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Relationship: 2469
Title
Activation, EGFR leads to Goblet cell metaplasia
Upstream event
Downstream event
Key Event Relationship Overview
AOPs Referencing Relationship
Taxonomic Applicability
Sex Applicability
Sex | Evidence |
---|---|
Mixed | Moderate |
Life Stage Applicability
Term | Evidence |
---|---|
All life stages | Low |
Key Event Relationship Description
Airway epithelial injury can be caused by various inhalation exposures (e.g. cigarette smoke, sulfur dioxide, endotoxin, viruses). Subsequent tissue repair processes are thought to initiate the transdifferentiation process, whereby ciliated epithelial cells first dedifferentiate and then redifferentiate to goblet cells, without an apparent increase in the total number of epithelial cells resulting in goblet cell metaplasia (Lumsden et al., 1984; Shimizu et al., 1996; Reader et al., 2003). EGFR was shown to be a key player in this process in both murine and human airway epithelia (Tyner et al., 2006; Hao et al., 2011; Habibovic et al., 2016).
Evidence Collection Strategy
Evidence Supporting this KER
Goblet cell metaplasia was shown to occur following the activation of EGFR-mediated anti-apoptotic signaling in ciliated epithelial cells (Tyner et al., 2006). Subsequent stimulation by proinflammatory stimuli such as the Th2 cytokines interleukin (IL)-4 and IL-13 then promotes transdifferentiation of ciliated cells into goblet cells, thereby increasing the number of goblet cells (“second hit hypothesis”) in mouse tracheal epithelium and airway epithelia of COPD patients (Curran and Cohn, 2010). In vitro, EGFR can be activated by ROS or IL-13 to lead to ciliated cell transdifferentiation. IL-13 stimulates transdifferentiation of ciliated epithelial cells to goblet cells through EGFR activation increasing MMP/ADAM activity and ERK/MAPK activation (Casalino-Matsuda et al., 2006; Yoshisue and Hasegawa, 2004; Tyner et al., 2006).
Biological Plausibility
Two studies showed EGFR involvement in a decrease in goblet cell and increase in ciliated cell numbers or cell-specific marker expression (Yoshisue and Hasegawa, 2004; Casalino-Matsuda et al., 2006). Other studies demonstrated ciliated cell transdifferentiation in response to ILß13 in an EGFR-dependent manner in a mouse viral infection model and mouse tracheal epithelial cells in vitro (Tyner et al., 2006), rat nasal epithelial cells (Lee et al., 2000), and human airway epithelial cells (Kim et al., 2002; Hao et al., 2011). The KER is therefore highly plausible.
Empirical Evidence
Cigarette smoke exposure resulted in increased goblet cell numbers and extensive AB/PAS staining, which were considered signs of goblet cell metaplasia (the outcome of transdifferentiation), in the airways of Sprague-Dawley rats. These effects were greatly diminished in animals pre-treated daily with the EGFR kinase inhibitor BIBX 1522 (Takeyama et al., 2001). Similarly, plugging of F344 rat bronchi with agarose resulted in goblet cell metaplasia as evidenced by histology and increased AB/PAS staining, and the extent of airway remodeling was markedly lower in animals treated concurrently with BIBX 1522 (Lee et al., 2000). EGFR-dependent goblet cell metaplasia was also observed in primary human bronchial epithelila cells exposed to ROS (Casalino-Matsuda et al., 2006), nasal epithelial cells of asthmatics and lungs of patients with diffuse panbronchiolitis that exhibited goblet cell metaplasia and concomitant EGFR expression or activation (Habibovic et al., 2016; Kim et al., 2004).
Uncertainties and Inconsistencies
It is not well known how transdifferentiation of ciliated cells occurs in humans. Under normal conditions, lung epithelial cells (except basal cells) are terminally differentiated (Donnelly et al., 1982; Breuer et al., 1990; Rawlins and Hogan, 2008), and which signals initiate the dedifferentiation/redifferentiation process is not well understood. The available evidence is indirect or correlative. It also is not in agreement with other studies, which showed that ciliated cells do not give rise to goblet cells during airway remodeling in rodents and humans and with studies that provide evidence for increased goblet cell proliferation and goblet cell hyperplasia (Pardo-Sargenta et al., 2013; Hays et al., 2006; Lawson et al., 2002; Tesfaigzi et al., 2004; Taniguchi et al., 2011; Park et al., 2006; Turner et al., 2011).
Known modulating factors
Unknown
Quantitative Understanding of the Linkage
Response-response Relationship
A 30-min treatment of primary human bronchial epithelial cells at the air-liquid interface with 0.6 mM xanthine and 0.5 units xanthine oxidase resulted in a 2-fold increase in EGFR phosphorylation. Daily 30-min treatments of primary human bronchial epithelial cells at the air-liquid interface with 0.6 mM xanthine and 0.5 units xanthine oxidase for 3 days resulted in goblet cell metaplasia as evidenced by an increase in the numbers of MUC5AC-positive cells from 3.3 ± 1.2%to 21.6 ± 3.4%, a decrease in ciliated cell numbers, and increased MUC5AC protein expression (32.5 + 9.3% above PBS control). This effect could be inhibited by EGFR blockade with neutralizing antibodies (Casalino-Matsuda et al., 2006).
