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Oliw Faculty opponent Professor Leif Bertilsson Members of the examining board Dr. Maria Norlin Docent Inger Johansson Professor Leif Jansson List of Original Papers Th is thesis is based on the following original papers, which will be referred to by their Roman numerals in the text. Four articles are reprinted with permission from the copy- right holders. I. Katarina Stark, Hans Törmä, Mirela Cristea, and Ernst H. Oliw. Expression of CYP4F8 (prostaglandin H 19-hydroxylase) in human epithelia and prominent induction in epidermis of psoriatic lesions. Arch. Biochem. Biophys., 2003; 409;188-196. II. Katarina Stark, Hans Törmä and Ernst H. Oliw. Expression of Cyclooxygenase-2, Cytochrome P450 4F8, and Microsomal Prostaglandin E Synthase-1 in Psoriatic Lesions and in Epidermal Keratinocytes. 2005 (submitted). III. Katarina Stark, Stefan Seregard, Hans Törmä, and Ernst H. Oliw. CYP4F8 in Plexiform Layers of the Human Retina and CYP4F8 Expression in Corneal Epithelium. 2005 (submitted). IV. Katarina Stark, Johan Bylund, Hans Törmä, Göran Sahlén, and Ernst H. Oliw. On the mechanism of biosynthesis of 19-hydroxyprostaglandins of human seminal fl uid and expression of cyclooxygenase-2, PGH 19-hydroxylase (CYP4F8) and microsomal PGE synthase-1 in seminal vesicles and vas deferens. Prostaglandins Other Lipid Mediat., 2005; 75;47-64. V. Ernst H. Oliw, Katarina Stark, Johan Bylund. Oxidation of prostaglandin H(2) analogs by human cytochrome P450; analysis of omega side chain hydroxymetabolites and four stereoisomers of 5-hydroxyprostaglandin I(1) by mass spectrometry. Biochem. Pharmacol., 2001; 62; 407-415. VI. Katarina Stark, Lydia Schauer, Göran E. Sahlén, Gunnar Ronquist, and Ernst H. Oliw. Expression of CYP4F12 in Gastrointestinal and Urogenital Epithelia. Basic Clin. Pharmacol. Toxicol., 2004; 94; 177-183. VII. Katarina Stark, Buanus Wongsud, Robert Burman, and Ernst H. Oliw. Oxygenation of Polyunsaturated Long Chain Fatty Acids by Recombinant CYP4F8 and CYP4F12. Catalytic Importance of Gly-328 and Tyr-125 of CYP4F8 in Oxidation of Arachidonic acid and PGH2 Analogs. 2005 (manuscript). Contents Metabolism of Arachidonic acid and Eicosanoids .......................................... 11 Arachidonic Acid Cascade .....................................................................................11 Th e Cyclooxygenase Pathway ...........................................................................12 Historical Background ............................................................................... 12 Cyclooxygenases ......................................................................................... 13 Prostaglandin H2 Transformations .............................................................13 Prostaglandins .............................................................................................14 Th e Lipoxygenase Pathway ...............................................................................15 Th e Cytochrome P450 Pathway ........................................................................15 Th e Cytochrome P450 Family ........................................................................16 Background ...........................................................................................................16 Nomenclature ...................................................................................................16 Th e Evolution of the Cytochrome P450 Family ................................................16 P450 Metabolism of Exogenous Compounds ...................................................17 P450s Expression and Function in Extra Hepatic Tissues .................................18 P450s in Prostate and Seminal Vesicles .......................................................18 P450s in Skin ..............................................................................................18 P450s in Ocular Tissues ..............................................................................18 Th e CYP4 Family ..........................................................................................20 Binding of the Prosthetic Heme .............................................................................21 CYP4F subfamily ...........................................................................................22 Overview .............................................................................................................. 22 CYP4F8 ........................................................................................................... 23 CYP4F12 ......................................................................................................... 23 Aims .............................................................................................................. 25 Comments on Materials and Methods ...........................................................26 Tissue samples ...................................................................................................... 26 Isolation of total RNA and transcription .............................................................. 26 DNA sequencing .................................................................................................. 26 RT-PCR and real-time PCR ................................................................................. 27 Northern blotting ................................................................................................. 27 In situ hybridization ..............................................................................................28 Immunization ....................................................................................................... 28 Western blot analysis ............................................................................................ 28 Immunohistological analysis .................................................................................29 Immunofl uorescence analysis ................................................................................29 Site directed mutagenesis and protein expression in yeast ..................................... 30 Keratinocyte cell cultures ..................................................................................... 30 Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry ..........................................................................................................31 Experimental ....................................................................................................31 Liquid chromatography-mass spectrometry analysis (LC-MS) .........................31 Gas chromatography-mass spectrometry analysis (GC-MS) .............................31 Bioinformatic Resources ....................................................................................... 32 Results and Discussion ...................................................................................33 Tissue Distribution of CYP4F8 (paper I) .............................................................. 33 Prominent Expression of CYP4F8 in Psoriatic Skin (papers I and II) .............. 33 CYP4F8 in Ocular Tissues (paper III) ............................................................. 36 COX-2, CYP4F8, and mPGES-1 in Genital Glands and Biosynthesis of 19R-hydroxy-Prostaglandin Es (papers IV and V) ............................................ 37 Tissue Distribution of CYP4F12 (paper VI) ......................................................... 40 Catalytic Properties of CYP4F8 and CYP4F12 (papers IV, VI, and VII) ...............41 Polymorphism of CYP4F8 ....................................................................................42 Analysis of the CYP4F8 Promoter Region .......................................................43 General Discussion ........................................................................................44 Function of CYP4F8 in Biosynthesis of 19-Hydroxy Prostaglandins ....................44 Possible Function of CYP4F8 in Normal and Psoriatic Skin .................................45 Other Functional Roles of CYP4F8 ......................................................................47 Biological Function and Catalytic Properties
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