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Relationship: 970

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Activation, EGFR leads to Increased goblet cell proliferation

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Activation, EGFR

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increased goblet cell proliferation

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	Moderate	NCBI
mouse	Mus musculus	Moderate	NCBI
rat	Rattus norvegicus	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Mixed	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

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

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

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).

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

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.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

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

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

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

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

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).

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

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

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

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).

References

List of the literature that was cited for this KER description. More help

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.

Booth, B.W., Adler, K.B., Bonner, J.C., Tournier, F., and Martin, L.D. (2001a). Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha. Am. J. Respir. Cell Mol. Biol. 25, 739–743.

Booth, B., J. C. Bonner, K. B. Adler, and L. D. Martin. (2001b). Autocrine production of TGF mediates interleukin 13-induced proliferation of human airway epithelial cells during development of a mucous phenotype in vitro. Am. J. Respir. Crit. Care Med. 163, A738.

Booth, B.W., Sandifer, T., Martin, E.L., and Martin, L.D. (2007). IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17. Respir. Res. 8, 51.

Casalino-Matsuda, S., Monzón, M., and Forteza, R. (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.

Duh, G., Mouri, N., Warburton, D., and Thomas, D.W. (2000). EGF regulates early embryonic mouse gut development in chemically defined organ culture. Pediatr. Res. 48, 794–802.

Gu, J., Chen, L., Shatos, M.A., Rios, J.D., Gulati, A., Hodges, R.R., and Dartt, D.A. (2008). Presence of EGF growth factor ligands and their effects on cultured rat conjunctival goblet cell proliferation. Exp. Eye Res. 86, 322–334.

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.

Harris, J.F., Fischer, M.J., Hotchkiss, J.R., Monia, B.P., Randell, S.H., Harkema, J.R., and Tesfaigzi, Y. (2005). Bcl-2 sustains increased mucous and epithelial cell numbers in metaplastic airway epithelium. Am. J. Respir. Crit. Care Med. 171, 764-772.

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.

Hirota, N., Risse, P.A., Novali, M., McGovern, T., Al-Alwan, L., McCuaig, S., Proud, D., Hayden, P., Hamid, Q., and Martin, J.G. (2012). Histamine may induce airway remodeling through release of epidermal growth factor receptor ligands from bronchial epithelial cells. FASEB J. 26, 1704-1716.

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.

Lamb, D., and Reid, L. (1968). Mitotic rates, goblet cell increase and histochemical changes in mucus in rat bronchial epithelium during exposure to sulphur dioxide. J. Pathol. Bacteriol. 96, 97–111.

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.

Li, D., Shatos, M.A., Hodges, R.R., and Dartt, D.A. (2013). Role of PKCα activation of Src, PI-3K/AKT, and ERK in EGF-stimulated proliferation of rat and human conjunctival goblet cells. Invest. Ophthalmol. Vis. Sci. 54, 5661-5674.

Lumsden, A.B., McLean, A., and Lamb, D. (1984). Goblet and Clara cells of human distal airways: evidence for smoking induced changes in their numbers. Thorax 39, 844-849.

Reader, J.R., Tepper, J.S., Schelegle, E.S., Aldrich, M.C., Putney, L.F., Pfeiffer, J.W., et al. (2003). Pathogenesis of mucous cell metaplasia in a murine asthma model. Am. J. Pathol. 162, 2069-2078.

Rock, J.R., Onaitis, M.W., Rawlins, E.L., Lu, Y., Clark, C.P., Xue, Y., et al. (2009). Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. U.S.A. 106, 12771-12775.

Shan, X., Zhang, Y., Chen, H., Dong, L., Wu, B., Xu, T., et al. (2017). Inhibition of epidermal growth factor receptor attenuates LPS-induced inflammation and acute lung injury in rats. Oncotarget 8, 26648-26661.

Shatos, M.A., Gu, J., Hodges, R.R., Lashkari, K., and Dartt, D.A. (2008). ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture. Invest. Ophthalmol. Vis. Sci. 49, 3351-3359.

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.

Shimizu, T., Takahashi, Y., Kawaguchi, S., and Sakakura, Y. (1996). Hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium induced by endotoxin. Am. J. Respir. Crit. Care Med. 153, 1412–1418.

Silva, M.A., and Bercik, P. (2012). Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells. Lab. Invest. 92, 937-948.

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., 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.

Takezawa, K., Ogawa, T., Shimizu, S., and Shimizu, T. (2016). Epidermal growth factor receptor inhibitor AG1478 inhibits mucus hypersecretion in airway epithelium. Am. J. Rhinol. Allergy 30, e1-e6.

Tamaoki, J., Isono, K., Takeyama, K., Tagaya, E., Nakata, J., and Nagai, A. (2004). Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L1127–L1133.

Taniguchi, K., Yamamoto, S., Aoki, S., Toda, S., Izuhara, K., and Hamasaki, Y. (2011). Epigen is induced during the interleukin-13-stimulated cell proliferation in murine primary airway epithelial cells. Exp. Lung Res. 37, 461–470.

Tesfaigzi, J., Hotchkiss, J.A., and Harkema, J.R. (1998). Expression of the Bcl-2 protein in nasal epithelia of F344/N rats during mucous cell metaplasia and remodeling. Am. J. Respir. Cell Mol. Biol. 18, 794-799.

Tesfaigzi, Y., Harris, J.F., Hotchkiss, J.A., and Harkema, J.R. (2004). DNA synthesis and Bcl-2 expression during development of mucous cell metaplasia in airway epithelium of rats exposed to LPS. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, L268-L274.

Turner, J., Roger, J., Fitau, J., Combe, D., Giddings, J., Heeke, G.V., and Jones, C.E. (2011). Goblet cells are derived from a FOXJ1-expressing progenitor in a human airway epithelium. Am. J. Respir. Cell Mol. Biol. 44, 276–284.

Tyner, J., Tyner, E., Ide, K., Pelletier, M., Roswit, W., Morton, J., Battaile, J., Patel, A., Patterson, G., Castro, M., et al. (2006). Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals. J. Clin. Invest. 116, 309–321.

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, 443-459.