Cigarette smoke exposure at 8 cigarettes (nonfiltered cigarettes; 1.2 mg nicotine, 12 mg condensate) per day for 5 days markedly increased AB/PAS staining in airway epithelia of male Sprague-Dawley rats and goblet cell numbers from 40 ± 19 to 167 ± 19 cells/mm of epithelium, while decreasing the number of ciliated cells (not quantified). Treatment with the EGFR inhibitor BIBX1522 during exposure dose-dependently decreased goblet cell numbers, with a maximal decrease seen for 3 mg/kg inhibitor (51 ± 19 cells/mm epithelium) (Takeyama et al., 2001).
Intranasal insitillation of 0.1 mg LPS (E.coli 0111:B4) once a day for 3 consecutive days induced goblet cell metaplasia in the nasal epithelium (as judged by histopathology), with an approx. 50% increase in AB/PAS-stained epithelium compared to untreated controls. Intranasal insitllation of AG1478 1 hr after LPS instillation dose-dependently decreased the % AB/PAS-stained epithelium, with a maximal decrease seen at 10 mg/kg (Takezawa et al., 2016).
Induction of airway inflammation with 50 µg house dust mite (1.27 endotoxin units/mg) for 5 days/week for 3 weeks resulted in a 3-fold increase of pEGFR-positive cells in the bronchiolar epithelium of C57Bl/6 mice. Six-week treatment led to goblet cell metaplasia as evidenced by extensive AB staining and an approx. 10-fold increase in Clca3-positive cells in the animals' airways. Concomitant treatment with 100 mg/kg erlotinib six times a week for 6 weeks reduced the number of Clca3-positive cells by ca. 5-fold (Le Cras et al., 2011). Using the same model with a 3-week treatment demonstrated goblet cell metaplasia as judged by increased PAS staining in the airway epithelium and ca. 10-, 5-, and 4-fold increases in expression of goblet cell metaplasia-related genes Muc5ac, Clca1, and Postn, respectively (Habibovic et al., 2016).
Pyocyanin, a redox-active exotoxin of Pseudomonas aeruginosa, caused goblet cell metaplasia in C57Bl/6 mice after 3-week treatment (25 µg/day). PAS staining increased by ca. 30%; the percentage of Muc5ab-positive cell in bronchial epithelium increased 6.4-fold and in bronchiolar epithelium 11.4-fold. This was accompanied by increased EGFR phosphorylation coincident with AB/PAS staining. Moreover, 24-h pyocyanin treatment of H292 lung cancer cells and immortalized human bronchial epithelial 16-HBE cells with physiologically relevant concentrations from 1.3 to 25 µg/mL pyocyanin significantly increased MUC5B mRNA expression 3.8- to 13.4-fold and increased levels of pEGFR 11.8- to 18.3-fold (1.6 to 12.5 µg/mL pyocyanin) (Hao et al., 2012).
Male Sprague–Dawley rats that were exposed to 3 ppm acrolein for 6 h a day, for 12 days developed goblet cell metaplasia (as judged by histopathology), increasing the % AB/PAS-positive stained epithelium from ca. 5% (in air controls) to 35%. This was accompanied by a 1.6-fold change in EGFR phosphorylation, a nearly 15% increase in Muc5ac-positive stained cells, a ca. 3-fold increase in Muc5ac mRNA expression and a ca. 4-fold increase in protein expression (Chen et al., 2010).
Exposure of BALB/c mice to 1.0 ppm O3 , but not lower concentrations, for 3 h a day, for 7 days, caused goblet cell metaplasia in the bronchial epithelium (as judged by histopathology). Exposure to 0.25, 0.5, and 1.0 ppm O3 also increased levels of p-EGFR (Y1068) in the bronchial epithelium and lung tissues in a dose-dependent manner, with the maximal (an approx. 2-fold) increase over controls reached at 0.5 ppm (Feng et al., 2016).
Exposure of female Sprague-Dawley rats to wood smoke (40 g of China fir sawdust was smoldered) for 1 h four times per day, five days per week, for three months caused goblet cell metaplasia in the airways (as judged by histopathology), a 2-fold increase in Muc5ac gene expression, an increase in the % AB/PAS-positive stained epithelium from approx. 6% (air controls) to ca. 17%, an increase in Muc5ac-positive stained cells from approx. 5% (air controls) to ca. 25%. EGFR activation by wood smoke particle exposure was confirmed in vitro: Treatment of H292 lung cancer cells with wood smoke particulate matter (0.5 to 24 μg/mL) for 24 h increased MUC5AC gene and protein expression in a dose-dependent manner, with maximal increases of ca. 40-fold and 5-fold, respectively, seen for 8 μg/mL. Treatment of H292 lung cancer cells with 8 μg/mL wood smoke particulate matter for various times (2–36 h) increased MUC5AC gene and protein expression in a time-dependent manner, with significant increases starting at 24 h. Treatment of H292 lung cancer cells with 8 μg/mL wood smoke particulate matter for up to 24 h also increased EGFR phosphorylation in a time-dependent manner, with a significant and sustained increase (approx. 3-fold compared to control) seen from 1 h onwards. Pretreatment with 0.5 μg/mL neutralizing anti-EGFR antibody for 1 h completely abrogated the increases in EGFR phosphorylation and MUC5AC gene expression (Huang et al., 2017).
