Véronique Lefebvre

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.

A new long form of Sox5 (L‐Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene

Transcripts for a new form of Sox5, called L‐Sox5, and Sox6 are coexpressed with Sox9 in all chondrogenic sites of mouse embryos. A coiled‐coil domain located in the N‐terminal part of L‐Sox5, and absent in Sox5, showed >90% identity with a similar domain in Sox6 and mediated homodimerization and heterodimerization with Sox6. Dimerization of L‐Sox5/Sox6 greatly increased efficiency of binding of the two Sox proteins to DNA containing adjacent HMG sites. L‐Sox5, Sox6 and Sox9 cooperatively activated expression of the chondrocyte differentiation marker Col2a1 in 10T1/2 and MC615 cells. A 48 bp chondrocyte‐specific enhancer in this gene, which contains several HMG‐like sites that are necessary for enhancer activity, bound the three Sox proteins and was cooperatively activated by the three Sox proteins in non‐chondrogenic cells. Our data suggest that L‐Sox5/Sox6 and Sox9, which belong to two different classes of Sox transcription factors, cooperate with each other in expression of Col2a1 and possibly other genes of the chondrocytic program.

Transcriptional mechanisms of chondrocyte differentiation

With the goal of identifying master transcription factors that control the genetic program of differentiation of mesenchymal cells into chondrocytes, we first delineated a 48-bp chondrocyte-specific enhancer element in the gene for proalpha1(II) collagen (Col2a1), an early and abundant marker of chondrocytes. Our experiments have demonstrated that the HMG-box-containing transcription factor, Sox9 which binds and activates this enhancer element, is required for chondrocyte differentiation and for expression of a series of chondrocyte-specific marker genes including Col2a1, Col9a2, Col11a2 and Aggrecan. In the absence of Sox9 the block in differentiation occurs at the stage of mesenchymal condensation, suggesting the hypothesis that Sox9 might also control expression of cell surface proteins needed for mesenchymal condensation. Since Sox9 also contains a potent transcription activation domain, it is a typical transcription factor. Two other members of the Sox family, L-Sox5 and Sox6, also bind to the 48-bp Col2a1 enhancer and together with Sox9 activate this enhancer as well as the endogenous Col2a1 and aggrecan genes. L-Sox5 and Sox6 have a high degree of sequence identity to each other and are likely to have redundant functions. Except for the HMG-box, L-Sox5 and Sox6 have no similarity to Sox9 and, hence, are likely to have a complementary function to that of Sox9. Our experiments suggest the hypothesis that, like Sox9, Sox5 and Sox6 might also be needed for chondrocyte differentiation. Other experiments, have provided evidence that the Sox9 polypeptide and the Sox9 gene are targets of signaling molecules that are known to control discrete steps of chondrogenesis in the growth plate of endochondral bones. Protein kinase A (PKA) phosphorylation of Sox9 increases its DNA binding and transcriptional activity. Since PKA-phosphorylated-Sox9 is found in the prehypertrophic zone of the growth plate, the same location where the gene for the receptor of the parathyroid hormone-related peptide (PTHrP) is expressed and since PTHrP signaling is mediated by cyclic AMP, we have hypothesized that Sox9 is a target for PTHrP signaling. Other experiments have also shown that fibroblast growth factors (FGFs) increase the expression of Sox9 in chondrocytes in culture and that this activation is mediated by the mitogen-activated protein kinase pathway. These results favor the hypothesis that in achondroplasia, a disease caused by activating mutations in FGF receptor 3, there might also be an abnormally high Sox9 expression.

Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis

To assess the role of the transcription factor Sox9 in cartilage formation we have compared the expression pattern of Sox9 and Col2a1 at various stages of mouse embryonic development. Expression of Col2a1 colocalized with expression of Sox9 in all chondroprogenitor cells. In the sclerotomal compartment of somites the onset of Sox9 expression preceded that of Col2a1. A perfect correlation was also seen between high levels of Sox9 expression and high levels of Col2a1 expression in chondrocytic cells. However, no Sox9 expression was detected in hypertrophic chondrocytes; only low levels of Col2a1 RNA were found in the upper hypertrophic zone. Coexpression of Sox9 and Col2a1 was also seen in the notochord. At E11.5 Sox9 expression in the brain and spinal neural tube was more widespread than that of Col2a1 although at E14.5 Sox9 and Col2a1 transcripts were colocalized in discrete areas of the brain. Distinct differences between Sox9 and Col2a1 expression were observed in the otic vesicle at E11.5. At E8.5, expression of Sox9 but not of Col2a1 was seen in the dorsal tips of the neural folds and after neural tube closure also in presumptive crest cells emigrating from the dorsal pole of the neural tube. No Col2a1 expression was detected in gonadal ridges in which high levels of Sox9 expression were detected. Together with our previous results showing that the chondrocyte‐specific enhancer element of the Col2a1 gene is a direct target for Sox9, these results suggest that Sox9 plays a major role in expression of Col2a1. The correlation between high expression levels of Sox9 and high expression levels of Col2a1 in chondrocytes suggests the hypothesis that high levels of Sox9 are needed for full expression of the chondrocyte phenotype; lower levels of Sox9 such as in neuronal tissues which are also associated with lower expression levels of Col2a1 would be compatible with other cell specifications. Dev. Dyn. 209:377–386, 1997.

