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Activation, EGFR leads to Increased goblet cell proliferation
Key Event Relationship Overview
AOPs Referencing Relationship
Life Stage Applicability
Key Event Relationship Description
The EGF receptor family comprises 4 members, EGFR (also referred to as ErbB1/HER1), ErbB2/Neu/HER2, ErbB3/HER3 and ErbB4/HER4, all of which are transmembrane glycoproteins with an extracellular ligand binding site and an intracellular tyrosine kinase domain. Receptor-ligand binding induces dimerization and internalization, subsequently leading to activation of the receptor through autophosphorylation (Higashiyama et al., 2008). Following EGFR activation, classical downstream activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, also known as Raf/Ras/MAPK/ERK pathway, increases airway epithelial cell proliferation (Lemjabbar et al., 2003; Kim et al., 2005; Hackel et al., 1999) and facilitates epithelial wound repair (Burgel & Nadel, 2004; Van Winkle et al., 1997; Allahverdian et al., 2010). While there is evidence that increased goblet cell proliferation may be the underlying cause of goblet cell hyperplasia (Silva et al., 2012), the key players mediating an increase in airway goblet cell numbers following EGFR activation are still largely unexplored.
Evidence Collection Strategy
Evidence Supporting this KER
Activation of EGFR through direct binding of its ligands EGF, TGFA or epigen or indirectly by oxidative stress following exposure to endotoxin, ozone, ultrafine particles or cigarette smoke induces airway epithelial cell proliferation. While not all studies specifically identify goblet cells as the proliferating cell population, others do - at least indirectly by quantifying the increase in MUC5AC expressing cells (Booth et al., 2001b; Booth et al., 2007; Taniguchi et al., 2011; Sydlik et al., 2006; Tamaoki et al., 2004; Tesfaigzi et al., 1998; Tesfaigzi et al., 2004; Harris et al., 2005; Tamiguchi et al., 2001).
Although there are no studies providing direct evidence for proliferation of goblet cells in the lung following EGFR activation, there is direct in vitro evidence in conjunctival goblet cells (Gu et al., 2008; Shatos et al., 2008) and in murine embryonic colon (Duh et al., 2000). However, multiple studies indirectly demonstrate a link between exposure to stressors known to activate EGFR and increases in goblet cell numbers.
Studies specific to goblet cells have been performed in cultured rat conjunctival goblet cells where EGFR ligands EGF, TGFA, HB-EGF stimulate an increase in phosphorylated EGFR as well as proliferation (Gu et al., 2008) and in human cultured conjunctival goblet cells where EGF caused a dose-dependent increase in proliferation (Shatos et al., 2003). In lungs, studies either look generally at epithelial cell proliferation, not specifically goblet cells, or measure an increase in goblet cells, not specifically proliferation (Casalino-Matsuda et al., 2006). IL13-induced proliferation is mediated by EGFR in mouse primary airway epithelial cells and human bronchial epithelial cell, and this proliferation could be inhibited by EGFR blockade (Booth et al., 2001; Booth et al., 2007; Taniguchi et al., 2011). Proliferation of rat lung epithelial cells increased dose-dependently following treatment with both ultrafine carbon black and amorphous silica particles (14 nm diameter), but not larger carbon black particles (260 nm diameter) (Sydlik et al., 2006).
Uncertainties and Inconsistencies
The majority of studies supporting this KER did not specifically measure goblet cell proliferation. Instead, many studies measured an increase in mucin production upon EGFR activation, equating this with an increase in goblet cell numbers (Takeyama, et al. 2008; Shim et al. 2001; Casalino-Matsuda et al. 2006).
Basal epithelial cells which exhibit stem cell-like properties have also been postulated to function as a source of goblet cells in injured airways, utilizing cell fate pathways that favor secretory cells over other cell populations (Rock et al., 2009). However, both in vitro studies and studies in mouse models of asthma, COPD, and viral infection indicate that EGFR activation leads to transdifferentiation of ciliated or club cells into goblet cells, which is referred to as goblet cell metaplasia, and that goblet cell metaplasia more likely contributes to the expansion of this cell population in the airways (Tyner et al., 2006; Lumsden et al., 1984; Reader et al., 2003; Turner et al., 2011; Evans et al., 2004; Lamb et al., 1968; Shimizu et al., 1998).
Known modulating factors
Quantitative Understanding of the Linkage
Daily xanthine/xanthine oxidase treatment of human primary bronchial epithelial cells grown at the at the air-liquid interface for 3 days increased the number of MUC5AC-positive cells (from 3.3 ± 1.2% (PBS) to 21.6 ± 3.4%). This increase was prevented by pretreatment with anti-EGFR antibodies (Casalino-Matsuda et al., 2006).
