API

Aop: 148

AOP Title

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EGFR Activation Leading to Decreased Lung Function

Short name:

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Decreased lung function

Authors

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Philip Morris International: Karsta Luettich (Karsta.Luettich@pmi.com); Marja Talikka; Julia Hoeng

British American Tobacco: Frazer Lowe; Linsey Haswell; Marianna Gaca

 

Point of Contact

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Karsta Luettich

Contributors

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  • Karsta Luettich

Status

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Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development 1.51 Included in OECD Work Plan


This AOP was last modified on June 27, 2017 06:26

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Revision dates for related pages

Page Revision Date/Time
Occurrence, Transdifferentiation of ciliated epithelial cells September 16, 2017 10:16
Occurrence, Metaplasia of goblet cells September 16, 2017 10:16
Occurrence, Hyperplasia of goblet cells September 16, 2017 10:16
Increase, Proliferation of goblet cells September 16, 2017 10:16
Activation, SP1 September 16, 2017 10:16
Decrease, Apoptosis of ciliated epithelial cells September 16, 2017 10:16
Activation, EGFR September 16, 2017 10:16
Increase, Mucin production September 16, 2017 10:16
Decrease, Lung function June 19, 2017 01:21
Chronic, Mucus hypersecretion September 16, 2017 10:16
Occurrence, Transdifferentiation of ciliated epithelial cells leads to Occurrence, Metaplasia of goblet cells November 29, 2016 20:46
Increase, Proliferation of goblet cells leads to Goblet cell hyperplasia November 29, 2016 20:46
Activation, EGFR leads to Increase, Mucin production November 29, 2016 20:48
Activation, SP1 leads to Increase, Mucin production November 29, 2016 20:48
Decrease, Apoptosis of ciliated epithelial cells leads to Occurrence, Transdifferentiation of ciliated epithelial cells July 21, 2017 08:45
Activation, EGFR leads to Decrease, Apoptosis of ciliated epithelial cells November 29, 2016 20:47
Activation, EGFR leads to Activation, SP1 November 29, 2016 20:47
Activation, EGFR leads to Occurrence, Transdifferentiation of ciliated epithelial cells November 29, 2016 20:47
Activation, EGFR leads to Increase, Proliferation of goblet cells November 29, 2016 20:47
Goblet cell hyperplasia leads to Increase, Mucin production December 03, 2016 16:38
Occurrence, Metaplasia of goblet cells leads to Increase, Mucin production December 03, 2016 16:38
Chronic, Mucus hypersecretion leads to Decrease, Lung function December 03, 2016 16:38
Increase, Mucin production leads to Chronic, Mucus hypersecretion December 03, 2016 16:38
Reactive oxygen species August 15, 2017 10:43

Abstract

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Provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: (1) the background/purpose for initiation of the AOP’s development (if there was a specific intent); (2) a brief description of the MIE, AO, and/or major KEs that define the pathway; (3) a short summation of the overall weight of evidence supporting the AOP and identification of major knowledge gaps (if any); (4) a brief statement about how the AOP may be applied. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance.

Instructions

To add or edit the Abstract, click Edit in the upper right hand menu on the AOP page, bringing the user to a page where they can edit fields of the AOP. Under the Abstract field, a text editable form provides ability to edit the Abstract.  Clicking ‘Update AOP’ will update these fields.


Background (optional)

