mean ± SEM of five independent assays.

 
We analyzed whether BMP-2 had any effect on Sox9 expression levels. As shown in Fig. 3BGo, either Western (data not shown) or semiquantitative RT-PCR analysis showed no significant changes in Sox9 expression after BMP-2 addition (Fig. 3BGo). Furthermore, in C3H10T1/2 cells transfected with a reporter containing a 6.8-kb promoter region of Sox9, BMP-2 was unable to induce activation of the reporter (Fig. 3CGo). Interestingly, in the same assays, retinoic acid (RA) induced a 2.5-fold activation of the reporter, which correlates with the previously reported activation of Sox9 expression by RA in chondrocytic cells (41). Figure 3DGo shows that in C3H10T1/2 cells overexpressing SOX9, addition of BMP-2 further increased transcription from the Col2a1 enhancer. These data suggest either the existence of a BMP-2-inducible postranslational modification of SOX9 or the expression of an additional factor responsible for this BMP-2-induced transcriptional increase.

BMP-2 Induction of Sox6 Gene Expression
Previous experiments showed that L-SOX5 and SOX6 coexpress with SOX9 and bind to the same HMG sites cooperating in the expression of chondrocyte-specific genes (4, 7, 34). Taking these data into account, we then examined the BMP-2 effects on L-SOX5 and SOX6 expression. Sox6 mRNA expression showed time-dependent BMP-2-induction (3-fold induction after 24–48 h) (Fig. 4AGo). In contrast, there was no increase in Sox6 mRNA expression in response to RA treatment, and neither RA nor BMP-2 affected the expression of L-Sox5 (Fig. 4AGo). We confirmed the induction of SOX6 expression by BMP-2 by means of Western blot analysis with affinity-purified antibodies generated against the HMG domain of murine SOX6 (Fig. 4BGo).



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Figure 4. BMP-2 Induces SOX6 Transcription Factor

A, Semiquantitative RT-PCR analysis was performed with total RNA from C3H10T1/2 cells treated for different periods of time with 3 nM BMP-2, as described in Materials and Methods. Quantification from three separate assays was performed using RT-PCR of actin as normalization and were expressed as fold-induction over control samples at time zero. B, Cell extracts were obtained from C3H10T1/2 treated with 3 nM BMP-2 for 0, 4, 8, 12, and 24 h in differentiation media. HEK 293 T cells were transfected with SOX6 expression vector as a control. Each protein extract (50 µg) was analyzed by Western blot analysis using antitubulin antibody as loading control. Results shown are representative of at least three separate experiments.

 
To investigate whether higher expression of SOX6 correlated with higher SOX6 function, we performed EMSAs using C3H10T1/2 nuclear extracts stimulated with BMP-2 and the Col2a1 enhancer sequence as a probe. We first examined whether the HMG domain of SOX6 could bind on its own to the HMG sites. As shown in Fig. 5AGo, purified fusion glutathione-S-transferase (GST)-SOX6[HMG] protein specifically bound to the 48-bp Col2a1 enhancer. This binding could be efficiently competed with 25-fold molar excess of cold probe. Similar assays with nuclear extracts of C3H10T1/2 cells treated with 3 nM BMP-2 revealed two bands, the intensity of which increased upon BMP-2 treatment and the appearance of a slower migrating band (Fig. 5BGo, arrow). Preincubation with antibodies against SOX6 significantly reduced the slower migrating BMP-2-induced band, whereas it did not affect the two major faster migrating bands. However, antibodies against SOX9 reduced the intensity of all shifted bands (Fig. 5BGo). None of the preimmune serums diminished the BMP-2-induced bands (data not shown). These data suggested that SOX proteins assemble into different complexes bound to the 48-bp Col2a1 enhancer and that SOX6 is probably present in the slower migrating complex.



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Figure 5. Binding of HMG Factors to the Col2a1 Enhancer Is Increased in C3H10T1/2 Cells Treated with BMP-2

A, GST alone or GST-SOX6[HMG] domain fusion proteins were purified and tested to bind DNA. Gel shift was performed using 32P-labeled probes for the Col2a1 enhancer. The binding was also competed with 25-fold excess of cold probe. B, Nuclear extracts were obtained from cells treated with 3 nM BMP-2 for the indicated times in differentiation media. After addition of 32P-labeled probe, extracts (10 µg of protein) were incubated with anti-SOX6 or anti-SOX9 where indicated.