Treatment of human primary bronchial epithelial cells with 1 ng/mL amphiregulin or HB-EGF for 24 h significantly increased the proportion of proliferating cells by approx. 20% (Ki-67 staining and flow cytometry). Treatments with higher concentrations of amphiregulin and HB-EGF did not further increase this proportion (Hirota et al., 2012).
Treatment of murine primary airway epithelial cells growing at the air-liquid interface with 20 ng/mL IL-13 for 14 days doubled the numbers of cells per 5 high-power fields (cell count on hematoxylin/eosin-stained culture sections) and the numbers of PCNA-positive cells by ca. 20% (immunohistochemistry) compared to PBS controls. In addition, [3H]-thymidine incorporation was significantly increased following IL-13 treatment (from ca. 22000 cpm in controls to ca. 33000 cpm). Co-treatment of cultures with IL-13 and the EGFR inhibitor AG1478 (0.25 µg/mL or 0.5 µg/mL) prevented the proliferative response, with greater effects seen at the higher AG1478 concentration (Taniguchi et al., 2011).
Treatment of rat conjunctival goblet cells with 0.1 µM EGF significantly increased phosphorylation of the EGFR by 28.6 ± 7.6- and 29.2 ± 3.2-fold at 1 and 5 minutes, respectively. At the same concentration, 24-hour EGF treatment increased proliferation 4.9 ± 1.8-fold. Pre-treatment with 0.1 µM AG1478 significantly inhibited the EGF response by 87% ± 8%. (Shatos et al., 2008). Similar observations were made with human conjunctival goblet cells: Treatment with 0.1 µM EGF significantly increased proliferation 1.5 ± 0.3-fold above basal (Li et al., 2013). In another study in rat conjunctival goblet cells, treatment with 0.1 µM EGF, TGF-α, or HB-EGF for 5 min significantly stimulated the phosphorylation of EGFR by 21.1 ± 2.5, 22.2 ± 6.7, and 19.9 ± 6.0 fold above basal level, and 24-h treatment stimulated cell proliferation 1.3 ± 0.1 fold, 1.2 ± 0.02 fold, and 1.1 ± 0.04 fold compared to untreated cells (WST-1 assay). These latter results were also confirmed by Ki-67 immunofluorescent staining, showing increases in positive cells by 61.4%, 38.1%, 27.8% following EGF, TGF-α, and HB-EGF treatment compared to untreated cells (Gu et al., 2008).
Approx. 50% of AB/PAS-positive cells were BrdU-positive in the airways of rats at day 2 following instillation of F344 rats with 1 mg LPS, suggesting that they may have been derived from proliferating cells (Tesfaigzi et al., 2004). Intranasal and intratracheal administration of LPS to rats have previously been shown to activate EGFR (Takezawa et a., 2016; Shan et al., 2017).
Treatment of primary human bronchial epithelial cells, grown at the air-liquid interface for 9 days, with 5 ng/mL TGFa or 10 ng/mL IL-13 for 24 h resulted in 1.5- to 2-fold increases in cell numbers (by [3H]thymidine incorporation). These increases were prevented by co-incubation with the EGFR inhibitor AG1478, with maximal decreases (approx. 30%) in cell numbers seen at 5 ng/mL AG1478 (Booth et al., 2001a). Although this study did not specify the affected cell type as goblet cells, another study by the same group using the same model showed that the percentage of AB/PAS–positive, mucus-producing cells increased following IL-13 treatment (Booth et al., 2001b).
Proliferation of rat conjunctival goblet cells was observed following stimulation with 100 µM EGF, TGFa or HB-EGF after 14 h, peaking at 18 h (Gu et al., 2008).
The average height of epithelial cells did not increase and hypertrophic airway remodeling was not seen until 48 h after instillation of F344 rats with 1 mg LPS, 46 mucous cells per millimeter of basal lamina (vs 17 in controls) of which 24 mucous cells per millimeter of basal lamina were BrdU-positive (vs 1 in controls) (Tesfaigzi et al., 2004). Intranasal and intratracheal administration of LPS to rats have previously been shown to activate EGFR (Takezawa et a., 2016; Shan et al., 2017).
Treatment of rat lung epithelial RLE-6TN cells with 10 μg/cm2 ultrafine particles moderately but significantly increased DNA synthesis after 24 h (approx. 1.6-fold, BrdU incorporation; approx. 1.5-fold, PCNA staining). Longer incubation did not increase proliferation further. A significant increase in pEGFR (approx. 3-fold compared to untreated) was observed as early as 2 min following addition of ultrafine particles (10 μg/cm2), and a second more persistent signal was observed from 120 up to 480 min (Sydlik et al., 2006).
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Epithelial cell proliferation mediated by EGFR has been studied in human (Booth et al., 2001; Booth et al., 2007), mouse (Taniguchi et al., 2011) and rat (Sydlik et al., 2006).
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