Activation, EGFR leads to Decreased ciliated cell apoptosis

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.

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. 

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).

Unknown