 
SOX6 Cooperates with SOX9 in the Transcriptional Induction of Col2a1 Expression
The need for SOX6 binding in the transcriptional activation of the Col2a1 enhancer was also tested. Overexpression of SOX6, mimicking the BMP-2 effect, induced an approximately 2-fold increase in reporter activity, whereas SOX9 overexpression led to about a 20-fold increase (Fig. 6BGo). Interestingly, whereas addition of BMP-2 further increased SOX9 activation of the reporter, it did not modify that corresponding to SOX6-overexpressing cells. Cotransfection of both SOX6 and SOX9 had a synergistic effect (~140-fold induction) on reporter gene activity, which was not further increased by BMP-2 treatment. Furthermore, transfection of a truncated SOX6 protein lacking the DNA binding domain (Fig. 6AGo), either alone or in combination with SOX9, resulted in a loss of induction of reporter activity by BMP-2, suggesting it behaves, at least partially, as a dominant negative. Similarly, a truncated form of SOX9, lacking both transactivation domains, also reduces, to some extent, BMP-2 inducibility of the Col2a1 enhancer. Taken together, these results suggest the involvement of the SOX6 transcription factor downstream of BMP-2 in the activation of chondrocyte-specific genes.



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Figure 6. Synergistic Activation of Col2a1 Chondrocyte-Specific Enhancer by SOX6 and SOX9

A, Schematic comparison of SOX6 and its deletion mutant. HMG for DNA binding and the two coiled-coil domains are boxed. B, C3H10T1/2 cells were cotransfected with the Col2a1-enhancer reporter and 1 µg of the wild-type or mutant SOX6 and/or SOX9 expression vectors, as indicated. One day later, they were split and incubated with 3 nM BMP-2 for 24 h in differentiation media. Results are shown as mean ± SEM of five independent assays.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BMPs and BMP signal transduction components are expressed in the mesenchyme before the aggregation of prechondrogenic condensations and, later, in the mature chondrocytes (18, 39, 42). In addition, accumulating evidence suggests that BMP family members participate in the induction of both early-phase prechondrogenic condensations and terminal chondrocytic differentiation (3, 19, 20, 21, 39). The pluripotent mesenchymal cell line C3H10T1/2 constitutes a useful model for assaying early events involving the commitment of undifferentiated cells to a particular lineage. BMP-2 has been shown to cause osteoblastic and chondroblastic differentiation of this cell line (24, 25, 43). C3H10T1/2 cells express Bmp2 (data not shown) as well as about 5000 BMP-2 receptors per cell (44), which are able to stimulate SMAD1/5-dependent transcriptional responses (31).

Our studies of dose- and time-dependent appearance of Alcian blue-positive material, and time-dependent activation of type-II collagen gene expression, further verified the chondroinductive action of this cytokine in these mesenchymal cells as a model for early steps of chondrogenic differentiation. BMP-2 effects on Col2a1 expression rely on protein synthesis, because addition of cycloheximide completely abolished its induction. This fact indicates that, although activation of Col2a1 transcription likely depends on the BMP receptor-SMAD signaling pathway (Fig. 2BGo), direct activation of the Col2a1 promoter elements requires synthesis of additional protein/s. We also show that a 48-bp chondrocyte-specific enhancer, within the intron I, is sufficient to induce transcriptional activation of type-II collagen expression by BMP-2. C3H10T1/2 cells express L-SOX5, SOX6, and SOX9, although only mRNA and protein levels of SOX6 are up-regulated as early as 8–12 h after BMP-2 addition, a time that correlates with the induction of type-II collagen expression in these cells. It is noteworthy that, so far, all direct transcriptional targets of either TGF-ß or BMP signaling pathways display induction profiles that begin at 30 min and reach maximal levels at 1–3 h after stimulation (31, 45). Thus, the observed induction profiles of either Col2a1 or Sox6 suggest that they are not direct targets of SMADs or that efficient activation of their promoters by SMADs would require new synthesis of a coactivator.

It has been shown that individual members of the SOX family are required for the initiation of cartilage differentiation during embryogenesis (4). SOX9 was the first transcription factor proved to play a central role in the specification of the chondrocyte lineage (16). It binds to the chondrocyte-specific enhancer, a 48-bp enhancer region of type-II collagen and other genes of the chondrocytic program (7, 8, 11). However, our results showed no changes in SOX9 expression soon after BMP-2 addition. We also used a 6.8-kb Sox9-promoter construct (10) to conclude that there is no BMP-2 transcriptional regulation of Sox9 expression, because RA addition, but not BMP-2 stimulation, up-regulated its transcriptional activity.

