University of Bath PHD The role of the reactive oxygen species-generating enzyme, xanthine oxidoreductase, in cytokine- and hormone-induced bone resorption Kanczler, Janos Michael Award date: 1999 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 THE ROLE OF THE REACTIVE OXYGEN SPECIES- GENERATING ENZYME, XANTHINE OXIDOREDUCTASE, IN CYTOKINE- AND HORMONE-INDUCED BONE RESORPTION. Submitted by: Janos Michael Kanczler For the degree of PhD of the University of Bath 1999 Copyright: Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from this thesis and no information derived from it may be published without prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purpose of consultation. Signed: UMI Number: U536556 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U536556 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 UNIVERSITY OF BATH LIBRARY H BB»ncununnwt(T.ii' *30 1 7 MAY 2G33 Acknowledgements. During my time in studying for my thesis there are a number of people I would like to thank who have given their valuable time and effort to provide help, ideas, inspiration and assist in the studies of this thesis and its preparation. Dr. Cliff Stevens, Dr. Tulin Bodamyali and Professor David Blake for their informative discussion, constant support and advice on the preparation of this thesis. Mr. Tim Millar for advice on experimental techniques, ideas and preparation of this thesis over the last 3 years. Ms. Hazel Finney for the technical help provided in assaying my calcium samples on the Monarch 2000 analyser. Dr. Ian James for analysing my calcium samples from the resorption experiments for pyridinium crosslinks. Mrs. Leslie Moore from providing and looking after the MF-1 mouse pups. Dr Bishay (Royal United Hospital, Bath) & Dr. Gareth Scott (Royal London Hospital, London) for kindly providing human bone tissue samples. The Arthritis & Rheumatism Council for the funding of this study. Finally, I would like to thank Fiona Twomey for her encouragement, advice, support and great patience while I carried out my studies. Dedication. To my Mother, Father, Sister and Fiona. Abstract. Bone resorption underlies the pathology of many disabling diseases, ranging from the inflammatory condition, rheumatoid arthritis to the hormonally controlled, metabolic disorder, osteoporosis. It is known that various agents, including cytokines, prostaglandins, parathyroid hormone (PTH), la ,25 dihydroxy vitamin D 3 and free radicals influence the bone resorptive process. Pro-inflammatory cytokines such as TNFa and IL-lp have been ascribed a pivotal role in rheumatoid arthritis (RA). Bone resorptive mechanisms employ complex cellular signalling pathways between osteoblasts and osteoclasts that involve reactive oxygen (ROS) and nitrogen species (RNS). Recent studies have demonstrated that one of these species, hydrogen peroxide (H 2O2) can increase osteoclast differentiation and bone resorption. One possible source of these ROS is xanthine oxidase. This thesis explores the role of XO- derived ROS directly and as mediators of cytokine- and hormone-induced bone resorption in vitro. The results of this thesis demonstrated that calvarial osteoblasts contain XO, which can be upregulated by TNFa and IL -ip. Consequently, induction of XO by these cytokines led to the generation of hydrogen peroxide. TNFa and IL-lp caused a dose-related increase in resorption of mouse calvariae in culture, which was inhibited by lOU/ml catalase. The competitive inhibitor of XO, allopurinol, also caused a dose related (0.05-50p,M) inhibition of TNF-a (O.l-lOpM) and IL-lp-induced resorption respectively. Additionally, IFN-y inhibition of bone resorption could be reversed by the addition of superoxide dismutase to the cultures. PTH- and la ,25 (OH) 2 D3-induced bone resorption could be inhibited by catalase (lOU/ml) but was unaffected by allopurinol, implying