Exposure of male Sprague-Dawley rats to smoke from five cigarettes (2R4F, University of Kentucky) a day for 5 days resulted in goblet cell metaplasia in the airways (as judged by histopathology) and an approx. 70% increase in AB/PAS-stained epithelium. EGFR activation by cigarette smoke exposure was confirmed in vitro: Treatment of NCI-H292 lung cancer cells with cigarette smoke extract (2R4F cigarette smoke was withdrawn into a 35-mL polypropylene syringe at a rate of one puff/min and then bubbled slowly into 20 mL of RPMI 1640 medium containing 50mM HEPES buffer) for 15 min increased EGFR phosphorylation, and dose-dependently increased MUC5AC protein expression, with a significant increase starting from 3 puffs (ca. 130% over control) and a maximum increase of nearly 250% over control seen with 9 puffs (Lee et al., 2006).
Intratracheal instillation of LPS (P. aeruginosa serotype 10; 200 or 300 μg in 300 μL PBS) in male Sprague-Dawley rats caused goblet cell metaplasia in the airways, with 42.31 ± 3.36, 45.46 ± 2.24, and 63.13 ± 4.6% AB/PAS-positive staining at 3, 5, and 7 days after low-dose LPS instillation, respectively, and 71.6 ± 2.56% AB/PAS-positive staining at 7 days after high-dose LPS instillation. MUC5AC protein expression in the bronchial epithelium of the control and LPS groups (300 μg, 7 days post-instillation) were 5.46 ± 4.68 and 75.32 ± 4.53, respectively, and the mean % area of bronchiolar epithelium showing EGFR-positive staining in the LPS group was 24.54 ± 5.78% (compared to absent staining in the control) (Kim et al., 2004).
Time-scale
Induction of airway inflammation with 50 µg house dust mite (1.27 endotoxin units/mg) for 5 days/week for 3 weeks resulted in a 3-fold increase of pEGFR-positive cells in the bronchiolar epithelium of C57Bl/6 mice. Six-week treatment led to goblet cell metaplasia as evidenced by extensive AB staining and an approx. 10-fold increase in Clca3-positive cells in the animals' airways. Concomitant treatment with 100 mg/kg erlotinib six times a week for 6 weeks reduced the number of Clca3-positive cells by ca. 5-fold (Le Cras et al., 2011). Using the same model with a 3-week treatment demonstrated goblet cell metaplasia as judged by increased PAS staining in the airway epithelium and ca. 10-, 5-, and 4-fold increases in expression of goblet cell metaplasia-related genes Muc5ac, Clca1, and Postn, respectively (Habibovic et al., 2016).
Instillation of agarose plugs (0.7-0.8 mm diameter, 4% agarose II) in Fischer rats caused a time-dependent increase in goblet cell area (by AB/PAS staining), which was detectable as early as 24 h and was greatest 72 h post-instillation. The AB/PAS-stained area increased from 0.1 ± 0.1% in control animals to 4.7 ± 1.4, 13.3 ± 0.7, and to 19.1 ± 0.7% at 24, 48, and 72 h post-instillation, respectively. Goblet cell numbers increased from 0 to 13.1 ± 5.6, 25.7 ± 15.0, and 51.5 ± 9.0 cells/mm basal lamina at 24, 48, and 72 h post-instillation, respectively. Treatment of the animals prior and after instillation with 80 mg/kg/day BIBX1522 resulted in a marked decrease in the AB/PAS-stained area (<5% at 72 h). Of note, the AB/PAS staining in the airway epithelia coincided with EGFR staining (Lee et al., 2000).
Intratracheal instillation of LPS (P. aeruginosa serotype 10; 200 or 300 μg in 300 μL PBS) in male Sprague-Dawley rats caused goblet cell metaplasia in the airways, with 42.31 ± 3.36, 45.46 ± 2.24, and 63.13 ± 4.6% AB/PAS-positive staining at 3, 5, and 7 days after low-dose LPS instillation, respectively, and 71.6 ± 2.56% AB/PAS-positive staining at 7 days after high-dose LPS instillation. MUC5AC protein expression in the bronchial epithelium of the control and LPS groups (300 μg, 7 days post-instillation) were 5.46 ± 4.68 and 75.32 ± 4.53, respectively, and the mean % area of bronchiolar epithelium showing EGFR-positive staining in the LPS group was 24.54 ± 5.78% (compared to absent staining in the control) (Kim et al., 2004).
Known Feedforward/Feedback loops influencing this KER
Unknown
Domain of Applicability
Two mouse studies demonstrated ciliated cell transdifferentiation and goblet metaplasia in response to viral infection and/or IL-13 treatment (Tyner et al., 2006; Fujisawa et al., 2008). Indirect evidence is also available from rat studies and studies on human cells and clinical samples.
References
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