Benoit de Crombrugghe

The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation

We have identified a novel zinc finger-containing transcription factor, called Osterix (Osx), that is specifically expressed in all developing bones. In Osx null mice, no bone formation occurs. In endochondral skeletal elements of Osx null mice, mesenchymal cells, together with osteoclasts and blood vessels, invade the mineralized cartilage matrix. However, the mesenchymal cells do not deposit bone matrix. Similarly, cells in the periosteum and in the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. These cells do, however, express Runx2/Cbfa1, another transcription factor required for bone formation. In contrast, Osx is not expressed in Runx2/Cbfa1 null mice. Thus, Osx acts downstream of Runx2/Cbfa1. Because Osx null preosteoblasts express typical chondrocyte marker genes, we propose that Runx2/Cbfa1-expressing preosteoblasts are still bipotential cells.

Sox9 is required for cartilage formation.

Chondrogenesis results in the formation of cartilages, initial skeletal elements that can serve as templates for endochondral bone formation. Cartilage formation begins with the condensation of mesenchyme cells followed by their differentiation into chondrocytes. Although much is known about the terminal differentiation products that are expressed by chondrocytes, little is known about the factors that specify the chondrocyte lineage. SOX9 is a high-mobility-group (HMG) domain transcription factor that is expressed in chondrocytes and other tissues. In humans, SOX9 haploinsufficiency results in campomelic dysplasia, a lethal skeletal malformation syndrome, and XY sex reversal. During embryogenesis, Sox9 is expressed in all cartilage primordia and cartilages, coincident with the expression of the collagen α1(II) gene (Col2a1; refs 8,11, 12). Sox9 is also expressed in other tissues, including the central nervous and urogenital systems. Sox9 binds to essential sequences in the Col2a1 and collagen α2(XI) gene (Col11a2) chondrocyte-specific enhancers and can activate these enhancers in non-chondrocytic cells. Here, Sox9 is identified as a regulator of the chondrocyte lineage. In mouse chimaeras, Sox9-/- cells are excluded from all cartilages but are present as a juxtaposed mesenchyme that does not express the chondrocyte-specific markers Col2a1, Col9a2, Col11a2 and Agc. This exclusion occurred cell autonomously at the condensing mesenchyme stage of chondrogenesis. Moreover, no cartilage developed in teratomas derived from Sox9-/- embryonic stem (ES) cells. Our results identify Sox9 as the first transcription factor that is essential for chondrocyte differentiation and cartilage formation.

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.

The Transcription Factors L-Sox5 and Sox6 Are Essential for Cartilage Formation

L-Sox5 and Sox6 are highly identical Sry-related transcription factors coexpressed in cartilage. Whereas Sox5 and Sox6 single null mice are born with mild skeletal abnormalities, Sox5; Sox6 double null fetuses die with a severe, generalized chondrodysplasia. In these double mutants, chondroblasts poorly differentiate. They express the genes for all essential cartilage extracellular matrix components at low or undetectable levels and initiate proliferation after a long delay. All cartilages are thus extracellular matrix deficient and remain rudimentary. While chondroblasts in the center of cartilages ultimately activate prehypertrophic chondrocyte markers, epiphyseal chondroblasts ectopically activate hypertrophic chondrocyte markers. Thick intramembranous bone collars develop, but the formation of cartilage growth plates and endochondral bones is disrupted. L-Sox5 and Sox6 are thus redundant, potent enhancers of chondroblast functions, thereby essential for endochondral skeleton formation.

Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9+/− mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9. Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.

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.

Regulatory mechanisms in the pathways of cartilage and bone formation.

Three transcription factors of the Sox family have essential roles in different steps of the chondrocyte differentiation pathway. Because the transcription factor Cbfa1, which is needed for osteoblast differentiation, also stimulates hypertrophic chondrocyte maturation, it links the chondrocyte and osteoblast differentiation pathways in endochondral bone formation. Signaling molecules, including Indian Hedgehog, PTHrP and FGFs, also establish essential links either between these pathways, between steps in these pathways or between signaling molecules and transcription factors, so that a more comprehensive view of endochondral bone formation is emerging.

NFAT and Osterix cooperatively regulate bone formation

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

Role of the CCAAT-binding protein CBF/NF-Y in transcription

The CCAAT motif is one of the common promoter elements present in the proximal promoter of numerous mammalian genes transcribed by RNA polymerase II. CBF (also called NF-Y and CP1) consists of three different subunits and interacts specifically with the CCAAT motif. In each CBF subunit, the segment needed for formation of the CBF-DNA complex is conserved from yeast to human and, interestingly, the conserved segment of two CBF subunits, CBF-A and CBF-C, are homologous to the histone-fold motif of eukaryotic histones and archaebacterial histone-like protein HMf-2. The histone fold motifs of CBF-A and CBF-C interact with each other to form a heterodimer that associates with CBF-B to form a heterotrimeric CBF molecule, which then binds to DNA.