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Activation, EGFR leads to Decreased ciliated cell apoptosis
Key Event Relationship Overview
AOPs Referencing Relationship
Life Stage Applicability
Key Event Relationship Description
Exposure to airborne toxicants and pathogens causing oxidative stress as well as oxidative stress induced by inflammatory responses to environmental exposures 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 Collection Strategy
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. Oxidative stress following inhalation exposure increased Bcl-2 mRNA and protein levels in human and rat airway epithelial cells (Casalino-Matsuda et al., 2006; Foster et al., 2003; Lee et al., 2011; Tesfaigzi et al., 1998; Tesfaigzi et al., 2000; Petecchia et al., 2009). Neutralization of Bcl-2 expression in rat nasal epithelium reduced goblet cell metaplasia (Harris et al., 2005), and treatment of OVA-sensitized Balb/c mice with the EGFR inhibitor gefitinib decreased Bcl-2 expression and increased apoptosis (Song et al., 2016).
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 decreased the number of ciliated cells and increased Bcl-2 mRNA expression, and this could be prevented by pre-incubation with anti-EGFR antibodies (Casalino-Matsuda et al., 2006). 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, although it is unclear whether only ciliated cells were affected (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 the latter study 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 provide indirect support of a variety of stressors known to activate EGFR causing apoptosis of airway epithelial cells, although the identity of the affected cells is not always specified (Tesfaigzi et al., 2000; Tesfaigzi et al., 1998; Song et al., 2016; Sydlik et al., 2006). Other studies make no reference to the airway epithelial cell type that is affected by apoptosis.
Known modulating factors
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 (not quantified) 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 (not quantified), and EGFR blockade with PD153035 caused a dose-dependent decrease in ciliated cell numbers, with a maximum decrease (50%) seen at 0.3 µM 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 rat alveolar epithelial cells (RLE-6TN) exposed to 10 µg/cm2 ultrafine carbon black particles for as little as 2 min. After an 8-h 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).
Treatment of NCI-H292 lung cancer cells with 10 ng/mL the EGFR ligand TGFa for 24 h increased Bcl-2 protein expression by 50%, which was prevented by pretreatment with AG1478 (Takeyama et al., 2008).
Treatment of immortalized human bronchial epithelial BEAS-2B cells with 0.1 mM Ni2+ for 24 h significantly increased EGFR mRNA expression (1.59 ± 0.04-fold compared to untreated), EGFR phosphorylation and Bcl-2 protein expression levels (Giunta et al., 2012).
Treatment of primary human airway epithelial cells, grown as a monolayer, with 10 µg/mL cigarette smoke extract for 48 h increased EGFR phosphorylation by approx. 50%, Bcl-2 mRNA expression ca. 2.5-fold and Bcl-2 protein expression ca. 4.5-fold (Hussain et al., 2018).
In the small airways of male Sprague-Dawley rats that were exposed to cigarette smoke generated from 5 unfiltered cigarettes for 30 min twice daily for 4 weeks, the rate of apoptosis, as assessed by TUNEL staining, increased by 50%, and phosphorylation of EGFR at Tyr1068 increased 5.1-fold (Ning et al., 2013).
EGFR was persistently activated in ciliated cells in 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 (approx. 10% increase in ß-tubulin-positive cells), a decreased number of goblet cells (approx. 10% decrease in Muc5ac-positive cells), but not with the expression of the proliferation markers BrdU, Ki67 or PCNA (Tyner et al., 2006).
In rat alevolar epithelial cells, treatment with ultrafine carbon black particles resulted in phosphorylation of EGFR after 2 minutes and a second, more persistent activation of the receptor from 120 to 480 min. Caspase 3 activity increased in a time-dependent manner, starting at 4 h and reaching a maximum after 8 h (Sydlik et al., 2006).
Intranasal influenza virus infection (7.5 PFU of H1N1) led to the loss of ciliated epithelial cells (acetylated tubulin–positive) by day 3, with recovery of the epithelial barrier by day 14 (Fujino et al., 2019). EGFR promotes uptake of influenza viruses and is activated (ca. 2-fold increase in phosphorylated EGFR) at 10 min following infection of immortalized human bronchial epithelial BEAS-2B cells (Ueki et al., 2013).
Chronic, 6-month exposure of immortalized human bronchial epithelial BEAS-2B cells to 0.25 μm Cr(VI) activated EGFR constitutively, beginning from month 2, and permanently elevated Bcl-2 protein levels (Kim et al., 2015).
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The studies that support epithelial cell apoptosis induced by EGFR activation include rat and mouse in vivo experiments and in vitro experiments in human and rat cells.
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.
Fujino, N., Brand, O.J., Morgan, D.J., Fujimori, T., Grabiec, A.M., Jagger, C.P., et al. (2019). Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases. J. Exp. Med. 216, 2184-2201.
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? Respirol. 20, 722-729.
Giunta, S., Castorina, A., Scuderi, S., Patti, C., and D’agata, V. (2012). Epidermal growth factor receptor (EGFR) and neuregulin (Neu) activation in human airway epithelial cells exposed to nickel acetate. Toxicol. In Vitro 26, 280-287.
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.
Hussain, S.S., George, S., Singh, S., Jayant, R., Hu, C.-A., Sopori, M., et al. (2018). A Small Molecule BH3-mimetic Suppresses Cigarette Smoke-Induced Mucous Expression in Airway Epithelial Cells. Sci. Rep. 8(1), 13796-13796.
Kim, D., Dai, J., Fai, L.Y., Yao, H., Son, Y.-O., Wang, L., et al. (2015). Constitutive activation of epidermal growth factor receptor promotes tumorigenesis of Cr(VI)-transformed cells through decreased reactive oxygen species and apoptosis resistance development. J. Biol. Chem. 290, 2213-2224.
Ning, Y., Shang, Y., Huang, H., Zhang, J., Dong, Y., Xu, W., et al. (2013). Attenuation of cigarette smoke-induced airway mucus production by hydrogen-rich saline in rats. PLoS One 8, e83429.
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.
Petecchia, L., Sabatini, F., Varesio, L., Camoirano, A., Usai, C., Pezzolo, A., et al. (2009). Bronchial airway epithelial cell damage following exposure to cigarette smoke includes disassembly of tight junction components mediated by the extracellular signal-regulated kinase 1/2 pathway. Chest 135, 1502-1512.
Song, L., Tang, H., Liu, D., Song, J., Wu, Y., Qu, S., et al. (2016). The chronic and short-term effects of gefinitib on airway remodeling and inflammation in a mouse model of asthma. Cell. Physiol. Biochem. 38, 194-206.
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.
Ueki, I.F., Min-Oo, G., Kalinowski, A., Ballon-Landa, E., Lanier, L.L., Nadel, J.A., et al. (2013). Respiratory virus-induced EGFR activation suppresses IRF1-dependent interferon λ and antiviral defense in airway epithelium. J. Exp. Med. 210, 1929-1936.