Bone Abstracts

Formation and growth of long bones occur through the process of endochondral ossification, which depends on proliferation and hypertrophy of chondrocytes in growth cartilage. In a subtractive hybridization study of equine cartilage, we recently identified a number of genes, the roles of which in growth cartilage have not been characterized. A subset of these genes was found to be differentially expressed between the zones of equine growth cartilage. The genes encoding ATPase H⁺ transporting lysosomal d2 subunit (Atp6v0d2), DEAD box polypeptide-5 (Ddx5), triose phosphate isomerase-1 (Tpi1) and thymosin β 4 (Tmsb4) were more highly expressed in the hypertrophic zone than in the reserve and proliferation zones of equine growth cartilage, while Foxa3 was more highly expressed in the reserve zone than in the hypertrophic zone. We examined the expression of these genes during maturation of ATDC5 cells (a murine teratocarcinoma-derived chondrocyte-like cell line), with the aim of using this cell line to identify the roles of the genes of interest in chondrocyte hypertrophy. ATDC5 cells were cultured to confluence, then changed to medium containing insulin-transferrin-sodium selenite, triiodothyronine and ascorbate-2-phosphate to induce expression of hypertrophy-associated genes. The expression of the genes of interest was examined by quantitative PCR. After 4 days of treatment, expression of the hypertrophy associated genes Col10, Runx2, and Mmp13 was significantly higher than before treatment. At this time point, of the genes up-regulated with hypertrophy in vivo, expression of Atp6v0d2, Ddx5 and Tpi1 was also found to be significantly up-regulated in ATDC5 cells, but expression of Tmsb4 was down-regulated. Expression of Foxa3 was down-regulated under these conditions, in keeping with its suppression during hypertrophy in vivo. This study identifies novel hypertrophy-associated genes and indicates that ATDC5 cells will provide an appropriate vehicle for manipulation of gene expression to investigate the functions of these genes in chondrocyte hypertroph