The vacuolar H ATPase V subunit d is associated with chondrocyte hypertrophy and supports chondrocyte differentiation.

Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (, , , , , , , , , , and ) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas and were expressed at lower levels in the hypertrophic zone than in the reserve zone. , which encodes vacuolar H ATPase (V-ATPase) V subunit d (ATP6V0D2), was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including and (differentiation medium). In ATDC5 cells cultured in control medium, knockdown of or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not knockdown, prevented the upregulation of , and by differentiation medium, while knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.