Upstream eventActivation, EGFR
Decrease, Apoptosis of ciliated epithelial cells
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|EGFR Activation Leading to Decreased Lung Function||adjacent||Moderate||Low|
Life Stage Applicability
Key Event Relationship Description
Exogenous oxidative stress arising from e.g. the exposure to airborne toxicants and pathogens as well as oxidative stress induced by inflammatory responses mediate proteolytic cleavage of membrane-bound EGFR ligand precursors (Burgel and Nadel, 2004; Gao et al., 2015; Øvrevik et al., 2015). Subsequent ligand binding then activates the receptor tyrosine kinase in an autocrine fashion. Downstream of EGFR activation, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling elicits an anti-apoptotic response in ciliated cells favoring their survival (Tyner et al., 2006).
Evidence Supporting this KER
Tyner et al. (2006) reported that ciliated cell survival in a mouse airway infection model is promoted via EGFR-dependent PI3K/Akt signaling. Other studies provide indirect support of a variety of stressors known to activate EGFR causing apoptosis of airway epithelial cells, although the identity of cells is not always specified (Casalino-Matsuda et al., 2006; Tesfaigzi et al., 2000; Tesfaigzi et al., 1998; Song et al., 2016; Sydlik et al., 2006).
Downstream EGFR signaling involving the PI3K/AKT pathway regulating cell survival is well-documented, in particular in cancer cells where this pathway is often deregulated (e.g. Hennessey et al., 2005). However, to date, very few studies reported on the direct link between EGFR activation and the identity of airway epithelial cells undergoing apoptosis, so biological plausibility is only moderate.
In C57BL/6J mice infected with Sendai virus, persistent EGFR activation was seen in ciliated epithelial cells that coincided with increased ciliated and goblet cell numbers and could be suppressed by treatment with the EGFR inhibitor EKB-569. Moreover, in mouse tracheal epithelial cells in vitro, blockade of EGFR-PI3K signaling resulted in loss of ciliated cells that was accompanied by activation of caspase 3 (Tyner et al., 2006). Additional, indirect evidence shows that increased oxidative stress in primary bronchial epithelial cells grown at the air-liquid interface increased Bcl-2 mRNA expression, and this could be prevented by pre-incubation with anti-EGFR antibodies (Casalino-Matsuda et al., 2006). Similarly, instillation of LPS in Brown Norway rats or exposure of F/N344 rats to ozone also resulted in increased Bcl-2 expression in the airways (Tesfaigzi et al., 2000; Tesfaigzi et al., 1998). Moreover, injection of Bcl-2 antisense oligonucleotides prior to and following LPS instillation resulted in a significant reduction in Bcl-2-positive cells in rat nasal and lung epithelia (Harris et al., 2005). While these studies reported apoptosis to occur in goblet cells, the described mechanism may also apply to ciliated cells.
In summary, direct evidence with respect to the identity of airway epithelial cells undergoing proliferation or apoptosis is limited, therefore the empirical support for this KER weak.
Uncertainties and Inconsistencies
Some evidence available to date is correlative, demonstrating increased ciliated cell numbers following EGFR or EGFR/PI3K blockade. Other studies make no reference to the airway epithelial cell type that is affected by apoptosis.
Quantitative Understanding of the Linkage
In primary bronchial epithelial cells, treatment with 0.6 mM xanthine and 0.05 units xanthine oxidase (X/XO) for 30 min doubled the pEGFR/EGFR ratio and increased the Bcl-2/Actin mRNA ratio by ca. 5-fold. Both effects could be at least partially suppressed by pretreatment of cells with anti-EGFR neutralizing antibodies. Of note, this study was focused on goblet cells; however, the described mechanism may also apply to ciliated cells (Casalino-Matsuda et al., 2006).
