Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PP) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PP decreased osteoclast formation and resorptive activity by ≤50 %. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PP present for the entire (0-21 days) or latter stages of culture (7-21/14-21 days) decreased bone mineralisation by ≤95 %. However, PP present for the differentiation phase only (0-7/0-14 days) increased bone formation (≤70 %). Prolonged treatment with PP resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PP (≤3-fold). PP levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PP reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35 %) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PP. ATP release from osteoclasts and osteoblasts was decreased ≤60 % by PP and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gα subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PP. The actions of PP on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PP, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PP to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PP levels and respond accordingly. Our data also raise the possibility that some actions of PP on bone cells could be mediated by a Gα-linked GPCR.