PURINERGIC SIGNALLING IN SKIN AINA VH GREIG MA FRCS Autonomic Neuroscience Institute Royal Free and University College School of Medicine Rowland Hill Street Hampstead London NW3 2PF in the Department of Anatomy and Developmental Biology University College London Gower Street London WCIE 6BT 2002 Thesis Submitted for the Degree of Doctor of Philosophy University of London ProQuest Number: U643205 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. uest. ProQuest U643205 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Purinergic receptors, which bind ATP, are expressed on human cutaneous kératinocytes. Previous work in rat epidermis suggested functional roles of purinergic receptors in the regulation of proliferation, differentiation and apoptosis, for example P2X5 receptors were expressed on kératinocytes undergoing proliferation and differentiation, while P2X? receptors were associated with apoptosis. In this thesis, the aim was to investigate the expression of purinergic receptors in human normal and pathological skin, where the balance between these processes is changed. A study was made of the expression of purinergic receptor subtypes in human adult and fetal skin. Functional studies using primary human keratinocyte cultures were performed, using specific agonists and antagonists to purinergic receptor subtypes. Purinergic signalling in non-melanoma skin cancer was investigated by immunohistochemical analysis of human basal cell and squamous cell carcinomas. A human cutaneous squamous cell carcinoma cell line (A431) was grown in culture. Specific agonists and antagonists to purinergic receptor subtypes were applied to these cells and changes in cell number were quantified. The expression of purinergic receptors in warts was investigated because of the relationship between human papilloma virus and the development of cutaneous squamous cell carcinomas. Changes in purinergic receptor expression were examined in a rat model of normal and delayed wound healing. The possibility is raised that purinergic signalling within the skin may open the pathway for the development of new treatment modalities for skin cancers, wound healing and perhaps other skin diseases. TABLE OF CONTENTS ABSTRACT ..................................................................................................................................................... 2 ACKNOWLEDGEMENTS......................................................................................................................14 PREFACE ........................................................................................................................................................ 15 CH APTER 1 ................................................................................................................................................... 17 GENERAL INTRODUCTION .............................................................................................................. 17 I. PURINERGIC RECEPTORS AND PURINERGIC SIGNALLING ........................... 18 H is t o r y .......................................................................................................................................................18 PI RECEPTORS............................................................................................................................................................ 19 P2X RECEPTORS....................................................................................................................................... 20 P 2Y RECEPTORS....................................................................................................................................... 24 S ources a n d fate of nucleotides ...............................................................................................27 Sources of ATP ................................................................................................................ 28 The purinergic cascade ................................................................................................... 29 Co-existence of ATP-consuming and ATP-generating pathways ..............................30 Physiological roles of A T P.......................................................................................................... 31 S hort -term purinergic sig n a l lin g ............................................................................................ 31 ATP as a co-transmitter in the nervous system ........................................................... 31 Exocrine and endocrine secretion .......................................................... 31 Regulation of vascular tone ............................................................................................32 Regulation of immune cell function ...............................................................................32 Lo n g -term purinergic s ig n a l l in g ............................................................................................. 32 Cell proliferation and growth .........................................................................................32 Interactions of purines and pyrimidines with growth factors .................................... 33 Purinergic signalling in embryonic development ........................................................33 Purinergic signalling in apoptosis and cancer ............................................................ 34 Su m m a r y of features of purinerg ic s ig n a l l in g ...............................................................39 II. S K I N ...................................................................................................................................................... 40 The e p id e r m is ..........................................................................................................................................40 Epidermal architecture ................................................................................................... 40 Keratinocyte proliferation and stem cells .....................................................................42 Keratinocyte differentiation ............................................................................................45 3 Agents that ajfect keratinocyte function ......................................................................47 Ha ir ......................................................................................................................................52 Hair follicle morphogenesis ......................................................................................... 52 Hair follicle cycle...........................................................................................................52 Structure and properties of the hair follicle ............................................................... 53 Skin Embryology and Development .........................................................................57 The epidermis.................................................................................................................. 57 Epidermal stratification .................................................................................................57 The periderm ................................................................................................................... 58 Cutaneous wound healing ............................................................................................60 Wound epithelialisation ................................................................................................. 60 Migrating kératinocytes change phenotype ................................................................ 60 Model of keratinocyte motility ...................................................................................... 60 Kératinocytes dissect the wound ................................................................................... 61 Kératinocytes at the wound margin proliferate .......................................................... 61 Skin CANCER........................................................................................................................62 Non-melanoma skin cancer ...........................................................................................62 Human papilloma virus .................................................................................................. 66 HPV and human non-melanoma skin cancer ............................................................. 67 III. ROLE OF PURINES IN SKIN................................................................................... 69 Short term purinergic signalling ..............................................................................69 Purinergic signalling in cutaneous blood vessels ......................................................69 Long term purinergic signalling ................................................................................69 Purinergic signalling