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Activation, EGFR leads to Increase, Mucin production
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||non-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 Do not cite||Under Development|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
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). Classical EGFR downstream signaling involves activation of Ras which subsequently initiates signal transduction through the Raf-1/MEK/ERK pathway. MAP kinase activation in turn promotes airway epithelial cell proliferation and differentiation (Lemjabbar et al., 2003; Kim et al., 2005; Hackel et al, 1999) and facilitates epithelial wound repair (Burgel and Nadel, 2004; van Winkle et al., 1997; Allahverdian et al., 2010). EGFR signal transduction via the MAPK cascade also activates the transcription factor Sp1 (Di et al., 2012; Hewson et al., 2004; Lee et al., 2011; Perrais et al., 2002; Barbier et al., 2012; Oyanagi et al, 2016).
Evidence Supporting this KER
EGFR can be activated by bacterial infection, EGFR ligands, exposure to cigarette smoke and other sources of ROS, leading to increased mucin production via Ras/Raf-1/MEK/ERK-mediated activation of the Sp1 transcription factor, which can be suppressed at least partially in the presence of EGFR inhibitors (Sydlik et al., 2006; Casalino-Matsuda et al., 2006; Takeyama et al., 2008; Perrais et al., 2002; Hewson et al., 2004; Wu et al., 2007; Barbier et al., 2012; Lee et al., 2011).
Multiple studies showed that ligand- or ROS-mediated EGFR activation leads to increased goblet cell numbers and mucin production in rat airway epithelia (Shim et al., 2001; Lee et al., 2000), normal human bronchial epithelial cells grown at the air liquid interface (Casalino-Matsuda et al., 2006; Hao et al., 2014), and in a human pulmonary mucoepidermoid carcinoma cell line (NCI-H292) (Takeyama et al., 2008; Takeyama et al., 1999).
Uncertainties and Inconsistencies
The described Sp1-mediated mechanism for this KER may specifically apply in the context of increased MUC5AC gene and protein expression, but not to that of MUC5B, another gel-forming mucin associated with mucus hypersecretion in the airways (Wu et al., 2007).
Daily treatment of primary human bronchial epithelial cells with 0.6 mM xanthine and 0.5 units xanthine oxidase for 3 days nearly doubled the levels of phosphorylated EGFR and increased expression of MUC5AC mRNA by approx. 2-fold and that of MUC5AC protein by ca. 30%, and these responses could be at least partially prevented by pre-incubation with anti-EGFR antibodies (Casalino-Matsuda et al., 2006).
Increased MUC5AC expression (ca. 5-fold) was seen in the tracheal, but not the lung epithelium of C57Bl/6 mice 48 h after instillation of PM2.5. This effect was significant with 50 µg, but not with 10 µg PM2.5. PM2.5 dose-dependently increased MUC5AC expression in human H292 lung cancer cells and primary human bronchial epithelial cells grown as monolayer, with significant increases of ca. 10- and 8-fold above that of control at PM2.5 concentrations > 5 µg/cm2. At a concentration of 10 µg/cm2, MUC5AC mRNA level in H292 cells peaked at >30 times that of control after 24 h of treatment and decreased to approx. 20-fold that of control at 36 h. When primary bronchial epithelial cells differentiated the air-liquid interface were treated with PM2.5, MUC5AC expression also increased in a dose-dependent manner. However, 10 µg/cm2 PM2.5 were necessary to induce a significant, maximal response (ca. 3-fold increase), and pretreatment with 10 µM AG1478 or 0.5 µg/µL neutralizing anti-EGFR antibody reduced this response by ca. 50% (Val et al., 2012).
Mycoplasma pneumoniae M129 infection of H292 lung cancer cells at MOIs of both 25:1 and 50:1 resulted in a ca. 1.5- and 2.2-fold increase, respectively, in MUC5AC mRNA expression. Similarly, MUC5B mRNA was induced approx. 2.2- and 2.5-fold. Under the same conditions, EGFR phosphorylation increased 4- and 6-fold in H292 cells, and treatment with 10 µM AG1478 attenuated the induction of MUC5AC and MUC5B by M. pneumoniae M129 infection. In addition, in human 3D organotypic bronchial tissues, infection with M. pneumoniae M129 caused 6.8- and 5-fold increases in MUC5AC and MUC5B protein expression (Hao et al., 2011).
