Bone Abstracts

In a recent unbiased transcriptomic study of genes associated with equine osteochondrosis, we identified several novel cartilage genes. The current study was undertaken to determine whether these genes are regulated during chondrocyte hypertrophy, and to identify novel hypertrophy-associated genes for further study in vitro. Gene expression was investigated by quantitative PCR (qPCR) in different zones of growth cartilage microdissected from equine foetal metatarsal bones (n=22). Nine novel cartilage genes (ATP6V0D2, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1 and WSB2) were found to be more highly expressed in the hypertrophic zone than in reserve or proliferative zones. Regulation of expression of three of these genes by conditions inducing expression of hypertrophy-associated genes (ITS/AA/T3) was investigated by qPCR in the mouse ATDC5 chondrocyte cell line; Atp6v0d2, Ddx5 and Tpi1 were all upregulated under these conditions. Atp6v0d2, the gene encoding the vacuolar H⁺ ATPase V₀ subunit d₂, was selected for further study. Immunochemical staining for ATP6V0D2 protein in equine foetal growth cartilage was more intense in the hypertrophic than reserve or proliferative zones; in cultures of ATDC5 cells, staining was more intense in ITS/AA/T3-treated than control cells. Knockdown of Atp6v0d₂ gene and protein expression in ATDC5 cells was achieved using Atp6v0d₂-targetting siRNA, as confirmed by qPCR (>50% knockdown, P<0.05, in both ITS/AA/T3-treated and control cells) and by immunocytochemistry. In control cells but not ITS/AA/T3-treated cells, the siRNA consistently suppressed expression of Col2a1 (60%, P<0.05). Hypertrophy-associated genes were not differentially expressed between siRNA- and sham-transfected cultures. Knockdown of Atp6v0d2 expression in ATDC5 cells cultured under hypertrophy-inducing conditions caused a decrease (18%, P<0.0001) in nuclear area as well as increases in the number of cells (28%, P<0.05) and percentage containing mitotic default (59%, P<0.0001). These observations suggest that ATP6V0D2 expression (and presumably vacuolar H⁺ATPase activity) supports differentiation and suppresses proliferation of chondrocytes.