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C-Met Initiates Epithelial Scattering Through Transient Calcium Influxes and NFAT-Dependent Gene Transcription

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C-Met Initiates Epithelial Scattering Through Transient Calcium Influxes and NFAT-Dependent Gene Transcription Brigham Young University BYU ScholarsArchive Theses and Dissertations 2011-12-13 c-Met Initiates Epithelial Scattering through Transient Calcium Influxes and NFAT-Dependent Gene Transcription Peter R. Langford Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Cell and Developmental Biology Commons, and the Physiology Commons BYU ScholarsArchive Citation Langford, Peter R., "c-Met Initiates Epithelial Scattering through Transient Calcium Influxes and NFAT- Dependent Gene Transcription" (2011). Theses and Dissertations. 3186. https://scholarsarchive.byu.edu/etd/3186 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. c-Met Initiates Epithelial Scattering through Transient Calcium Influxes and NFAT-dependent Gene Transcription Peter Ronald Langford A dissertation submitted to the faculty of Brigham Young University In partial fulfillment of the requirements for the degree of Doctor of Philosophy Marc D. H. Hansen, Chair Merritt B. Andrus Michael D. Brown Allan M. Judd Michael R. Stark Department of Physiology and Developmental Biology Brigham Young University December 2011 Copyright © Peter R. Langford All Rights Reserved Abstract c-Met Initiates Epithelial Scattering through Transient Calcium Influxes and NFAT-dependent Gene Transcription Peter Ronald Langford Department of Physiology and Developmental Biology, BYU Doctor of Philosophy Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and migrating as solitary cells, a process that parallels epithelial- mesenchymal transition. HGF binds and activates the c-Met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. This study addresses this shortcoming in a number of ways. A high-throughput in vitro drug screen was employed to identify proteins necessary in this HGF-induced signaling. Cells were tested for reactivity to HGF stimulation in a Boyden chamber assay. This tactic yielded several small molecules that block HGF-induced scattering, including a calcium channel blocker. Patch clamping was used to determine the precise effect of HGF stimulation on Ca2+ signaling in MDCK II cells. Cell-attached patch clamping was employed to detect Ca2+ signaling patterns, and channel blockers were used in various combinations to deduce the identity of Ca2+ channels involved in EMT. The results of these experiments show that HGF stimulation results in sudden and transient increases in calcium channel influxes. These increases occur at predictable intervals and rely on proper tubulin polymerization to appear, as determined through the use of a tubulin polymerization inhibitor. Though multiple channels occur in the membranes of MDCK II cells, noticeably TRPV4 and TrpC6, it is TrpC6 that is specifically required for HGF-induced scattering. These HGF-induced calcium influxes through TrpC6 channels drive a transient increase in NFAT-dependent gene transcription which is required for HGF-induced EMT. This was determined through the use of luciferase-based NFAT reporter assays and confirmed through confocal immunofluorescence. Using a small-molecule inhibitor of WNK kinase, it was determined that loss of WNK kinase function is sufficient to prevent HGF-induced EMT. Furthermore, patch-clamp analysis demonstrated that WNK kinase significantly increases channel opening at the surface of MDCK cells, indicating a possible mechanism of action for c-Met inhibition, but leaving doubt as to whether WNK kinase is in fact normally involved in c-Met signaling, or whether it is simply permissive. Keywords: cancer, c-Met, HGF, calcium, NFAT, tubulin, epithelial-mesenchymal transition, WNK kinase Acknowledgements I would like to thank Dr. Marc Hansen for his guidance, resourcefulness and creativity in helping me to perform this project. None of this would have been possible without his willing support at each step. Thanks also to all of the members of my committee for making their labs available to me as well as for their confidence, instruction and good humor that helped to make this all seem possible. I would also like to thank Dr. David Busath, who helped me begin patch clamping and loaned me his electrophysiology equipment for so many months. Thanks also to the many other professors in the BYU Department of Physiology and Developmental Biology, who were always on hand to help and who opened their labs to me as needed, and to the professors in the BYU Department of Chemistry and Biochemistry, who contributed their time, materials and expertise to help us begin our drug screen. In particular, I would like to thank my family, namely my parents, Richard and Marie Langford, for their support, both financial and moral, in helping me through school and for encouraging me to continue my education as far as I wanted to go. Thanks especially to my wife, Karyn, for her constant support and encouragement, her continued interest in my research, and her willingness to continue to believe that my work is worthwhile, even when it seems to hardly be moving. Finally, thanks to all of the individuals, anonymous and otherwise, who have contributed to Brigham Young University. This work was supported extensively by BYU and its generous donors, including two Summer Research Fellowships from the BYU Cancer Research Center, the BYU Women’s Studies and Gerontology programs, two travel grants from the BYU Graduate Student Society, and a BYU Graduate Student Grant. Thank you to all of the individuals who made this possible. Contents List of Tables ............................................................................................................................. vii List of Figures .......................................................................................................................... viii Chapter 1: Introduction ............................................................................................................... 1 A review of c-Met signaling and other receptor tyrosine kinases ........................................... 2 c-Met as a receptor tyrosine kinase ...................................................................................... 2 HGF-induced epithelial-mesenchymal transition ................................................................ 8 Induction of actin rearrangement ......................................................................................... 8 Induction of cell spreading................................................................................................... 9 Induction of cell-cell detachment....................................................................................... 10 Induction of increased cell migration ................................................................................ 12 Induction of cell invasion................................................................................................... 13 c-Met signaling in processes independent of EMT ............................................................ 13 Overall network structure .................................................................................................. 14 Summary ............................................................................................................................ 15 Established methods for identifying key components of signal transduction pathways in cancer ..................................................................................................................................... 15 Background of calcium signaling .......................................................................................... 17 The role of calcium conductance in cancer progression ........................................................ 19 Tubulin as a mediator of ion channel function ...................................................................... 20 NFAT involvement in cancer signaling................................................................................. 21 The role of WNK kinase in cancer and cation channel regulation ........................................ 22 MDCK as a model system ..................................................................................................... 24 MDCK as a model system in the high-throughput drug screen ......................................... 24 MDCK as a model system to determine upstream regulators of Ca2+ influx in HGF signaling ............................................................................................................................. 24 MDCK as a model system for calcium channel classification and NFAT testing ............. 25 MDCK as a model for WNK kinase signaling .................................................................. 25 Summary of literature review and my specific aims ............................................................. 26 Specific aim 1: Identify proteins essential to HGF signal transduction and classify them according to function ......................................................................................................... 27 Specific aim 2: Determine the mechanism of Ca2+ signaling in HGF signal transduction 28 iv Specific aim 3: Characterize the interaction between HGF-induced Ca2+ influx and NFAT activation
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