Novel Insights into Tace Regulation of Autocrine and Paracrine Signaling in Epithelial Cells

Bunker, Eric Newman

Graduate Thesis Or Dissertation

Novel Insights into Tace Regulation of Autocrine and Paracrine Signaling in Epithelial Cells Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/xd07gs902

Abstract

A multitude of cellular functions are controlled by the activity of the membrane-boundprotease TACE, including immune response and development. Through cleavage of itsmembrane anchored substrates, TACE can activate a myriad of signaling pathways inan auto- or paracrine manner, transferring signals across a tissues and systems.Despite its importance, it is highly debated how TACE is regulated. I helped generateTSen, a fluorescence-based sensor of TACE activity, a critical tool that provednecessary to interrogate the underlying signaling that leads to TACE activation. TSenenabled us to uncover a wide variation between cell lines and their underlying signalingin regards to TACE which may account for much of the confusion about its regulation.Furthermore, we used this sensor to find that TACE is intimately tied actin cytoskeletaldynamics, which appears to be a novel mechanism of TACE activation. I then usedTSen to monitor TACE activity in single cells in conjunction with an ERK reporter tomeasure both activities simultaneously for the first time. I was able to confirm pastresults showing the propagation of ERK pulses from cell to cell, as well as identify anew type of activity pulse: spikes in ligand shedding. These pulses of TACE activityoccur in neighbor cells prior to an ERK pulse, which is followed by another spike inligand shedding, validating the model that ERK pulses propagate between cells throughligand shedding. I then probed cell junctions for their role in TACE signaling. Becausethe aggressiveness of an epithelial-derived cancer is highly correlated with loss of celljunctions, I generated an α-catenin knockdown in HaCaT cells, recreating a phenotypeof mesenchymal morphology and hyper proliferation. I found that silencing α-catenin inthis system generates a large increase in ligand shedding and subsequent proliferationand that the hyper proliferative phenotype does not occur in the absence of TACE. Myfindings contribute to the overall understanding of TACE regulation, as well as thedownstream effects of this signaling molecule in ERK pulses and proliferation.

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