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Activation, EGFR leads to Increase, goblet cell number
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|EGFR Activation Leading to Decreased Lung Function||adjacent||High||High||Karsta Luettich (send email)||Under development: Not open for comment. Do not cite||Under Development|
Life Stage Applicability
Key Event Relationship Description
Mucus hypersecretion in the airway epithelium is a key characteristic of many lung diseases, including chronic obstructive pulmonary disease (COPD), asthma and cystic fibrosis (Yoshida & Tuder, 2007). Epidermal growth factor receptor (EGFR)-mediated signaling has been identified as the key pathway that leads to increased mucus production in the airways (Burgel & Nadel, 2004). The KER is restricted to EGFR activation that results in the increase in the number of goblet cells based on evidence of goblet cell metaplasia, hyperplasia, or increased proliferation/differentiation of goblet cells. In metaplasia, the increase in goblet cells occurs at the expense of other cell types, such as ciliated cells (Curran & Cohn, 2010). EGFR-mediated metaplasia or hyperplasia has been demonstrated in response to allergens (Song et al, 2016; Takeyama et al, 1999), viruses (Tyner et al, 2006), reactive oxygen species (ROS) (Casalino-Matsuda et al, 2006), and cigarette smoke (Takeyama et al, 2001a) in the respiratory epithelium. The EGFR mediated proliferation of the goblet cells has been demonstrated in the mucus and tear producing conjunctiva of the rat and human eye (Gu et al, 2016; Li et al, 2013; Shatos et al, 2008). Finally, EGFR has been shown to also augment goblet cell maturation in colon organ cultures (Duh et al, 2000) and favor goblet cell differentiation from human airway basal cells (Parker et al, 2015).
Evidence Collection Strategy
The relevant papers supporting the KER were identified using keywords: “EGFR” AND “goblet”. Only the studies showing increased number of goblet cells (i.e., not only mucin production), either in the form of metaplasia, hyperplasia, increased proliferation, or differentiation of goblet cells, by the stressor were chosen. The studies that did not demonstrate involvement of EGFR in the increase of the number of goblet cells, either by stimulation with EGFR ligands or by blocking EGFR signaling (e.g., chemicals, antibodies) were omitted as weak evidence.
Evidence Supporting this KER
The involvement of EGFR in regulating the number of goblet cells has been demonstrated in both in vitro and in vivo studies. The treatment of human bronchial epithelial cells differentiated and cultured in air-liquid interphase with EGFR ligands (e.g., amphiregulin or HB-EGF) significantly increased the number of goblet cells (Hirota et al., 2012; Jia et al., 2021). Moreover, ALI cultures of bronchial epithelia from asthmatic children differentiated without EGF had less goblet cells than those differentiated in the presence of EGF (Parker et al., 2015). EGFR ligands also stimulated proliferation of cultured rat and human conjunctival goblet cells (Gu et al., 2008; Li et al., 2013; Shatos et al., 2008). EGFR augments goblet cell maturation and increases colonic goblet cell index during the colonic organ culture (Duh et al., 2000). EGFR also mediates the increase in goblet cells number by a variety of stressors. Xanthine oxidase increased the number of Muc5Ac positive cells in bronchial epithelial ALI cultures, and this increase was blocked by anti-EGFR antibodies (Casalino-Matsuda et al., 2006). Moreover, EGFR mediated the increase in goblet cells in the mice airways challenged with allergens (Jia et al., 2021; Le Cras et al., 2011; Song et al., 2016), and viruses (Tyner et al., 2006). Finally, challenging the rat airway with allergens (Takeyama et al., 1999), bacterial endotoxin (Takezawa et al., 2016), agarose plug (Lee et al., 2000), or cigarette smoke (Takeyama et al., 2001b) increased goblet cell numbers in a EGFR dependent manner.
The studies that provide strong empirical evidence for the KER are listed below:
In vitro systems
Treatment of bronchial epithelial cells grown in ALI with xanthine plus xanthine oxidase increased EGFR activation (measured by pEGFR/EGFR ratios) and the number of Muc5Ac positive cells. The increase in goblet cell numbers was prevented by anti-EGFR monoclonal antibodies (Casalino-Matsuda et al., 2006).