that a different mediator, other than XO, is required for hormone-induced bone resorption. In conclusion, this thesis demonstrates that modulation of the redox balance in the bone microenvironment can have profound effects on the bone resorbing process. Our results show that XO may play a pivotal role in affecting this redox balance and if manipulated appropriately, could be used to have a therapeutic benefit in inflammatory bone disorders such as RA. Abbreviations and acronyms. ABC. Avidin biotin complex. la,25-(OH)2D3. l a , 25(OH)2 vitamin D 3 (calcitriol). ALP. Alkaline phosphatase. AMPS. Ammonium persulphate. AP-1. Activator protein-1. BMP. Bone morphogenic protein. BOF-4272. Sodium-8-(3-methoxy-4-phenylsulfynilphenyl) pyrzolo[l,5,-a]- l,3,5-tria-zine-4-olate monohydrate. Ca2+. Calcium. Caio (P0 4 )6 (0 H)2. Hydroxyapatite. ECL. Enhanced chemiluminescense. CO2. Carbon dioxide. COX. Cyclooxygenase. CT. Calcitonin. DDW. Distilled deionised water. DMEM. Dulbecco’s modified eagles medium. DPI. Diphenyliodonium chloride DTT. Dl-Dithioreitol. ECACC. European Collection of Animal Cell Culture. ECM. Extracellular matrix. EDTA. Ethylenediamine tetracetic acid disodium salt. EGF. Epidermal growth factor. FAD. Flavin adenine dinucleotide. FCS. Foetal calf serum. Fe-S. Iron-Sulphur. FGF. Fibroblast growth factor. GAGs. Glycosaminoglycans. GI. Gastrointestinal. GM-CSF. Granulocyte/Macrophage-colony stimulating factor. H20 . Water. HBSS. Hanks balanced salt solution. HCL. Hydrochloric acid. HFBA. Heptafluorobutyric acid. v Abbreviations and acronyms. HIF. Hypoxia-Inducible factor. HIV. Human immunodeficiency virus. HO-1. Haemoxygenase-1. H2O2. Hydrogen peroxide. HPLC. High-performance liquid chromatography. IFN-y. Interferon-gamma. IGF. Insulin-like growth factor. IgG. Immunoglobulin. IL-ip. Interleukin 1 beta. IL-2. Interleukin-2. IL-6 . Interleukin- 6 . IMS. Industrial methylated spirits. iNOS. Inducible nitric oxide synthase. iNOS-HRE. Inducible nitric oxide synthase-hypoxic response element. kD. Kilodalton. KPO4. Potassium phosphate. LIF. Leukaemia inhibitory factor. /■'i LMMA. L-N -monomethyl arginine. M. Molarity. Mab. Monoclonal antibody. M-CSF. Macrophage-colony stimulating factor. Mg2+. Magnesium. mg/dL. Milligram per decilitre. MMP. Metalloproteinase. MNGC. Multi-nucleated giant cells. Mn2+. Manganese. Mo. Molybdenum. MT1-MMP. Membrane-type-1 matrix metalloproteinase. NAC. N-acetyl cysteine. NAD+. Nicotinamide adenine dinucleotide. NADH. Nicotinamide adenine dinucleotide, reduced form. NADPH. Nicotinamide adenine dinucleotide phosphate, reduced form. (N H ^ SO4. Ammonium sulphate. Abbreviations and acronyms. NFkB. Nuclear factor kappa B. NIBSC. National Institute for Biological Standards and Control. NMR. Nuclear magnetic resonance. NO. Nitric oxide. N 0 3'. Nitrate. n o 2‘. Nitrite. 0 2*\ Superoxide. OA. Osteoarthritis. OB. Osteoblast. OC. Osteoclast. OH*. Hydroxyl radical. ONOO'. Peroxynitrite. ORSA. Osteoclast resorption stimulating activity. PAGE. Polyacrylamide gel electrophoresis. PBS. Phosphate buffered saline. PDGF. Platelet derived growth factor. p g e 2. Prostaglandin (E2). PMN. Polymorophonuclear leukocytes. pmoles. Pico (xlO'12) moles. PMSF. Phenylmethylsulphonyl fluoride. PTH. Parathyroid hormone. RA. Rheumatoid arthritis. RCOBs. Rat calvarial osteoblasts. RGD. (Arg-Glys-Asp). ROS. Reactive oxygen species. RNS. Reactive nitrogen species. SCF. Stem cell factor. SCID. Severe combined immune deficient. SDS. Sodium dodecyl sulphate (Lauryl Sulphate). SOD. Superoxide dismutase. std. Standard deviation. S.E.M. Standard error of mean. TCM. Tissue culture medium. vii Abbreviations and acronyms. TEMED. N, N, N', N'-Tetramethylethylene diamine. TGFp. Transforming growth factor beta. TNFa. Tumour necrosis factor-alpha. TRAP. Tartrate resitant acid phosphatase. Tris-HCL. Tris[hydroxymethyl] aminomethane hydrochloride.