Diacetyl Induces Amphiregulin Shedding in Pulmonary Epithelial Cells and in Experimental Bronchiolitis Obliterans

Abstract

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air-liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-a-converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-a protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.

Expression and Function of the Epidermal Growth Factor Receptor in Physiology and Disease

“…Expression of both EGF and EGFR markedly increases in asthmatic airways (13) and has been shown to provide protection against epithelial injury by stimulating mucin production and inflammatory cell recruitment to the site of injury to initiate repair (64). However, other studies suggest that amphiregulin-dependent activation of EGFR can mediate development of pulmonary fibrosis through activation of fibroblast proliferation (260,580,642), that overexpression TGF-␤ correlates with development of idiopathic or bleomycin-induced pulmonary fibrosis (271,325,326,336), and that EGFR inhibitors reduce pulmonary fibrosis (209,239,562). Local release of AREG by human mast cells plays an important role in lung fibrosis by promoting the proliferation of primary human lung fibroblasts.…”

Models of toxicity of diacetyl and alternative diones

“…It is thought that because of these chemical features α-diketones including DA can affect protein structure and function as a consequence (Miller and Gerrard 2005). There are four representative in vitro studies that address the possible nature of the cellular injury that DA may cause (Zaccone, Goldsmith et al, Fedan, Dowdy et al 2006, More, Raza et al 2012, Kelly, Sun et al 2014). For example, it has been shown that exposure to 10 mM DA increased epithelial layer permeability as determined by decreased measures of transepithelial resistance (Fedan, Dowdy et al 2006).…”

Assessment of biological responses of EpiAirway 3-D cell constructs versus A549 cells for determining toxicity of ambient air pollution

“…There are 7 studies where EpiAirway ™ cells were exposed at ALI conditions (Balharry et al, 2008, Iskandar et al, 2013, Kelly et al, 2014, Künzi et al, 2013, Mathis et al, 2013, Seagrave et al, 2007, Seagrave et al, 2010, Sexton et al, 2011). Only one study that used diesel exhaust (DE) was relevant to ambient air pollution (Seagrave et al, 2007).…”