Treatment of human primary bronchial epithelial cells with amphiregulin or HB-EGF significantly induced goblet cell differentiation demonstrated by CLCA1 (marker of goblet cells) expression in NHBE cells cultured in ALI (Hirota et al., 2012).
Differentiated human bronchial epithelial ALI cultures treated basolaterally with human bronchial epithelial growth factor (HBEGF) resulted in a remarkable increase in the number of cells expressing MUC5AC (Jia et al., 2021).
EGFR inhibitor AG1478 treatment of bronchial epithelial cell ALI cultures from asthmatic children showed significant reductions in goblet cell numbers. In addition, the cultures differentiated in EGF-negative conditions had reduced goblet cell numbers compared with the cultures differentiated in the presence of EGF (Parker et al., 2015).
The treatment of rat and human conjunctival goblet cells with EGF significantly increased proliferation via PI3K/AKT pathway (Li et al., 2013).
Treatment of rat conjunctival goblet cells with EGF significantly increased phosphorylation of EGFR and increased proliferation. Pre-treatment of goblet cells stimulated with EGF with AG1478 significantly inhibited the EGF-induced proliferation (Shatos et al., 2008).
Treatment of rat conjunctival goblet cells with EGF, TGF-α, or HB-EGF stimulated the phosphorylation of EGFR and resulted in increased proliferation, demonstrated with the WST-1 assay and the increase of Ki-67 positive cells (Gu et al., 2008).
In colon culture, EGF treatment increased the goblet cell index (number of goblet cells/total colonic epithelial cells) compared with untreated cultures (Duh et al., 2000).
In vivo systems
The irreversible EGFR inhibitor, EKB-569, blocked goblet cell metaplasia in the Sendai virus-infected mouse model (Tyner et al., 2006).
Induction of airway inflammation with house dust mite resulted in goblet cell metaplasia as evidenced by extensive AB staining and increase in Clca3-positive cells in mouse airways. Concomitant treatment with EGFR inhibitor, erlotinib, reduced the number of Clca3- (Le Cras et al., 2011) Muc5AC- positive cells (Jia et al., 2021) in the mouse lung.
Chronic treatment with EGFR inhibitor, gefitinib, decreased goblet cell hyperplasia in the airways of ovalbumin-challenged mice (Song et al., 2016).
Agarose plug instillation in the rat bronchial epithelium decreased the basal, ciliated and secretory cells, and increased the number of pregoblet and goblet cells. Intraperitoneal injection of the EGFR inhibitor, BIBX1522, reduced epithelium stained with Alcian blue-PAS in the agarose plug-induced rat airway (Lee et al., 2000).
Intranasal instillation of LPS induced increase in the number of goblet cells (AB/PAS staining) in the rat nasal epithelium, and the AB/PAS staining was reduced by injection of EGFR inhibitor, AG1478 (Takezawa et al., 2016).
Ovalbumin sensitization increased the numbers of goblet and pre-goblet cells in the rat airway epithelium, and EGFR inhibitor, BIBX1522, prevented the observed goblet cell increase (Takeyama et al., 1999).
Cigarette smoke exposure increased the number of goblet cells (AB-PAS staining) in the rat airway epithelum and this increase was blocked by concomitant treatment with EGFR tyrosine kinase inhibitor, BIBX1522 (Takeyama et al., 2001b).
Uncertainties and Inconsistencies
Known modulating factors
Quantitative Understanding of the Linkage
Below we list some quantitative aspects of the response relationship.
Rats who underwent small bowel resection had significantly more goblet cells (number of cells/mm villus/crypt in the ileum compared with sham operated animals. The rats that were treated with an EGFR inhibitor, ZD1839 50 mg/kg per day) starting at the day of surgery did not show the increase in goblet cells following small bowel resection (Jarboe et al, 2005).
The removal of EGF (human recombinant, 0.5 ng/ml) from the culture medium of human bronchial epithelial cells during differentiation in ALI reduced the number of goblet cells to 6.7 ± 0.8% from 15.5 ± 1.5 in cultures differentiated in the presence of EGF (Atherton et al, 2003).
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).