Sendai virus, delivered at 2 × 105 PFUs intranasally to C57Bl/6J mice, caused EGFR activation in ciliated cells 21 days after inoculation, which was accompanied by an approx. 40-fold increase in ciliated cell number in the absence of proliferation as evidenced by the lack of BrdU staining. Complementary experiments in mouse tracheal epithelial cells grown at the air-liquid interface stimulated with 1 or 10 ng/mL EGF also demonstrated EGFR activation, and EGFR blockade with PD153035 caused a dose-dependent decrease in ciliated cell numbers, with a maximum decrease (50%) seen at 0.3 microM inhibitor, while the number of TUNEL- and caspase 3-positive cells nearly tripled and quadrupled, respectively (Tyner et al., 2006).
EGFR phosphorylation was increased approx. 3-fold in alveolar epithelial cells (RLE-6TN) exposed to 10 microg/cm2 ultrafine carbon black particles for as little as 2 minutes. After an 8-hour exposure, DNA fragmentation had doubled and caspase 3 activity tripled, but the latter could be almost completely suppressed by pretreatment with the EGFR inhibitor AG1478 (Sydlik et al., 2006).
EGFR was persistently activated in ciliated cells C57Bl/6J mouse lungs at day 21, but not day 12, post-inoculation with Sendai virus, which coincided with an increased number of ciliated cells but not with proliferation markers BrdU, Ki67 or PCNA (Tyner et al., 2006).
In rat alevolar epithelial cells, treatment with ultrafine caron black particles results in phosphorylation of EGFR after 2 minutes and a second, more persistent activation of the receptor from 120 to 480 minutes. Caspase 3 activity increases in a time-dependent manner, starting at 4 hours and reaching a maximum after 8 hours (Sydlik et al., 2006).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The studies that support epithelial cell apoptosis induced by EGFR include rat, mouse and human in vitro experiments.
Burgel, P., and Nadel, J. (2004). Roles of epidermal growth factor receptor activation in epithelial cell repair and mucin production in airway epithelium. Thorax 59, 992-996.
Casalino-Matsuda, S., Monzón, M., and Forteza, R. (2006). Epidermal Growth Factor Receptor Activation by Epidermal Growth Factor Mediates Oxidant-Induced Goblet Cell Metaplasia in Human Airway Epithelium. Am J Respir Cell Mol Biol 34, 581–591.
Curran, D., and Cohn, L. (2010). Advances in mucous cell metaplasia: a plug for mucus as a therapeutic focus in chronic airway disease. Am J Respir Cell Mol Biol 42, 268–275.
Gao, W., Li, L., Wang, Y., Zhang, S., Adcock, I.M., Barnes, P.J., Huang, M., and Yao, X. (2015). Bronchial epithelial cells: The key effector cells in the pathogenesis of chronic obstructive pulmonary disease? Respirology 20, 722-729.
Hennessy, B.T., Smith, D.L., Ram, P.T., Lu, Y. and Mills, G.B., 2005. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4, 988-1004.
Øvrevik, J., Refsnes, M., Låg, M., Holme, J.A., and Schwarze, P.E. (2015). Activation of proinflammatory responses in cells of the airway mucosa by particulate matter: Oxidant- and non-oxidant-mediated triggering mechanisms. Biomolecules 5, 1399-1440.
Sydlik, U., Bierhals, K., Soufi, M., Abel, J., Schins, R.P.F., and Unfried, K. (2006). Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R. Am J Physiol Lung Cell Mol Physiol 291, L725–L733.
Tesfaigzi, J., Hotchkiss, J.A., and Harkema, J.R. (1998). Expression of the Bcl-2 protein in nasal epithelia of F344/N rats during mucous cell metaplasia and remodeling. Am J Resp Cell Mol Biol 18, 794-799.
Tesfaigzi, Y., Fischer, M.J., Martin, A.J., and Seagrave, J. (2000). Bcl-2 in LPS- and allergen-induced hyperplastic mucous cells in airway epithelia of Brown Norway rats. Am J Physiol Lung Cell Mol Physiol 279, L1210-L1217.
Tyner, J., Tyner, E., Ide, K., Pelletier, M., Roswit, W., Morton, J., Battaile, J., Patel, A., Patterson, G., Castro, M., et al. (2006). Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals. J Clin Invest 116, 309–321.