Molecular Characterization of Mc3r and Evaluation of Its Potential Role As a Modifier of Lung Function in Cystic Fibrosis
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MOLECULAR CHARACTERIZATION OF MC3R AND EVALUATION OF ITS POTENTIAL ROLE AS A MODIFIER OF LUNG FUNCTION IN CYSTIC FIBROSIS By Jeenah Park A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD March 2014 © Jeenah Park All rights Reserved Abstract Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, affects approximately 30,000 individuals in the United States. CF is caused by loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene. Once the disease-causing gene for CF was identified, numerous studies attempted to correlate CFTR mutations with specific CF phenotypes because genotype-phenotype correlations can predict a course of the disease and lead to the design of a genotype- specific therapeutic strategy. Unfortunately, distilling correlation has been challenging for several reasons. First, CF is a multi-system disease that involves different organs and its phenotype is analyzed in context of its various clinical components. Second, patients exhibit a wide range of disease severity although CF is considered a classic Mendelian disorder. In fact, the degree of variability observed in 293 individuals with the identical CFTR mutations suggests that factors other than the CFTR genotype contribute to lung function variation. Given that obstructive lung disease is the cause of death in 90% of CF patients, there has been continued interest in determining these factors that influence the severity of pulmonary disease in CF patients. MC3R has been identified as a compelling candidate for modifying CF lung disease. Before exploring its role as a modifier, we aimed to understand the molecular organization of MC3R. Using 5’ RACE, we discovered a novel upstream exon that extends the length of the 5’ UTR in MC3R without changing the ORF. The full-length 5’ UTR directs utilization of an evolutionarily conserved second in-frame ATG as the primary translation start site. MC3R synthesized from the second ATG is localized to apical membranes of polarized Madin Darby Canine Kidney (MDCK) cells, consistent ii with its function as a cell surface mediator of melanocortin signaling. Expression of MC3R causes re-localization of a known accessory factor for MC2R, MRAP2, to the apical membrane, which is coincident with the location of MC3R. In contrast, protein synthesized from MC3R cDNAs lacking the 5’ UTR displayed diffuse cytosolic distribution and no effect upon the distribution of MRAP2. Our findings demonstrate that a previously unannotated 5’ exon directs translation of MC3R protein that localizes to apical membranes of polarized cells. To define the mechanism of lung disease in CF that underlies the linkage signal at chr20q13.2, we evaluated MC3R as a potential modifier of lung function. Three rare variants in MC3R were significantly associated with lung function in individuals with CF, suggesting that variation in MC3R contibutes to CF lung disease severity. In silico prediction tools and in vitro studies were performed to understand the functional effect of the rare variants. We demonstrated that antagonist (siRNA) or agonist ([D-Trp8]-γ-MSH) of MC3R can modulate pro-inflammatory cytokine expression. Evidence for the presence of MC3R in primary airway epithelial cells of the lungs further supported our hypothesis that MC3R is a modifier of lung function in CF. These studies support the role of MC3R as a mediator of the inflammatory response and a potential therapeutic target in the treatment of CF. Advisor & 1st Reader: Garry R. Cutting, MD Professor, Department of Pediatrics McKusick-Nathans Institute for Genetic Medicine Johns Hopkins University School of Medicine 2nd Reader: Pamela Zeitlin, MD/PhD Professor, Department of Pediatrics Director, Eudowood Division of Pediatric Respiratory Sciences Deputy Director, Institute for Clinical and Translational Research Johns Hopkins University School of Medicine iii In honor of my mother, who encouraged me to pursue my Ph.D. and helped me believe that I could do anything iv Acknowledgements I would like to thank my PhD advisor, Garry Cutting, for his unconditional support and encouragement. I cannot thank him enough for his contagious enthusiasm about science. Garry has this uncanny ability to get us excited about our project – even when the experiments keep failing. I have learned so much from being in his lab. He has taught me how to properly set up my experiments, critically look at results, write with intent, and give a good presentation. I know that I have more learning to do in order to become the best scientist I can be, but I would like to extend gratitude to Garry for giving me a home in his lab and training me for past 4+ years. I also want to thank Garry for allowing me to pursue teaching opportunities. It really meant a lot to me that he understood how much I enjoy teaching and how important