ALI cultures from bronchial epithelial cells of asthmatic children supplemented with 10ng/ml EGF showed a higher percentage of goblet cells (mean 23.4%) compared with cultures without EGF (mean 13.9%). Treatment of the EGF-supplemented cultures simultaneously with 0.2μg/ml or 2μg/ml AG147 resulted in decrease in the percentage of goblet cells (mean 13.1% and 7.7%, respectively) compared with cultures supplemented with EGF alone (Parker et al., 2015).
Treatment of mouse gut in organ culture, which do not have goblet cells at day 6, with 10 ng/mL EGF increased the colonic goblet cell index (number of goblet cells/total colonic epithelial cells) by 1.8-fold compared with untreated cultures (Duh et al., 2000).
The OVA-induced goblet cell hyperplasia (~33 mm2/mm) was significantly attenuated by 50 mg/kg gefitinib treatment for 12 h each day during days 14–20 (~12 mm2/mm, as measured by goblet cell area / perimeter of the bronchial basement membrane (Song et al., 2016).
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., 2001b).
Intranasal instillation of 0.1 mg LPS (E.coli 0111:B4) once a day for 3 consecutive days induced increased number of goblet cells in the nasal epithelium (as judged by histopathology), with an approx. 50% increase in AB/PAS-stained epithelium compared to untreated controls. Intranasal instillation of EGFR inhibitor 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).
Sensitization of rats with 10 mg ovalbumin (OVA) intraperitoneally (i.p.) on days 0 and 10 followed by intratracheal OVA instillations (0.1%, 100 ml) on days 20, 22, and 24 significantly increased the numbers of goblet cells in the airway epithelium on day 26. OVA sensitized rats were also treated with i.p. administration (10 mg/kg 1 h before the intratracheal instillation of ovalbumin), intratracheal instillation (1025M, 100 ml on days 20, 22 and 24), and i.p. injection (10 mg/kg of every 24 h until the day before the rats were euthanized) of BIBX1522. This treatment with the EGFR inhibitor reduced the Alcian blue/PAS stained area from ~18% to ~4 % (Takeyama et al., 1999).
Treatment of human primary bronchial epithelial cells with 1 ng/mL amphiregulin or HB-EGF for 24 h significantly induced goblet cell differentiation in NHBE cells cultured in ALI demonstrated by CLCA1 (marker of goblet cells) expression (Hirota et al., 2012).
Stimulation of human bronchial epithelial cells with human bronchial epithelial growth factor (20 ng/mL) for 24 h, resulted in a remarkable increase in the number of cells expressing MUC5AC. Treatment of the house dust mite-induced asthma mouse model with Erlotinib (during day 14–day 23)-resulted in decreased MUC5AC density in the lung (Jia et al., 2021).
105 PFUs of Sendai virus was intranasally administered to mice to reach maximal viral tissue levels at 3–5 days after inoculation and viral clearance by day 12. The blocking of EGFR by oral administration of 20 mg/kg EKB-569 daily during postinfection days 10–21 decreased the number of Muc5AC positive cells to ~2 compared with ~7 (cells/mm basement membrane) in lungs of virus only infected mice (Tyner et al., 2006).
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. Concurrently, the numbers of basal, ciliated, and secretory cells decreased. 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).
The following studies show that EGFR activation (upstream KE) occurs earlier than the increase of goblet cell (downstream KE), complying with temporal concordance terms of upstream KE occurring before downstream KE.
EGFR phosphorylation was detected in histamine stimulated NHBE cells in 2 hours and goblet cell differentiation marker CLCA1 mRNA was increased 5 days after histamine treatment (Hirota et al., 2012).
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. 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).
EGFR phosphorylation was observed in cultured rat conjunctival goblet cells after incubation with EGF for 1 or 5 minutes, and increase in number of goblet cells was measured at 24 hours after incubation with EGF for 20 minutes (Shatos et al., 2008).
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Evidences for this KER are derived from studies in human cell cultures as well as mouse and rat in vitro and in vivo systems. No sex-specific observations were noted.
