SUMMARYSOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

Supplementary material available online at

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.Elucidation of molecular mechanisms underlying chondrocyte differentiation is not only important for our understanding of skeletal development, but also of particular interest for our knowledge on the behavior of chondrocytes following articular cartilage damage during cartilage repair and treatment of degenerative cartilage diseases. Initial steps of chondrogenesis, i.e. the formation of a cartilage blastema from limb bud mesenchymal cells, include cell condensation and onset of chondrocyte differentiation marked by the expression of cartilage-specific matrix proteins such as aggrecan, collagen II, IX, and XI and others (1, 2). These events are regulated by the orchestrated action of several growth factors including BMPs, Wnt factors, FGFs, and the transcription factors Sox5,6, and 9 (3,4). Further steps of chondrocyte growth, maturation, and replacement by bone in the growth plate of long bones, ribs, and vertebrae during endochondral ossification can be defined by the stepwise onset or decline of differentially expressed genes: collagen II and Sox9 for resting and proliferating, FGFR3 for proliferating and prehypertrophic, Ihh and PTHrP receptor for prehypertrophic, collagen X for hypertrophic, and Runx2, osteocalcin, and MMP13 for late hypertrophic chondrocytes (...

Articular cartilage is an avascular, bradytrophic tissue in which the chondrocytes physiologically maintain their unique differentiation status throughout life. In contrast to chondrocytes of the fetal growth plate, articular chondrocytes are postmitotic cells that do not undergo terminal differentiation, and their extracellular matrix does not calcify above the tidemark. Bone marrow-stimulating techniques, such as microfracturing of the subchondral bone plate, are simple, minimally invasive, and cost-effective cartilage repair approaches that are frequently applied in clinical settings. Unfortunately, the ingrowing osteochondral progenitor cells often fail to undergo complete chondrogenic differentiation, which leads to the formation of inferior fibrocar-

No abstract