teaching is to me personally and professionally. I am thankful that I had an opportunity to teach undergraduate students at Towson for several semesters. Thank you for recommending me for a position in ASHG Information & Education Committee as well. I am forever grateful to have been part of the Cutting Lab. The lab was my second family. I loved coming to work everyday because I enjoyed working with everyone. Laura was the best across-the-bench mate I could’ve asked for. Briana is a master at handling large sequencing data. Melissa is a queen of R (thank you for beautiful graph). Arianna was my extra set of hands (and a weekly provider of cupcakes). Karen, our resident genetic counselor, was an awesome guest lecturer in my class. Most importantly, Neeraj taught me how to do science. I will miss our lunches, weddings, happy hours, v work outs, etc. Best of luck to potential new additions to the Cutting Lab: Ted Han and Ryan Longchamps. Maybe we will have extra Y chromosomes in lab soon. I would like to thank Dave Valle and Sandy Muscelli from the Human Genetics Program. Many thanks to Dave for giving me helpful tips about the job search. Thank you Sandy for always having an open door. The access to the candy bowl was crucial to many of us. In addition, I would like to sincerely thank Andy McCallion for his valuable help on the ASHG proposal. He let me barge into his office and ask thousands of naïve questions. He never gave up on me and gave me the encouragement I needed. Without his advice, I would not have been able to complete the proposal. The support of my family and friends has been incredible. This is especially true for my mom who encouraged me to pursue graduate school. Thank you to my parents-in- laws, Dick and Ellen, for sending care packages and treating me like their own daughter. Most importantly, I am thankful for my husband, Eben. He’s the source of my happiness, my inspiration, my everything. You have been beside me every step of this journey, and I am so happy that we crossed our paths at Hopkins. I look forward to the next chapter of our lives in Oakland, CA. vi Table of Contents Abstract ii Dedication iv Acknowledgements v Table of Contents vii List of Tables viii List of Figures ix Chapter 1 Introduction and literature review 1 Chapter 2 Molecular and cellular characterization of human 34 melanocortin-3-receptor (MC3R) Chapter 3 Evaluation of MC3R’s potential role as a modifier of lung 60 function in cystic fibrosis Chapter 4 Conclusion and Discussion 95 References 102 Curriculum Vitae 123 vii List of Tables Chapter 1: Table 1.1 Efficacy of the restoration of chloride transport in primary CF 24 bronchial epithelial cell culture Table 1.2 Biological distributions and functions of melanocortin receptor 26 family Table 1.3 Localization of MC3R expression by confocal microscopy 30 Chapter 3: Table 3.1 Association of MC3R variants with lung disease severity 66 Table 3.2 Association of MC3R haplotypes with lung disease severity 70 Table 3.3 In silico predictions for pathogenicity of the coding variants in 71 MC3R viii List of Figures Chapter 1: Figure 1.1 CF is a highly variable, multi-system disease caused by 3 mutations in CFTR Figure 1.2 CF is characterized by extensive allelic heterogeneity 9 Figure 1.3 The variability in lung function with an identical CFTR genotype 13 is considerable Figure 1.4 Multiple factors interact to determine the CF phenotype 15 Figure 1.5 20q13 contains a modifier for lung disease severity in CF 20 Figure 1.6 Schematic presentation of the general structure of GPCR 25 Figure 1.7 Anti-inflammatory effects of melanocortins 28 Figure 1.8 Effect of MRAP on MC2R and MC3R in a signaling pathway 32 Chapter 2: Figure 2.1 Mapping of the 5’ transcription start site and the gene structure 38 of MC3R Figure 2.2 MC3R translation begins at the second methionine 41 Figure 2.3 MC3R exhibits apical localization in polarized MDCK cells 43 Figure 2.4 In absence of the 5’ UTR, MC3R is mislocalized to the 45-46 cytoplasm of polarized cells Figure 2.5 MRAP2 is re-distributed in the presence of MC3R and MC2R 49-50 ix Chapter 3: Figure 3.1 Characteristics of common and rare variants in MC3R 64 Figure 3.2 Histogram of CF patients according to lung disease severity 67 Figure 3.3 LD plots across the MC3R locus generated by Haploview 68 Figure 3.4 Differential mRNA expression of MC3R in human transformed 73 lymphocytes Figure 3.5 Reduction in MC3R expression using siRNAs increases NF-κB- 75-76 mediated reporter gene expression in K562 cells Figure 3.6 Modulation in MC3R expression followed by PMA stimulation 78-79 alters NF-κB-driven cytokine expression in K562 cells Figure 3.7 Western blot and Immunohistochemical staining reveal that 81-82 MC3R is expressed in the airway epithelial cells of normal and CF lung tissues x Chapter 1: Introduction and literature review 1 Cystic Fibrosis Cystic fibrosis (CF) is the one of the most common lethal autosomal recessive disorder among Caucasians.