Acrolein exposure of FVB/NJ mice at 2 ppm for 6 h per day, 5 days a week, for 4 weeks increased lung Muc5ac RNA and protein levels approx. 4-fold. Gavage of 100 mg/kg erlotinib after every exposure abolished this effect (Deshmukh et al., 2008).
IL-13 instillation in F344 rats induced mucin expression in the carina in a dose-dependent fashion. 50 ng IL-13 were necessary to significantly raise the % mucin-expressing epithelial area significantly above background (<10% in controls vs 15% in treated rats), and % mucin-expressing epithelial area was maximal at the highest tested concentration, 500 ng. Treatment of animals with BIBX1522 prior to and following IL-13 instillation dose-dependently decreased mucin expression, with a maximal decrease to a level nearing that of control animals seen at 30 mg/kg (Shim et al., 2001).
At a concentration of 10 µg/cm2 PM2.5, MUC5AC mRNA level in H292 cells peaked at >30 times that of control after 24 h of treatment and decreased to approx. 20-fold that of control at 36 h (Val et al., 2012).
Infection of H292 cells with influenza A virus (IVA) at MOI=1 resulted in increased EGFR phosphorylation, peaking at 24 h. This was accompanied by activation of Sp-1 as shown by EMSA (Barbier et al., 2012).
Treatment of primary bronchial epithelial cells with 10 nM TCDD resulted in maximal EGFR phosphorylation after 30 min. TCDD treatment also led to a time-dependent increase in MUC5AC transcriptional promoter activity, peaking between 6 and 12 h. Sp1 involvement was demonstrated by treatment with the Sp1 inhibitor mithramycin A (Lee et al., 2011).
Treatment with 20 ng/mL EGF or TGFa induced phosphorylation of Sp1, corresponding to a 2-3-fold increase in MUC2 and MUC5AC promoter activity, after 24 hours which was inhibited by 100 nM mithramycin A (a Sp1 inhibitor) (Perrais et al., 2002).
Treatment of H292 cells with a combination of 4 ng/mL TGFa and 25 µg/mL polyI:C resulted in a ca. 3-fold increase in EGFR phosphorylation at 1 h. At 12 h, MUC5AC mRNA expression was induced, intracellular MUC5AC protein expression was increased by nearly 30% and secretion of MUC5AC into the cell culture medium rose approx. 4-fold. MUC5AC mRNA expression could be completely abolished by the Sp1 inhibitor mithramycin A (500 nM) (Oyanagi et al., 2016).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
EGFR-mediated activation of Sp1 leading to increased mucin production was reported in mouse (Hammoud et al., 2009; Lee et al., 2010), rat (Merchant et al., 1995; Mortensen et al., 1997), dog (Ikari et al., 2009; Ford et al., 1997) and human (Di et al., 2012; Hewson et al., 2004; Lee et al., 2011; Perrais et al., 2002; Barbier et al., 2012; Oyanagi et al, 2016).
Allahverdian, S., Wang, A., Singhera, G.K., Wong, B.W., and Dorscheid, D.R. (2010). Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair. Clin. Exp. Allergy 40, 607-618.
Barbier, D., Garcia-Verdugo, I., Pothlichet, J., Khazen, R., Descamps, D., Rousseau, K., Thornton, D., Si-Tahar, M., Touqui, L., Chignard, M., et al. (2012). Influenza A Induces the Major Secreted Airway Mucin MUC5AC in a Protease–EGFR–Extracellular Regulated Kinase–Sp1–Dependent Pathway. Am. J. Respir. Cell Mol. Biol. 47, 149–157.
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.M., Monzón, M.E., and Forteza, R.M. (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.
Deshmukh, H.S., Shaver, C., Case, L.M., Dietsch, M., Wesselkamper, S.C., Hardie, W.D., Korfhagen, T.R., Corradi, M., Nadel, J.A., and Borchers, M.T. (2008). Acrolein-activated matrix metalloproteinase 9 contributes to persistent mucin production. Am. J. Resp. Cell Mol. Biol. 38, 446-454.