Our data suggest that BMP-2 induces expression of an additional factor that is responsible for increased Col2a1 expression. Two other members of the SOX family, L-SOX5 and SOX6, play an essential role in chondrocytic differentiation (46). It has been shown that dedifferentiation and redifferentiation of chondrocytes in culture correlate with the expression levels of the Sox9, -5, and -6 gene products. In addition, whereas Sox5 or Sox6-null mutant mice have relatively mild skeletal phenotypes, Sox5/Sox6 double-knock out mice develop a severe chondrodysplasia characterized by the virtual absence of cartilage (17). Our data show that BMP-2, in parallel with increased SOX6 expression, also increases the Col2a1 enhancer-binding activity of some protein complexes. It is likely that L-SOX5 and SOX6, given its ability to bend DNA, have a function in both organizing an enhancer-protein complex and bringing this complex close to the basal transcriptional machinery (7). Preincubation with antibody against SOX9 suggests that SOX9 was present in the three BMP-2-induced complexes, whereas SOX6 was present in the slower migrating complex. L-SOX5 and SOX6 homo- and heterodimerize through their two coiled-coil domains, not found in SOX9 protein, and preferentially bind, as dimers, pairs of DNA recognition sites; whereas SOX9 binds single DNA sites as a monomer (4, 11). The proximity of several HMG-like sites in the enhancer probably promotes cooperation between the distinct Sox proteins in achieving transcriptional activation. Our functional assays confirmed the synergistic cooperation of SOX6 and SOX9 in increasing the transcriptional activity of a reporter plasmid containing the Col2a1 enhancer. In addition, forced expression of SOX6 makes C3H10T1/2 resistant to further activation of the Col2a1 enhancer by BMP-2. Furthermore, overexpression of a mutated form of Sox6, devoid of its HMG domain, is sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Because this mutant retains the two coiled-coil domains responsible for homo- and heterodimerization, this dominant negative effect could rely on its ability to sequester both endogenous SOX6 and L-SOX5 in protein complexes unable to bind efficiently to DNA.

Altogether, these observations raised the hypothesis that cooperation of distinct SOX transcription factors might mediate cross-talk between BMPs and other diffusible chondrogenic signals. BMP-2-induced signals regulate SOX6 expression and function, whereas other signals would control the expression and function of SOX9 as a chondrogenic transcription factor. Several data have reported that various stimuli that regulate SOX9 function, such as phosphorylation by protein kinase A, enhance SOX9 transactivation (47). In addition, SOX9 expression and function are up-regulated by RA or PTH [(41, 48 and our unpublished data). Furthermore, some evidence suggests that BMP-2 and RA signaling make fundamental coordinate contributions to cartilage development. BMP signaling is required for establishment and maintenance of precartilaginous condensations whereas RA controls their differentiation and maturation (18, 49, 50, 51).

In conclusion, the present results suggest that SOX6 is up-regulated by BMP-2 during chondrogenesis and contribute to our knowledge of the transcriptional mechanisms involved in type-II collagen expression in chondrocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Constructs
PI1P1L (27) and pCDNA3-Sox6 (4) were provided by Dr. de Crombrugghe; a 6.8-kb Sox9-promoter construct was provided by Dr. Koopman. pCDNA3-Sox9 wt and mutants (1–410 and 1–248) were as previously described (11). Mutant Sox6 (1–562) was generated by ApaI digestion of pCDNA3-Sox6. The 886-bp Col2a1 promoter construct, which includes the endogenous transcription initiation site, was performed by digestion of PI1P1L with SmaI and subcloning into PGL2-basic (Promega Corp., Madison, WI). The 1448-bp intron I construct was obtained by digestion of PI1P1L with BamHI and cloning into PGL2-fos, which contains the minimal c-fos promoter. The 465-bp intron I construct was obtained by removing 983 bp from the previous construct by digestion with SmaI. Col2a1 enhancer, wild type and mutant, was generated by complementary oligonucleotide annealing (wild type, 5'-GAT CTG TGA ATC GGG CTC TGT ATG CGC TTG AGA AAA GCC CCA TTC AT-3'; mutant, 5'-GAT CTG TGA ATC GGG CTC ACT ATG CGC TTG AGA TTA GCC CCA AAC AT-3') and cloning into PGL2-fos. To obtain the GST fusion protein used to generate SOX6 antibodies, we cloned a 1061-bp fragment of the Sox6 cDNA (+1596 to +2657) in the pGEX-4T plasmid (Amersham Pharmacia Biotech, Arlington Heights, IL).

Cell Culture and Transient Transfection
C3H10T1/2 fibroblasts derived from mouse embryo connective tissue were cultured in DMEM supplemented with 10% Fetal Bovine Serum (Life Technologies, Inc., Gaithersburg, MD), 0.2 mM glutamine, and antibiotics at 37 C in a humidified atmosphere of 10% CO2. For chondrocytic differentiation, the cultured media were replaced with DMEM containing 1% FBS, 3 nM human BMP-2, 50 µg/ml L-ascorbic acid and 10 mM ß-glycerophosphate (23, 25, 28, 29). Cell transfection was performed using FuGene6 (Roche, Indianapolis, IN). When different combination of plasmids were used, total DNA was kept constant by addition of empty vector.

