Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2017-03-01 Alterations in Tight Junctional Proteins and Their Effects on Pulmonary Inflammation Joshua B. Lewis Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Physiology Commons BYU ScholarsArchive Citation Lewis, Joshua B., "Alterations in Tight Junctional Proteins and Their Effects on Pulmonary Inflammation" (2017). All Theses and Dissertations. 6308. https://scholarsarchive.byu.edu/etd/6308 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Alterations in Tight Junctional Proteins and Their Effects on Pulmonary Inflammation Joshua B. Lewis A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Paul R. Reynolds. Chair Juan A. Arroyo Benjamin T. Bikman Brian D. Poole David A. Kooyman Department of Physiology and Developmental Biology Brigham Young University Copyright © 2017 Joshua B. Lewis All Rights Reserved ABSTRACT Alterations in Tight Junctional Proteins and Their Effects on Pulmonary Inflammation. Joshua B. Lewis Department of Physiology and Developmental Biology, BYU Doctor of Philosophy The lungs represent one of the earliest interfaces for pathogens and noxious stimuli to interact with the body. As such, careful maintenance of the permeability barrier is vital in providing homeostasis within the lung. Essential to maintaining this barrier is the tight junction, which primarily acts as a paracellular seal and regulator of ionic transport, but also contributes to establishing cell polarity, cell-to-cell integrity, and regulating cell proliferation and differentiation. The loss of these tight junctions has been documented to result in alterations in inflammation, and ultimately the development of many respiratory disorders such as COPD, Asthma, ARDS, and pulmonary fibrosis. One critical contributor that creates this permeability barrier is the tight junctional protein Claudin. While studies have begun to elucidate the various functions and roles of various Claudins, our understanding is still limited. To initially investigate these proteins, we looked at both temporal and spatial expression patterns for family members during development. A consistent pattern was demonstrated in mRNA expression for the majority of Claudin members. In general, Claudin expression underwent rapid increase during time periods that correlate with the pseudoglanduar/canalicular periods. One notable exception was Claudin 6 (Cldn6), which demonstrated decreasing levels of mRNA expression throughout gestation. We also sought to understand expression dynamics during the addition of maternal secondhand smoke (SHS) which resulted in an almost universal decrease in Claudin proteins. To more fully explore expression mechanisms that affect Claudin-6 (Cldn6), we exposed pulmonary alveolar type II (A549) cells to cigarette smoke extract (CSE) and found that it transcriptionally regulated Cldn6 expression. Using a luciferase reporter, we determined that transcription was negatively regulated at multiple promoter response elements by CSE, and transcription was equally hindered by hypoxic conditions. These findings identified Cldn6 as a potential target of SHS and other respiratory irritants such as diesel particulate matter (DPM). We next sought to assess whether an increase in Cldn6 was sufficient to provide a protective advantage against harmful exogenous exposure. To test this, we utilized a doxycycline induced Cldn6 over- expressing mouse, and subjected it to SHS for 30 days to stimulate an inflammatory state. Our findings demonstrated that Cldn6 transgenic animals have decreased inflammation as evidence by decreased total cell infiltration into the airways, decreased polymorphonuclocyte (PMNs) extravasation, total protein in bronchoalveolar lavage fluid (BALF), and decreased cytokine secretion. Anti-inflammatory advantages were also discovered during experiments involving acute exposure to DPM. In both cases, while stimulation of transgenic mice with SHS or DPM diminished Cldn6 expression, anti-inflammatory evidence emerged suggesting that genetic up- regulation of Cldn6 likely causes the recruitment of other tight junctional components during an organism’s response to environmental assault. Key Words: claudin 6, secondhand smoke, diesel particulate matter, hypoxia, lungs ACKNOWLEDGEMENTS Sir Isaac Newton is attributed with the quote, “If I have seen further than others, it is by standing on the backs of giants.” I echo his sentiments, but feel that my own personal success is more of a product of the love and support from the personal giants in my own life rather than my efforts. Indeed, I would be truly ungrateful not to acknowledge the many individuals who have helped me succeed. At this time I would like to formally extend my sincerest gratitude to my advisor and mentor, Dr. Paul Reynolds. In the time that I have known him, he has taught me valuable lessons in both science and research. More importantly however, his friendship, kindness, support, and example have given me the education, tools, and motivation to be the best I can. I also wish to thank Dr. Juan Arroyo for his kind friendship and continual guidance while at BYU. I consider Dr. Arroyo my second mentor, as he has given countless advice that has aided me not only in my scholastic endeavors but also in life. Additionally, I would like to thank Dr. Benjamin Bikman, who I was able to regularly interact with and learn from through various collaborations. To all the students in the Reynolds/Arroyo lab (and there has been many), I am indebted to you for your tremendous aid. I can honestly say I would not have been half as successful without your efforts. Lastly, I feel a profound sense of obligation to express my gratitude towards my family, and specifically my parents. My parents have always supported and encouraged my ambitions and aspirations, and have encouraged me in both my highs and my lows. Their example of perseverance and resiliency despite adversity has been inspiration for me in times of struggle. They may never fully understand the effects of their love and support, but nevertheless, I am eternally grateful. TABLE OF CONTENTS TITLE PAGE ................................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF FIGURES ..................................................................................................................... xiii LIST OF SYMBOLS AND ABBREVIATIONS ............................................................................1 CHAPTER 1: Introduction ..............................................................................................................4 Tight Junctions ........................................................................................................................ 6 Claudins .................................................................................................................................. 7 Claudin 6 ............................................................................................................................... 10 Preliminary Data ................................................................................................................... 11 References ............................................................................................................................. 17 CHAPTER 2: The Expression Profile of Claudin Family Members in the Developing Mouse Lung and Expression Alterations Resulting from Exposure to Secondhand Smoke (SHS). ................................................................................................................................24 Abstract ................................................................................................................................. 25 Introduction ........................................................................................................................... 26 Material and Methods ........................................................................................................... 28 Mice .................................................................................................................................. 28 qRT-PCR .......................................................................................................................... 28 iv Histology and Immunohistochemistry .............................................................................. 29 Statistical Analysis ............................................................................................................ 30 Results ................................................................................................................................... 30 Spatial and Temporal