Abstract
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. J. Cell. Physiol. 230: 3049–3056, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
Highlights
The negative actions of acidosis on the skeleton have long been known (Jaffe et al, 1932)
The present investigation provides the first evidence that the pyrophosphate-generating enzyme, NPP1, mediates the effects of acid on osteoblasts and bone mineralisation
This study demonstrated that mRNAs for six members of the ecto nucleoside triphosphate diphosphohydrolase (NTPdase) family (NTPdases 1–6) are expressed by primary rat osteoblasts
Summary
The negative actions of acidosis on the skeleton have long been known (Jaffe et al, 1932). Acidosis (pH 6.9) prevents the formation of mineralised bone nodules in primary cultures of osteoblasts Part of this inhibition can be attributed to physico-chemical dissolution of hydroxyapatite (Brandao-Burch et al, 2005); expression and activity of tissue non-specific alkaline phosphatase (TNAP) by osteoblasts is strikingly decreased in acid conditions, suggesting an additional cell-mediated component (Krieger et al, 1992; Brandao-Burch et al, 2005). Pyrophosphate (PPi) is a ubiquitous, potent inhibitor of mineralisation (Fleisch and Bisaz, 1962), and mineralisation in the bone microenvironment depends on the ratio of Pi to PPi concentrations (Felix and Fleisch, 1976; Kornak, 2011) Both Pi and PPi can be generated from extracellular nucleotide triphosphates (NTPs) such as ATP and UTP by the actions of.
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