Chondrocyte-specific enhancer elements in the Col11a2 gene resemble the Col2a1 tissue-specific enhancer

Type XI collagen and type II collagen are coexpressed in all cartilage, and both are essential for normal cartilage differentiation and skeletal morphogenesis. This laboratory has recently identified a 48-base pair (bp) enhancer element in the type II collagen gene Col2a1 that contains several HMG-type protein-binding sites and that can direct chondrocyte-specific expression in transient transfection and in transgenic mice. The present study has identified two short chondrocyte-specific enhancer elements within a region in the 5′ portion of the type XI collagen geneCol11a2 that has previously been shown to influence chondrocyte-specific expression in transgenic mice. TheseCol11a2 enhancer elements, like the Col2a1enhancer, contain several sites with homology to the high mobility group (HMG) protein-binding consensus sequence. In electrophoretic mobility shift assays, the Col11a2 elements formed a DNA-protein complex that was dependent on the presence of the HMG-like sites. It had the same mobility as the complex formed with theCol2a1 48-bp enhancer and appeared to contain the same or similar proteins, including SOX9. The Col11a2 elements directed gene expression in transient transfections of chondrocytes but not fibroblasts, and their activity was abolished by mutation of the HMG-like sites. Ectopically expressed SOX9 activated these enhancers in non-chondrocytic cells, as it also activates the Col2a1enhancer. Finally, the Col11a2 enhancer elements both directed transgene expression to cartilage in developing mouse embryos. Overall, our results indicate that the two Col11a2chondrocyte-specific enhancer elements share many similarities with theCol2a1 48-bp enhancer. These similarities suggest the existence of a genetic program designed to coordinately regulate the expression of these and perhaps other genes involved in the chondrocyte differentiation pathway.

Phosphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer

ABSTRACT Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Cα) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Cα in vivo. In COS-7 cells cotransfected with PKA-Cα and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S64 and S211) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1chondrocyte-specific enhancer; similarly, cotransfection of PKA-Cα increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Cα activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.

L-Sox5 and Sox6 Drive Expression of the Aggrecan Gene in Cartilage by Securing Binding of Sox9 to a Far-Upstream Enhancer

ABSTRACT The Sry-related high-mobility-group box transcription factor Sox9 recruits the redundant L-Sox5 and Sox6 proteins to effect chondrogenesis, but the mode of action of the trio remains unclear. We identify here a highly conserved 359-bp sequence 10 kb upstream of the Agc1 gene for aggrecan, a most essential cartilage proteoglycan and key marker of chondrocyte differentiation. This sequence directs expression of a minimal promoter in both embryonic and adult cartilage in transgenic mice, in a manner that matches Agc1 expression. The chondrogenic trio is required and sufficient to mediate the activity of this enhancer. It acts directly, Sox9 binding to a critical cis-acting element and L-Sox5/Sox6 binding to three additional elements, which are cooperatively needed. Upon binding to their specific sites, L-Sox5/Sox6 increases the efficiency of Sox9 binding to its own recognition site and thereby robustly potentiates the ability of Sox9 to activate the enhancer. L-Sox5/Sox6 similarly secures Sox9 binding to Col2a1 (encoding collagen-2) and other cartilage-specific enhancers. This study thus uncovers critical cis-acting elements and transcription factors driving Agc1 expression in cartilage and increases understanding of the mode of action of the chondrogenic Sox trio.

Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo.

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-α1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1–3) that were essential for its chondrocyte-specific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bpCol2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as largerCol2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocyte-specific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.

Sox9 Directs Hypertrophic Maturation and Blocks Osteoblast Differentiation of Growth Plate Chondrocytes

The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and β-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1. These findings thus reveal that Sox9 remains a central determinant of the lineage fate and multistep differentiation program of growth plate chondrocytes and thereby illuminate our understanding of key molecular mechanisms underlying skeletogenesis.

Characterization of primary cultures of chondrocytes from type II collagen/β-galactosidase transgenic mice

Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.