Di, Y.P., Zhao, J., and Harper, R. (2012). Cigarette smoke induces MUC5AC protein expression through the activation of Sp1. J. Biol. Chem. 287, 27948-27958.
Hackel, P.O., Zwick, E., Prenzel, N., and Ullrich, A. (1999). Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr. Opin. Cell Biol. 11, 184-189.
Hao, Y., Kuang, Z., Jing, J., Miao, J., Mei, L.Y., Lee, R.J., Kim, S., Choe, S., Krause, D.C., and Lau, G.W. (2014). Mycoplasma pneumoniae Modulates STAT3-STAT6/EGFR-FOXA2 Signaling To Induce Overexpression of Airway Mucins. Infect. Immun. 82, 5246–5255.
Higashiyama, S., Iwabuki, H., Morimoto, C., Hieda, M., Inoue, H., and Matsushita, N. (2008). Membrane-anchored growth factors, the epidermal growth factor family: Beyond receptor ligands. Cancer Sci. 99, 214-220.
Hewson, C., Edbrooke, M., and Johnston, S. (2004). PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J. Mol. Biol. 344, 683–695.
Kim, S., Schein, A.J., and Nadel, J.A. (2005). E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 289, L1049-L1060.
Lee, H.-M., Takeyama, K., Dabbagh, K., Lausier, J.A., Ueki, I.F., and Nadel, J.A. (2000). Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways. Am. J. Physiol. Lung Cell. Mol. Physiol. 278, L185–L192.
Lee, Y.C., Oslund, K.L., Thai, P., Velichko, S., Fujisawa, T., Duong, T., Denison, M.S., and Wu, R. (2011). 2,3,7,8-Tetrachlorodibenzo-p-dioxin–Induced MUC5AC Expression. Am. J. Respir. Cell Mol. Biol. 45, 270–276.
Lemjabbar, H., Li, D., Gallup, M., Sidhu, S., Drori, E., and Basbaum, C. (2003). Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-converting enzyme and amphiregulin. J. Biol. Chem. 278, 26202-26207.
Oyanagi, T., Takizawa, T., Aizawa, A., Solongo, O., Yagi, H., Nishida, Y., et al. (2017). Suppression of MUC5AC expression in human bronchial epithelial cells by interferon-γ. Allergol. Int. 66, 75-82.
Perrais, M., Pigny, P., Copin, M., Aubert, J., and Van Seuningen, I. (2002). Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1. J. Biol. Chem. 277, 32258–32267.
Shim, J.J., Dabbagh, K., Ueki, I.F., Dao-Pick, T., Burgel, P.R., Takeyama, K., Tam, D.C., and Nadel, J.A. (2001). IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am. J. Physiol. Lung Cell. Mol. Physiol. 280, L134–L140.
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.
Takeyama, K., Dabbagh, K., Lee, H., Agustí, C., Lausier, J., Ueki, I., Grattan, K., and Nadel, J. (1999). Epidermal growth factor system regulates mucin production in airways. Proc. Natl. Acad. Sci. U.S.A. 96, 3081–3086.
Takeyama, K., Tamaoki, J., Kondo, M., Isono, K., and Nagai, A. (2008). Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen. Clin. Exp. Allergy 38, 857–865.
Val, S., Belade, E., George, I., Boczkowski, J., and Baeza-Squiban, A. (2012). Fine PM induce airway MUC5AC expression through the autocrine effect of amphiregulin. Arch. Toxicol. 86, 1851-1859.
Van Winkle, L.S., Isaac, J.M., and Plopper, C.G. (1997). Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse. Am. J. Pathol. 151, 443.
Wu, D.Y., Wu, R., Reddy, S.P., Lee, Y.C., and Chang, M.M.-J. (2007). Distinctive epidermal growth factor receptor/extracellular regulated kinase-independent and -dependent signaling pathways in the induction of airway mucin 5B and mucin 5AC expression by phorbol 12-myristate 13-acetate. Am. J. Pathol. 170, 20–32.