Bone Abstracts

Extracellular nucleotides stimulate both the formation and resorptive activity of osteoclasts. Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse extracellular nucleotide triphosphates to their corresponding monophosphate and pyrophosphate (PP_i). We investigated if osteoclasts express functional NPPs and whether Enpp1 gene deletion influenced osteoclast formation and activity. Osteoclasts were formed from the bone marrow of 8 and 15 week old knockout (Enpp1^−/−) or wild-type (Enpp1^+/+) mice. RT-PCR demonstrated expression of Enpp1, Ennp3, Entpd1, Entpd3 and the PP_i transport molecule, Ank in early and mature osteoclasts. Enpp1 expression was increased in mature, resorbing osteoclasts relative to precursor cells, whilst Entpd3 expression was decreased. Significant total NPP activity indicated the expression of functional enzymes in wild-type osteoclasts. Cultured osteoclasts were activated to resorb by medium acidification to pH6.9. Enpp1 mRNA expression was upregulated in mature, resorbing osteoclasts compared to mature, inactive osteoclasts; total NPP activity was also increased 2-fold in acidified cells. Culture of cells from 8 and 15-week old Enpp1^−/− mice indicated that both osteoclast formation and resorptive function were unaffected by gene deletion. Analysis of the cortical bone from 8, 15 and 22-week Enpp1^−/− mice by microCT (0.9 μm) showed that whilst the periosteal diameter was unchanged, the endosteal diameter was increased ~20% at 15 and 22-weeks, suggesting increased endosteal osteoclast activity in vivo, particularly in older animals. ATP, a key substrate for NPPs, is constitutively released from osteoclasts. We found that baseline ATP release was unaffected in Enpp1^−/− osteoclasts; however, ATP release in response to fluid flow was 2-fold lower in Enpp1^−/− osteoclasts. The rate of ATP breakdown was unaffected in Enpp1^−/− osteoclasts. However, Enpp1^−/− osteoclasts displayed increased expression of other ecto-nucleotidases, such as Entpd1, Entpd3 and Enpp3 as well as ANK. These results suggest the possibility that NPPs may play a role in the regulation of osteoclast function.