PRO-FIBROGENIC AND ESTROGEN RECEPTOR SIGNALING PATHWAYS INTERACT IN THE LUNG: IMPLICATIONS FOR FIBROTIC LUNG DISEASE By LEY CODY SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Ley Cody Smith To my family and my friends ACKNOWLEDGMENTS I thank my advisor, Dr. Tara Sabo-Attwood, for her expert guidance, constructive advice, and patience throughout the dissertation. I would also like to thank my brilliant advisory committee for their support and guidance in the development of my project: Dr. Nancy Denslow, Dr. Paul Cooke, and Dr. Gregory Schultz. I would like to acknowledge the members of the Center for Environmental and Human Toxicology, past and present, for all their assistance and support. I especially want to thank Dr. Candice Lavelle, Dr. Joseph H. Bisesi, Jr., Dr. Georgia Roberts, and Dr. Erica Brockmeier for not only being excellent coworkers but also life-long friends. I thank Dr. Dale Porter of CDC NIOSH and Dr. Andrew Bryant of the UF Medical School for being enthusiastic collaborators. I thank Dr. Manjunatha Nanjappa and Terry Medrano of the UF Department of Physiological Sciences for their assistance with immunohistochemical staining. I thank members of the UF Interdisciplinary Center for Biotechnology Research including Dr. Alberto Riva of the Bioinformatics Core, Dr. Yanping Zhang of the Gene Expression & Genotyping Core, and Dr. David Moraga of the NextGen DNA Sequencing Core for assistance with the RNA-Seq analysis. I thank Dr. Navid Saleh and Dipesh Dah of the University of Texas at Austin Cockrell School of Engineering and Dr. Paul Carpinone of the UF Herbert Wertheim College of Engineering for their assistance with the nanotube experiments. I also want to thank the undergraduate researchers who have assisted me throughout the years including Santiago Moreno, Kristal Gant, Jennifer Knapp, and Kung Shroff. Last but not least, I thank my family and friends for their unconditional love and support throughout this journey. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 16 CHAPTER 1 BACKGROUND ...................................................................................................... 18 Pulmonary Fibrosis ................................................................................................. 18 TGF-β1 Signaling in Pulmonary Fibrosis ................................................................ 20 Multi-Walled Carbon Nanotube-Induced Pulmonary Fibrosis ................................. 22 Sex and Sex Hormones in Pulmonary Fibrosis ....................................................... 24 Estrogen Signaling in the Lung ............................................................................... 26 Interactions Between TGF-β1 and E2 Signaling ..................................................... 27 Cell-Specific Effects in Pulmonary Fibrosis ............................................................. 29 Research Objectives ............................................................................................... 30 2 MULTI-WALLED CARBON NANOTUBES INHIBIT ESTROGEN RECEPTOR ALPHA EXPRESSION IN THE LUNG VIA TGF-β1 ................................................ 32 Introduction ............................................................................................................. 32 Materials and Methods............................................................................................ 34 Chemicals ......................................................................................................... 34 Animal Exposure .............................................................................................. 35 Histopathology .................................................................................................. 35 Enzyme-Linked Immunosorbent Assay (ELISA) ............................................... 36 Immunohistochemistry (IHC) ............................................................................ 36 Cell culture ....................................................................................................... 37 Nanotube Dispersion and Characterization in vitro .......................................... 38 Total RNA Extraction and Purification .............................................................. 39 Quantitative Real-Time Polymerase Chain Reaction (qPCR)........................... 40 Intracellular Reactive Oxygen Species (ROS) Measurement ........................... 40 Statistics ........................................................................................................... 41 Results .................................................................................................................... 41 Mitsui MWCNTs Cause Histopathological Alterations in the Lungs of Mice ..... 41 Mitsui MWCNTs Increase TGF-β1 Levels in BALF .......................................... 42 Mitsui MWCNTs Reduce Estrogen Receptor mRNA Expression in Mouse Lungs ............................................................................................................ 42 5 ESR1 Protein is Expressed in Mouse Lung Epithelial Cells ............................. 43 Mitsui MWCNTs Reduce ESR1 mRNA Expression in BEAS-2Bs .................... 43 Mitsui MWCNT-Induced Reduction of ESR1 mRNA is Not a Function of Surface Chemistry ......................................................................................... 44 Mitsui MWCNTs Reduce ESR1 mRNA Expression in a TGF-β1-Dependent Manner in vitro .............................................................................................. 45 Mitsui MWCNT-Induced Reduction of ESR1 mRNA Expression is Likely Not a Consequence of ROS Generation .............................................................. 45 Discussion .............................................................................................................. 46 3 TRANSFORMING GROWTH FACTOR BETA1 TARGETS ESTROGEN RECEPTOR SIGNALING IN BRONCHIAL EPITHELIAL CELLS ........................... 67 Introduction ............................................................................................................. 67 Materials and Methods............................................................................................ 69 Chemicals ......................................................................................................... 69 Cell Culture ....................................................................................................... 70 Total RNA Extraction and Purification .............................................................. 71 Quantitative Real-Time Polymerase Chain Reaction (qPCR)........................... 71 Protein Extraction and Purification ................................................................... 72 Western Blot ..................................................................................................... 72 RNA-Seq Library Preparation ........................................................................... 73 RNA Sequencing .............................................................................................. 74 Bioinformatics ................................................................................................... 74 Statistics ........................................................................................................... 75 Results .................................................................................................................... 75 TGF-β1 Induces Changes Consistent with EMT .............................................. 75 E2 Does Not Significantly Affect TGF-β1-Induced EMT ................................... 76 TGF-β1 Reduces Estrogen Receptor mRNA and Protein Expression .............. 77 TGF-β1 and E2 Exhibit Unique Transcriptional Profiles ................................... 77 TGF-β1 and E2 Differentially Regulate Gene Sets ........................................... 79 Discussion .............................................................................................................. 80 4 GENE EXPRESSION ANALYSIS IN THE LUNGS OF PATIENTS WITH IDIOPATHIC PULMONARY FIBROSIS .................................................................. 98 Introduction ............................................................................................................. 98 Materials and Methods.......................................................................................... 100 Patient Samples ............................................................................................. 100 Handling of Lung Tissue ................................................................................. 101 Total RNA Extraction and Purification ............................................................ 101 Gene Expression Analysis in Whole Lung Tissue .........................................