Nucleotides as regulators of bone cell function and mineralisation Mark Omar Raimes Hajjawi A thesis submitted for the fulfilment of the degree of Doctor of Philosophy Department of Cell and Developmental Biology University College London 2014 Declaration of authorship Declaration of authorship I, Mark Omar Raimes Hajjawi, declare that this thesis titled ‘Nucleotides as regulators of bone cell function and mineralisation’ and the work presented in it are my own. I confirm that: This work was done wholly while in candidature for a research degree at this university. No part of this thesis has previously been submitted for a degree or any other qualification at this university or any other institution. Where I have consulted the published work of others, this is always clearly attributed. Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work. I have acknowledged all main sources of help. Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself. Signed: _________________________________________________ Date: ___________________________________________________ 2 Abstract 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. 3 Acknowledgments Acknowledgments I would like to thank Prof Tim Arnett and Dr Isabel Orriss for giving me the opportunity, and allowing me the privilege, to undertake my PhD under their supervision. Both have given me more that I could ever repay and share a part in any successes I have from this day forwards. I would also like to thank Prof Alan Boyde from QMUL for performing some of the electron microscopy in this thesis and offering sage words of advice, and Dr Chris Scotton from Exeter University for assisting me in the practical aspects of histology. Finally, I would like to thank my wife Rachel. 4 Contents Contents Declaration of authorship.......................................................................... 2 Abstract........................................................................................................... 3 Acknowledgments........................................................................................ 4 Contents.......................................................................................................... 5 Figures and tables......................................................................................... 10 Chapter 1......................................................................................................... 14 Introduction............................................................................................... 14 Development of the skeleton........................................................... 14 Endochondral ossification.................................................................... 14 Intramembranous ossification............................................................. 15 Bone cells............................................................................................... 15 Osteoblasts........................................................................................... 15 Osteocytes............................................................................................ 18 Osteoclasts........................................................................................... 22 Bone matrix........................................................................................... 27 The mineralisation of bone matrix…................................................ 28 Ecto-nucleotide pyrophosphatase / phosphodiesterase1.................... 28 Alkaline phosphatase........................................................................... 29 Phospho1………………………………………………………………............................. 30 Phosphate / pyrophosphate ratio........................................................ 31 ANK transport protein.......................................................................... 33 Factors that regulate bone cells and bone formation................. 33 The vascular system………………………………………………………………………….. 33 Contents Hydrogen ions...................................................................................... 34 Glucocorticoids..................................................................................... 36 Endocrine and paracrine regulation regulators of bone cells... 36 Bone morphogenetic protein signalling............................................... 36 Vitamin D.............................................................................................. 37 Parathyroid hormone (PTH)................................................................. 38 WNT signalling...................................................................................... 39 Extracellular nucleotide signalling............................................ 43 Purinoceptor signalling and osteoblasts.............................................. 44 Purinoceptor signalling and osteoclasts............................................... 46 Ecto-nucleotidases…................................................................. 48 Ecto-nucleoside triphosphate diphosphohydrolase............................. 48 Ecto-nucleotide pyrophosphatase/phosphodiesterase....................... 49 Ecto-5’nucleotidase.............................................................................. 54 Alkaline phosphatase........................................................................... 56 Nucleoside mono/di/tri-phosphate inter-conversion................. 57 Adenosine kinase................................................................................. 58 Adenylate kinase.................................................................................. 58 Nucleoside diphosphate kinase............................................................ 59 Adenosine triphosphate synthase........................................................ 60 Adenosine and adenosine receptors............................................... 61 Adenosine deaminase.......................................................................... 64 Adenine and adenine receptors........................................................... 65 Purine salvage pathway....................................................................... 66 Aims......................................................................................................... 68 Contents Chapter 2......................................................................................................... 69 Materials and methods.......................................................................... 69 Reagents................................................................................................