Previous work has shown that
acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of
phosphate (Pi ) to
pyrophosphate (PPi ) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi , a potent inhibitor of mineralisation, are generated from extracellular
nucleotides by the actions of ecto-
nucleotidases. This study investigated the expression and activity of ecto-
nucleotidases by osteoblasts under normal and
acid conditions. We found that osteoblasts express
mRNA for a number of ecto-
nucleotidases including NTPdase 1-6 (ecto-
nucleoside triphosphate diphosphohydrolase) and NPP1-3 (ecto-
nucleotide pyrophosphatase/
phosphodiesterase). The rank order of
mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 >
NTPdase 6 > NTPdase 5 >
alkaline phosphatase > ecto-5-nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2.
Acidosis (pH 6.9) upregulated NPP1
mRNA (2.8-fold) and
protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto-
nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in
acid conditions (P < 0.001). Release of
ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by
acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5-fold, P < 0.001) and was partially resistant to the inhibitory effect of
acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to
acid conditions.