Activation of Novel Signal Transduction Pathways by FP Receptors: The G-Protein Coupled Receptors for Prostaglandin F2alpha Item Type text; Electronic Dissertation Authors Xu, Wei Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 24/09/2021 06:48:12 Link to Item http://hdl.handle.net/10150/195223 ACTIVATIO OF OVEL SIGAL TRASDUCTIO PATHWAYS BY FP RECEPTORS: THE GPROTEI COUPLED RECEPTORS FOR PROSTAGLADI F2Α by WeiXu ADissertationSubmittedtothe Facultyofthe DEPARTMETOF PHARMACOLOGY & TOXICOLOGY InPartial FulfillmentoftheRequirements For theDegreeof DOCTOROF PHILOSOPHY IntheGraduateCollege THEUIVERSITYOFARIZOA 2007 2 THEUNIVERSITY OF ARIZONA® GRADUATECOLLEGE Asmembersofthe Final ExaminationCommittee,wecertifythatwehavereadthe dissertationpreparedby WeiXu_______________________________________________ Entitled: Activation of Novel Signal Transduction Pathways by FP Receptors: the G-Protein Coupled Receptors for Prostaglandin F2α and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _______________________________________________ Apr.12th,2007 John W.Regan Date _______________________________________________ Apr.12th,2007 John W.Bloom Date _______________________________________________ Apr.12th,2007 TimBowden Date _______________________________________________ Apr.12th,2007 RogerMiesfeld Date _______________________________________________ Apr.12th,2007 DanStamer Date Finalapprovalandacceptanceofthisdissertation iscontingentuponthecandidate’s submissionofthefinalcopyofthedissertationtotheGraduateCollege.I herebycertify that Ihavereadthisdissertationpreparedundermy directionandrecommendthatitbe acceptedasfulfillingthe dissertationrequirement. _________John W.Regan_________________________Apr.12th,2007 DissertationDirector Date 3 STATEMETBYAUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library tobemadeavailabletoborrowersunderrulesofthe Library. Brief quotations from this dissertation are allowable without special permission,provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances,however,permissionmustbeobtainedfromtheauthor. SIGNED: WeiXu 4 ACKOWLEDGEMETS First, I would like to thank Dr. John W. Regan for the support and guidance he has provided throughout my doctoral studies.Dr.Regan has given me many opportunities to expand my horizons by allowing me to have a lot of freedom in my research. I venerate Dr.Regan’s intelligence, work ethic,scientific integrity as well as attention to detail; they aresecondtononeandit iseasytoseewhyheis verysuccessful. I would also like to thank my committee members Doctors John Bloom,Tim Bowden, RogerMiesfeldand Dan Stamerfortheirscientific andprofessionalsupport. I would like to thank Dr. Chiling Chou for her time,experience and prospect in helping me with my studies. We worked together to get the results presented in this thesis. Withoutherhelp,Icannotimagehow Ican achievesomuchinmy graduatestudy. I would like to thank all the other folks in the Regan lab; Alfred, George, Dinesh, Hiromichi,Tony,Chi-ling and Amy for their help over the years. I would also like to thank Dr.Vallancourt for his help in my projects.It is his idea and vision to help this projecttakeoff. Last,and certainly not least,I would like to thank my parents, Kanglin Xu and Yixin Shenaswellasmywife ZhouandsonWilliamfortheirsupportoverthe years. 5 DEDICATIO This dissertation is dedicated to my family,especially my parents and parents-in-laws, who over the years have supported my efforts with great patience,support and love. I would also like to thank my wife Zhou and son William for their support over these years. 6 TABLEOFCOTETS LISTOF FIGURES ............................................................................................................ 8 LISTOFTABLES............................................................................................................ 10 LISTOFABBREVIATIONS........................................................................................... 11 ABSTRACT.............................................................................................................................................13 CHAPTERONE: INTRODUCTION,PURPOSEANDAIMS....................................... 15 1.1 Introduction............................................................................................................. 16 1.2Prostanoidreceptors................................................................................................ 19 1.3FPreceptors ............................................................................................................ 20 1.4Purposeandaims ................................................................................................... 24 CHAPTER TWO: IDENTIFICATION OF GENES REGULATED BY THE ACTIVATION OF FP PROSTANOID RECEPTOR UTILIZING CDNA MICROARRAYTECHNOLOGY .................................................................................. 27 2.1 Introduction ............................................................................................................ 28 2.2Experimentalprocedures ........................................................................................ 29 2.3Results..................................................................................................................... 32 2.4Discussion............................................................................................................... 34 CHAPTER THREE: CHARACTERIZATION OF THE SIGNALING PATHWAYS INVOLVED IN THE REGULATION OF EGR-1 EXPRESSION BY THE ACTIVATIONOF FPPROSTANOIDRECEPTORS .................................................... 43 3.1 Introduction ............................................................................................................ 44 3.2Experimentalprocedures ........................................................................................ 46 7 TABLEOFCOTETS-Continued 3.3Results...................................................................................................................... 47 3.4Discussion................................................................................................................ 50 CHAPTER FOUR: CHARACTERIZATION OF THE SIGNALING PATHWAYS INVOLVED IN THE REGULATION OF CYR61 EXPRESSION BY THE ACTIVATIONOF FPPROSTANOIDRECEPTORS .................................................... 57 4.1 Introduction............................................................................................................. 58 4.2Experimentalprocedures ........................................................................................ 59 4.3Results..................................................................................................................... 64 4.4Discussion............................................................................................................... 68 CHAPTER FIVE: CHARACTERIZATION OF THE SIGNALING PATHWAYS INVOLVED IN THE REGULATION OF CTGF EXPRESSION BY THE ACTIVATIONOF FPPROSTANOIDRECEPTORS .................................................... 80 5.1 Introduction............................................................................................................. 81 5.2Experimentalprocedures ........................................................................................ 83 5.3Results..................................................................................................................... 85 5.4Discussion...............................................................................................................89 CHAPTERSIX:CONCLUSIONSAND FUTURESTUDIES ..................................... 102 6.1Conclusions........................................................................................................... 103 6.2Futurestudies........................................................................................................ 107 REFERENCES ............................................................................................................... 110 8 LISTOFFIGURES Figure 1.1 Schematic pathways of eicosanoid biosynthesis from arachidonic acid release andmetabolism ............................................................................................................. 18 Figure1.2SignaltransductionpathwaysthroughtheFPreceptor ................................... 23 Figure2.1 FunctionClassificationofgenesidentifiedby cDNAmicroarray analysis ....39 Figure2.2Northern Blottinganalysisof genesidentifiedbycDNAmicroarrayanalysis41 Figure2.3Western Blottinganalysisof genesidentifiedbycDNAmicroarray analysis 42 Figure 3.1 MEK1/2 is involved in the gene regulation of EGR-1 through the FP receptor ......................................................................................................................................