Date of Award

2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Albert van der Vliet

Second Advisor

Matthew E. Poynter

Abstract

Cigarette smoke (CS) exposure is the leading cause of preventable death in the United States contributing to over 480,000 deaths a year with over 300 billion dollars in CS related costs spent per year. While the dangers of CS exposure have been studied and characterized for decades being largely attributed to reactive oxygen species and oxidative stress, increasing evidence suggests that reactive aldehydes in CS, specifically the α,β-unsaturated aldehyde acrolein, are responsible for many of the negative pathologies associated CS exposure. Previous work has shown that acrolein can bind directly to a number of cellular proteins containing redox sensitive cysteine residues. The non-receptor tyrosine kinase Src contains nine cysteine residues and is known to be activated in response to CS and oxidative stress. Despite being the first characterized and one of the most widely studied oncogenes, the exact mechanism for Src activation remains unclear. In the current studies we examined the effects of acrolein on Src activation and the resulting outcomes on the lung epithelium in an effort to better understand how reactive electrophiles in CS contribute to the development of lung disease.

To determine the effects of acrolein on Src activation, we first exposed NCI-H292 cells to acrolein and measured activity by western blot. We observed an increase in Src activity detected by an increase in Src phosphorylation at Y416 and an increase in phosphorylation of Src target proteins Caveolin1 and p120. Interestingly the increase in activation occurred without dephosphorylation of the inhibitory phosphorylated tyrosine Y527. Using biochemical-labeling strategies we identified Src as a direct target of acrolein adduction in vitro and in vivo, and we used mass spectrometry to confirm acrolein adduction to cysteine residues C245, C277 and C487, all which have been implicated in a redox dependent Src activation mechanism. Furthermore, increased Src activity following acrolein exposure was confirmed using an in vitro kinase activity assay and recombinant Src in a cell free system.

To study the effects of acute acrolein exposure on lung epithelial function we exposed cultured mouse tracheal epithelial cells (MTECs) to acrolein and show impaired epithelial barrier function, measured by a decrease in trans epithelial resistance (TER) and increased epithelial permeability to FITC-dextran, which could be prevented using the Src inhibitor PP2. Src inhibition also attenuated acrolein-induced loss of E-cadherin and ZO-1. Acute exposure of C57BL/6 mice to acrolein (5 ppm for 4 hrs) led to increased epithelial permeability, measured by enhanced leakage of i.v. injected FITC-dextran into the airspaces, and induction of HO-1 in the lung while chronic acrolein exposure resulted features of epithelial to mesenchymal transition including a reduction of E-cadherin, increased vimentin, increased expression of MMP9 and increased collagen deposition. Chronic acrolein exposure in vitro resulted in a reduction of E-cadherin that could be prevented using the Src inhibitor AZD0530.

Together our studies demonstrate that Src is a direct target for acrolein and plays an important role in epithelial alterations due to acrolein exposure. This work provides further insight into a potential mechanism involved in the development of cigarette smoke related disease and could provide a potential target for novel therapeutics.

Language

en

Number of Pages

97 p.

Available for download on Friday, May 25, 2018

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