Measurement of Chondrogenesis
Cultures were washed with PBS, fixed for 10 min with 4% paraformaldehyde, stained with 0.5% Alcian blue in 0.1 N HCl (pH 1.0) overnight, and rinsed with distilled water. Alcian blue-stained cultures were extracted with 200 µl 6 M guanidine-HCl for 2 h at room temperature. OD of the extracted dye was measured at 650 nm (23, 30).

Luciferase Assay
C3H10T1/2 cells were split after transient transfection, cultured in differentiation media, and treated with 3 nM BMP-2 (Genetics Institute, Cambridge, MA) for 24 h. Luciferase activities were quantified using the Luciferase Assay System (Promega Corp.). Luciferase values were standardized to ß-galactosidase as an internal control (Luminescent ß- Galactosidase Detection Kit II, CLONTECH Laboratories, Inc., Palo Alto, CA).

EMSAs. Nuclear Extracts
After different incubation times with 3 nM BMP-2, nuclear extracts were prepared as previously described (31). Complementary oligonucleotides (5'-GAT CTG TGA ATC GGG CTC TGT ATG CGC TTG AGA AAA GCC CCA TTC AT-3') corresponding to the enhancer of the Col2a1 gene were labeled with [{gamma}-32P]dATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (MBI Fermentas, Vilnius, Lithuania).

Ten micrograms of the nuclear protein were diluted to a final volume of 20 µl in a reaction mixture containing 20 mM Tris, pH 7.9, 50 mM NaCl, 10% glycerol, 0.1 mM dithiothreitol, and 1 µg of poly(dC-dG) (7). Labeled probe (0.2 pmol; 4 x 104 cpm) was added after 20 min in the presence or absence of antibodies. After an additional 30-min incubation at 37 C, the reaction mixture was separated in a 5% polyacrylamide gel, dried, and autoradiographed.

Northern Blot Analysis and RT-PCR
RNA isolation and Northern blot analysis were performed as previously described (31, 32) using a 752-bp PCR probe of the mouse Col2a1 gene (nucleotides 3562–4314). Total RNA (2 µg), isolated using the Ultraspec RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX), were reverse transcribed using a Ready-to-Go First Strand Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and oligo-dT as a primer. PCR amplification (15–25 cycles) was carried out using 2 µl of the reverse-transcribed RNA product and specific oligonucleotides: Sox9, forward, 5'-TTA ACA GTT TTA GAA GTC AGT AG-3'; and reverse, 5'-GCA ATG TAT ATT TAT TGT AAA CAA T-3'; L-Sox5, forward, 5'-GGG AAT AGA ATT CCA CCC CTC C-3'; and reverse, 5'-GCA CAC ACG CAC ACA CGG GTC A-3'; Sox6, forward 5'-TTC AGT GGG GGG TGT CAG TGA A-3' and reverse 5'-GTG GGG AAG CAT GAG GAG AAA C-3'; actin, forward, 5'-ATG GAT GAC GAT ATC GCT G-3' and reverse, 5'-ATG AGG TAG TCT GTC AGG T-3'. RT-PCR products were sequenced to confirm their identity.

Antibodies and Western Blot
SOX9 antibodies (Sam-1) were produced in rabbits as previously described (33). To produce SOX6 antibodies, purified chimeric protein GST-SOX6[HMG] (residues 474–828) was injected sc into rabbits to raise anti-SOX6 rabbit polyclonal antibodies. For Western blot, C3H10T1/2 cells were washed twice with cold PBS and lysed with sample buffer 1x [62.5 nM Tris (pH 6.8), 10% glycerol, 1% sodium dodecyl sulfate, 100 nM dithiothreitol]. Cell extracts were resolved on 10% (SOX9) or 7% (SOX6) SDS-PAGE and subjected to Western blot using a 1:1000 dilution of antibody for SOX9 and 1:500 for SOX6.


    ACKNOWLEDGMENTS
 
We thank the Genetics Institute for providing human BMP-2, Dr. de Crombrugghe for the Sox6 cDNA, and Dr. Koopman for the 6.8-kb Sox9-promoter reporter construct. We also thank Esther Adanero for her technical assistance.


    FOOTNOTES
 
This work was supported by grants from the Dirección General de Investigación Científica y Técnica (BMC 2002-00737). R.F.-L. received a predoctoral fellowship from the Generalitat de Catalunya.

Abbreviations: BMP, Bone morphogenetic protein; BMPR, BMP receptor; Col2a1, type-II collagen {alpha}1 chain; FBS, fetal bovine serum; GST, glutathione-S-transferase; HMG, high-mobility group; L-SOX5, alternatively spliced long form of SOX5; RA, retinoic acid; SOX, Sry-type HMG box proteins.

Received for publication July 19, 2002. Accepted for publication March 26, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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