ECTS2013 Oral Communications Mineralisation and energy metabolism (6 abstracts)
1University College London, London, UK; 2The Roslin Institute and Royal (Dick) School of Veterinary Studies, Edinburgh, UK; 3Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphate and the mineralisation inhibitor, pyrophosphate (PPi). This investigation examined the role of NPP1 in bone and soft tissue mineralisation using a mouse model lacking NPP1(Enpp1−/−). At physiological pH 7.35, cultured Enpp1−/− calvarial osteoblasts displayed ≥70% increase in bone mineralisation compared to wild types. Acidosis (pH 6.9), a well-known mineralisation inhibitor, completely abolished bone mineralisation in wild-type cells but only decreased mineralisation ~30% in Enpp1−/− osteoblasts. Differentiating and mature Enpp1−/− osteoblasts showed ≥70% reduction in constitutive release of ATP, a key NPP1 substrate; this was accompanied by a ~20% increase in total intracellular ATP levels. Fluid flow increased ATP release less than eightfold in wild-type osteoblasts; this response was impaired by ~60% in Enpp1−/− cells. Previous studies demonstrated significant changes in the bone structure of Enpp1−/− mice. Here, we used microCT (0. μm) to examine cortical bone changes in detail. Cortical bone volume was increased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30 and 60% at 15 and 22 weeks respectively. This was accompanied by ~13% decrease in pore diameter and ≤38% increase in inter-pore distance. However, cortical thickness was ≥33% lower in 15 and 22 weeks Enpp1−/− mice; thus, their bones were thinner but denser and less porous. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤50%. These animals also display ectopic joint calcification; in the knee this was accompanied by 30, 15 and 15% reductions in epiphyseal trabecular bone volume, thickness and number, respectively; tibial subchondral bone was reduced ≤17%. MicroCT and histological analysis of soft tissues revealed for the first time calcification of the whisker follicles, ear pinna and lungs of Enpp1−/− mice. Together, these data highlight the key role of NPP1 in regulating calcification of both skeletal and soft tissues.