Hajjawi, MOR; (2014) Nucleotides as regulators of bone cell function and mineralisation. Doctoral thesis , UCL (University College London).

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Abstract

Most cells, including bone cells, release ATP into the extracellular environment. A considerable body of previous work has shown that ATP, acting through the P2 receptors, inhibits bone formation by osteoblasts and increases bone resorption by osteoclasts. This work focuses on the action of two key breakdown products of ATP, pyrophosphate and adenosine on bone cell function. Pyrophosphate, a ubiquitous physicochemical inhibitor of mineralisation, is formed from extracellular ATP by the action of ecto-nucleotide pyrophosphatase phosphodiesterases (NPPs); in bone these enzymes act in opposition to alkaline phosphatase. Adenosine, which can be generated in a number of ways from ATP, has been previously reported to stimulate both osteoblast and osteoclast function. However, using in vitro cultures, I found that it had little or no effect on the differentiation and bone forming capacity of rat osteoblasts, nor on the formation and resorptive function of mouse osteoclasts. I investigated the possibility that osteocytes, which form an interconnected cellular network within bone, might regulate mineralisation via NPPs. I found that cultured, primary osteocyte-like cells derived from mouse bone expressed Enpp1 mRNA. Osteocyte lacunae in the femora of Enpp1-/- mice imaged by scanning electron microscopy were found to be reduced in area by about 35%; indirect estimates of lacunar size using microCT imaging were in agreement. These results are consistent with the notion that ATP-derived pyrophosphate is important for maintenance of osteocyte lacunae size. Enpp1-/- mouse bones (humerus) were found to have reduced cortical bone diameter, reduced cortical porosity and an increased endosteal diameter compared to wild types, suggesting that the knockout phenotype also involves increased bone resorption and decreased bone formation. Histology and microCT of Enpp1-/- mice confirmed inappropriate joint mineralisation and showed that cartilage in the trachea and ear pinna was also mineralised, as were whisker sheaths. Osteoblasts, osteoclasts and osteocytes cultured in vitro from Enpp1-/- mice were found to release less ATP compared to cells from Enpp1+/+ mice in static conditions and after fluid flow stimulation. Enpp1-/- osteoblasts and osteoclasts also contained higher levels of intracellular ATP. Enpp1-/- osteoblasts showed increased bone production in vitro compared to Enpp1+/+; no effects of Enpp1 knockout on the formation or resorptive activity of osteoclasts were noted. Sclerostin, an osteocyte-derived inhibitor of WNT signalling and bone formation, was found to increase Enpp1 mRNA expression and NPP activity of osteoblasts, without affecting ALP in vitro. These results emphasise the importance of ATP and its breakdown product pyrophosphate in regulating mineralisation.

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