Ank encodes a transmembrane protein that is involved in pyrophosphate (PPi) transport and mutations in the Ank gene have been associated with pathological mineralization in cartilage and bone. To understand how Ank works in normal skeletal development it is also important to know which cells within the developing skeleton express Ank. To this end, we examined the expression pattern of Ank mRNA during mouse embryonic development as well as in mouse hind limb joints with emphasis on the period when articular cartilage forms. Since it was previously shown that TGF-beta regulates PPi transport in cells in culture, we also tested the hypothesis that TGF-beta regulates Ank expression.
The localization of Ank mRNA was determined by radioactive in situ hybridization in E15.5 and E17.5 mouse embryos as well as in 1 and 3 week post-natal mice. Ank expression was compared to that of other cartilage markers. In situ hybridization and semi-quantitative RT-PCR were used to determine the effects of TGF-beta on Ank expression in metatarsal organ cultures.
Ank expression was detected at high levels at sites of both endochondral and intramembranous bone development. In endochondral bones, expression was detected in a subset of hypertrophic cells at ossification centers. Expression was also detected in osteogenic/chondrogenic cells of the perichondrium/periosteum lining the metaphysis, an area associated with the formation and extension of the bone collar. High levels of expression were also detected in non-mineralized tissues of the skeletal system including tendons and the superficial layer of the articular cartilage. Treatment with TGF-beta resulted in an approximately four-fold induction of Ank mRNA in prehypertrophic chondrocytes and perichondrium of metatarsal cultures.
The expression pattern of Ank suggests an important role both in inhibiting and regulating mineralization in the developing skeletal system. In addition, TGF-beta1 is able to mediate Ank mRNA expression in chondrocytes suggesting a possible role for TGF-beta and Ank in the regulation of normal mineralization.