ASPECTS OF THE TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF NITRIC OXIDE SYNTHASE 1 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shawn M. Pierson, B.S. ****** The Ohio State University 2005 Dissertation Committee Professor Anthony Young, Adviser Approved by: Assoc. Professor John Bauer Assoc. Professor Dale Hoyt _________________________ Adviser Professor Michael Ostrowski College of Pharmacy ABSTRACT The nitric oxide synthases (NOS) catalyze the formation of nitric oxide (NO) and citrulline from the reactants arginine and oxygen. Three isoforms of the enzyme have been discovered and designated NOS1, NOS2 and NOS3. NO produced by NOS1 plays a role in a variety of physiological processes including learning and memory, bronchial relaxation and GI motility. Excessive production of NO can be toxic to neurons and is associated with various pathophysiological conditions. This dissertation explores the regulation of the gene encoding for NOS1 in order to gain insights into its normal and abnormal regulation. Using PC12 cells, we studied the effect the peptide pituitary derived adenylate cyclase activating peptide (PACAP38), a 38 amino acid peptide hormone, had on endogenous NOS1 expression. We showed that PACAP led to an increase in both NOS1 mRNA and protein. Further, it was demonstrated that the transcript upregulated was a shortened transcript driven by a promoter located in what is normally exon 2. PACAP38 is able to activate a variety of second messenger pathways when binding to its receptors. Among these are the protein kinase A (PKA) and protein kinase C (PKC) pathways. Treatment of PC12 cells with forskolin, a drug that activates the PKA pathway led to a mild increase in NOS1 mRNA and protein - ii - that was smaller than that observed in cells treated with PACAP. When cells were treated with both forskolin and TPA, a drug that activates PKC, the increases in NOS1 protein and mRNA were similar to that observed with PACAP. Thus PACAP may have to activate both pathways to fully activate NOS1 expression. We next looked at the effect of PACAP38 had on the rat and human exon 2 promoters. We showed both of these promoters were stimulated by PACAP and required the activation of multiple signal transduction pathways for full activation. We next looked at the effect of PACAP38 on another human NOS1 promoter, the 5’2(1F) promoter. We demonstrated that the activation of the cAMP-PKA pathway was totally responsible for activation of this promoter by PACAP38. We then mapped the region of 5’2 necessary for PACAP stimulation to a 230 base pair region just upstream of the 5’2 alternate first exon and that a single CRE site within this region was essential for PACAP38 stimulation. Lastly, we cloned and characterized two novel promoters of NOS1. The first promoter drives the expression of a NOS1 transcript containing a novel alternate first exon discovered by Greg Hartt working in our lab. This exon was designated 5’3 by us and 1D by others. The second promoter drives the expression of a transcript containing a second alternate first exon discovered by Dr Hartt and designated 5’4 by us and 1C by others. iii ACKNOWLEDGMENTS I would like to thank my adviser, Tony Young, for his advice and encouragement in completing this project, for giving me the freedom to explore my own ideas and for pushing me when necessary. I would like to thank my family for their support in this endeavor. Even when they did not understand why I was doing this, they were always supportive. I thank my labmates past and present for their helpful discussions and for showing me how to use the equipment. In the beginning, Wei-Kang Chen, Terrie Rife and Greg Hartt were very helpful. I am grateful to Linda Zhang for showing me the tricks of cloning and to Deyu Zhang for his help with my first western blots. Kunyi Wu was quite helpful with the animal work and this is greatly appreciated as was Hua Wei’s assistance with the real time PCR. I would also like to thank the students, staff and faculty of the division of pharmacology, College of Pharmacy for their help and friendship. Finally, I want to thank the faculty, staff and students of the Center for Molecular Neurobiology. Whenever I needed to try a new technique, I could always find someone who had performed it before somewhere in the center and they were always willing to help (and sometimes even loan me equipment and supplies). This work was supported by a grant from the National Institutes of Health. iv VITA September 1, 1971....................................Born- Columbus, OH 1994..........................................................B.S. Pharmacy, The Ohio State University 1994-1997..................................................Staff Pharmacist Childrens Hospital Columbus, OH 1997-present.............................................Graduate Teaching and Research Associate, The Ohio State University FIELDS OF STUDY Major Field: Pharmacy v TABLE OF CONTENTS Abstract......................................................................................................................... ii Acknowledgments........................................................................................................ iv Vita............................................................................................................................... v List of figures...............................................................................................................viii List of Tables............................................................................................................... x Chapters: 1. Introduction................................................................................................. 1 The chemistry of nitric oxide............................................................... 1 The production of nitric oxide............................................................. 5 The NOS1 gene.................................................................................... 7 The NOS1 protein............................................................................... 12 Physiological roles of NO produced by NOS1................................... 17 Roles of NOS1 in disease.................................................................... 21 An introduction to PACAP................................................................. 24 2. NOS1 Regulation by PACAP................................................................... 39 Introduction......................................................................................... 39 Materials and Methods........................................................................ 41 Results................................................................................................. 52 Conclusions and discussion................................................................ 73 Chapter references............................................................................... 81 3. Effects of PACAP on various NOS1 promoters....................................... 84 Introduction......................................................................................... 84 Materials and Methods........................................................................ 87 Results................................................................................................. 93 Conclusions and Discussion............................................................... 107 vi Chapter references................................................................................ 112 4. Cloning and Characterization of 2 Novel Promoters of NOS1............... 113 Introduction........................................................................................ 113 Materials and Methods....................................................................... 115 Results................................................................................................ 126 Conclusions and discussion............................................................... 135 Chapter references.............................................................................. 144 List of References...................................................................................................... 146 vii LIST OF FIGURES Figure Page 1.1 Structure and biologically important reactions of NO..................................... 4 1.2 Reaction catalyzed by the Nitric Oxide Synthases (NOS)............................... 6 1.3 Genomic map of the human NOS1 alternate first exons.................................. 9 1.4 Map of the NOS1 protein domains................................................................. 14 1.5 Some of the Signal transduction Pathways Activated by PACAP.................. 27 2.1 Time Course of NOS1 protein expression in PC12 cells treated with PACAP................................................................................................... 53 2.2 Time Course of NOS1 protein expression in PC12 cells treated with forskolin.................................................................................................. 54 2.3 NOS Assay using extracts from PC12 cells treated with NGF and forskolin................................................................................................... 55 2.4 Time course of NOS1 expression in PC12 cells treated with forskolin and TPA.........................................................................................