Atherton HC, Jones G, Danahay H (2003) IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation. American Journal of Physiology-Lung Cellular and Molecular Physiology 285: L730-L739
Burgel P, 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 SM, Monzón ME, Forteza RM (2006) Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium. American journal of respiratory cell and molecular biology 34: 581-591
Curran DR, Cohn L (2010) Advances in mucous cell metaplasia: a plug for mucus as a therapeutic focus in chronic airway disease. American journal of respiratory cell and molecular biology 42: 268-275
Duh G, Mouri N, Warburton D, Thomas DW (2000) EGF regulates early embryonic mouse gut development in chemically defined organ culture. Pediatric research 48: 794-802
Gu F, Ma W, Meng G, Wu C, Wang Y (2016) Preparation and in vivo evaluation of a gel-based nasal delivery system for risperidone. Acta Pharmaceutica 66: 555-562
Gu J, Chen L, Shatos MA, Rios JD, Gulati A, Hodges RR, Dartt DA (2008) Presence of EGF growth factor ligands and their effects on cultured rat conjunctival goblet cell proliferation. Experimental Eye Research 86: 322-334
Hirota N, Risse PA, Novali M, McGovern T, Al-Alwan L, McCuaig S, Proud D, Hayden P, Hamid Q, Martin JG (2012) Histamine may induce airway remodeling through release of epidermal growth factor receptor ligands from bronchial epithelial cells. FASEB Journal 26: 1704-1716
Jarboe MD, Juno RJ, Stehr W, Bernal NP, Profitt S, Erwin CR, Warner BW (2005) Epidermal growth factor receptor signaling regulates goblet cell production after small bowel resection. Journal of pediatric surgery 40: 92-97
Jia Z, Bao K, Wei P, Yu X, Zhang Y, Wang X, Wang X, Yao L, Li L, Wu P (2021) EGFR activation-induced decreases in claudin1 promote MUC5AC expression and exacerbate asthma in mice. Mucosal Immunology 14: 125-134
Le Cras TD, Acciani TH, Mushaben EM, Kramer EL, Pastura PA, Hardie WD, Korfhagen TR, Sivaprasad U, Ericksen M, Gibson AM (2011) Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma. American Journal of Physiology-Lung Cellular and Molecular Physiology 300: L414-L421
Lee H-M, Takeyama K, Dabbagh K, Lausier JA, Ueki IF, Nadel JA (2000) Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways. American Journal of Physiology - Lung Cellular and Molecular Physiology 278: L185-L192
Li D, Shatos MA, Hodges RR, Dartt DA (2013) Role of PKCα activation of Src, PI-3K/AKT, and ERK in EGF-stimulated proliferation of rat and human conjunctival goblet cells. Investigative ophthalmology & visual science 54: 5661-5674
Parker JC, Douglas I, Bell J, Comer D, Bailie K, Skibinski G, Heaney LG, Shields MD (2015) Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model. PLoS One 10: e0129546
Shatos MA, Gu J, Hodges RR, Lashkari K, Dartt DA (2008) ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture. Investigative Ophthalmology & Visual Science 49: 3351-3359
Song L, Tang H, Liu D, Song J, Wu Y, Qu S, Li Y (2016) The chronic and short-term effects of gefinitib on airway remodeling and inflammation in a mouse model of asthma. Cellular Physiology and Biochemistry 38: 194-206
Takeyama K, Dabbagh K, Lee H-M, Agustí C, Lausier JA, Ueki IF, Grattan KM, Nadel JA (1999) Epidermal growth factor system regulates mucin production in airways. Proceedings of the National Academy of Sciences 96: 3081-3086
Takeyama K, Fahy J, Nadel J (2001a) Relationship of epidermal growth factor receptors to goblet cell production in human bronchi. American Journal of Respiratory and Critical Care Medicine 163: 511-516
Takeyama K, Jung B, Shim JJ, Burgel P-R, Dao-Pick T, Ueki IF, Protin U, Kroschel P, Nadel JA (2001b) Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. American Journal of Physiology - Lung Cellular and Molecular Physiology 280: L165-L172
Takezawa K, Ogawa T, Shimizu S, Shimizu T (2016) Epidermal growth factor receptor inhibitor AG1478 inhibits mucus hypersecretion in airway epithelium. American Journal of Rhinology & Allergy 30: e1-e6
Tyner JW, Kim EY, Ide K, Pelletier MR, Roswit WT, Morton JD, Battaile JT, Patel AC, Patterson GA, Castro M et al (2006) Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals. Journal of Clinical Investigation 116: 309-321
Yoshida T, Tuder RM (2007) Pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease. Physiological Reviews 87: 1047-1082