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The lungs’ mucous barrier is a natural defense against the harmful effects of inhaled xenobiotics, including respiratory toxicants and pathogens (Rubin, 2014). Under physiological conditions, foreign particles are trapped in mucus and eliminated from the airways via mucociliary clearance (Rose and Voynow, 2006). However, excessive mucus production can lead to impaired mucociliary clearance and airway obstruction and, eventually, result in decreased lung function (Nadel, 2013). Excessive mucus production, or mucus hypersecretion, is a characteristic feature of chronic diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, all of which pose a significant public health burden. Of note, exposure to cigarette smoke, occupational respiratory hazards, and air pollutants are clearly linked to the development of COPD, which is predicted to become the third leading cause of death worldwide by 2030 (Viegi et al., 2007; WHO, 2008). While regulation and public health measures seek to minimize exposures and thereby the incidence of the disease, airflow obstruction can be seen in approximately 25% of adults aged 40 and over globally (Diaz-Guzman and Mannino, 2014). Mucus hypersecretion in chronic bronchitis is characterized by an increase in the number of goblet cells, mucin synthesis and mucus secretion which can result in airway obstruction, decreased peak expiratory flow and respiratory muscle weakness (Kim & Criner, 2015; Yoshida & Tuder, 2007). Epidermal growth factor receptor (EGFR)-mediated signaling has been identified as the key pathway that leads to airway mucus hypersecretion (Burgel and Nadel, 2004), and redox signaling as the major initiator of receptor activation (Heppner and van der Vliet, 2016). Therefore, we believe that the molecular initiating event (MIE) of this AOP is oxidative stress leading to activation (phosphorylation) of EGFR on the surface of lung epithelial cells. Exogenous oxidative stress, e.g. arising from exposure to airborne toxicants and pathogens, as well as oxidative stress induced by inflammatory responses, mediates proteolytic cleavage of membrane-bound EGFR ligand precursors (Burgel and Nadel, 2004; Gao et al., 2015; Øvrevik et al. 2015). Subsequent ligand binding then activates the receptor tyrosine kinase in an autocrine fashion. Of note, ligand binding in itself has been identified as a source of reactive oxygen species (ROS), and specifically of hydrogen peroxide (H2O2), which function as second messengers potentially perpetuating the ensuing EGFR activation through chemical modification of the receptor (Paulsen et al., 2011; DeYulia et al., 2005). In addition, the presence of ROS may also contribute to EGFR activation by chemically modifying the receptor, thereby altering its structure and enhancing its kinase activity (Paulsen et al., 2011; Wu et al. 1999). Downstream of EGFR activation, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling elicits an anti-apoptotic response in ciliated cells, favoring their survival (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”; Curran and Cohn, 2010). Alternatively, 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 or Sp-1 transcription factor-mediated mucin gene and protein expression. Together these processes ultimately lead to goblet cell hyperplasia/metaplasia (GCH/GCM) and mucus hypersecretion (Rogers, 2007). If oxidative stress persists, e.g. under conditions of chronic exposure to respiratory toxicants, airway remodeling will cease being a physiological stress response aimed at eliminating the potential hazard and regaining the balance of a healthy airway epithelium. Instead, airway remodeling will result in airway narrowing, and in combination with GCH and chronic mucus production, lung function will begin to decline (Aoshiba and Nagai, 2004). Furthermore, over time, chronic mucus hypersecretion may contribute to a progressive deterioration in lung function (Kim & Criner, 2015).


Summary of the AOP

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Stressors

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Name Evidence Term
Reactive oxygen species Strong

Molecular Initiating Event

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Title Short name
Activation, EGFR Activation, EGFR

Key Events

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Title Short name
Occurrence, Transdifferentiation of ciliated epithelial cells Occurrence, Transdifferentiation of ciliated epithelial cells
Occurrence, Metaplasia of goblet cells Occurrence, Metaplasia of goblet cells
Occurrence, Hyperplasia of goblet cells Goblet cell hyperplasia
Increase, Proliferation of goblet cells Increase, Proliferation of goblet cells
Activation, SP1 Activation, SP1
Decrease, Apoptosis of ciliated epithelial cells Decrease, Apoptosis of ciliated epithelial cells
Increase, Mucin production Increase, Mucin production
Chronic, Mucus hypersecretion Chronic, Mucus hypersecretion

Adverse Outcome

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Title Short name
Decrease, Lung function Decrease, Lung function

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Life Stage Applicability

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Life stage Evidence
Adult Strong
Juvenile Weak

Taxonomic Applicability

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

Sex Applicability

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Sex Evidence
Unspecific Strong

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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This section addresses the relevant domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and weight of evidence for the overall hypothesised AOP (i.e., including the MIE, KEs and AO) as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). It draws upon the evidence assembled for each KER as one of several components which contribute to relative confidence in supporting information for the entire hypothesised pathway. An important component in assessing confidence in supporting information as a basis to consider regulatory application of AOPs beyond that described in Section 6 is the essentiality of each of the key events as a component of the entire pathway. This is normally investigated in specifically-designed stop/reversibility studies or knockout models (i.e., those where a key event can be blocked or prevented). Assessment of the overall AOP also contributes to the identification of KEs for which confidence in the quantitative relationship with the AO is greatest (i.e., to facilitate determining the most sensitive predictor of the AO).

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To edit the “Overall Assessment of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Overall Assessment of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page.  The new text should appear under the “Overall Assessment of the AOP” section on the AOP page.

Domain of Applicability

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Life Stage Applicability

EGFR activation leading to mucus hypersecretion is predominantly studied in adults; however, it has been shown to also occur in pediatric asthma and bronchitis (Rogers, 2003; Parker et al., 2015). Nevertheless, the environmental exposures that induce EGFR activation apply more to adults who are more likely to be exposed to these stimulants over time (cigarette smoke, particulate matter).

Taxonomic Applicability

There are extensive studies on mucus hypersecretion in humans from a clinical perspective. In vitro and in vivo mouse, rat and human studies have been performed to clarify the mechanisms of EGFR involvement in mucus hypersecretion by studying the increase in goblet cell numbers and subsequent mucus production.

Sex Applicability

There are no sex-specific differences in this AOP.


Essentiality of the Key Events

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Molecular Initiating Event Summary, Key Event Summary

EGFR signaling is considered critical for mucus hypersecretion and goblet cell hyperplasia (GCH)/goblet cell metaplasia (GCM)(Curran & Cohn, 2010), and numerous studies indicate that inhibition of EGFR decreases mucin production or goblet cell numbers (Tyner et al., 2006; Shim et al., 2001; Takeyama et al., 2008; Lee et al., 2011; Taniguchi et al., 2011; Song et al., 2016; Takeyama et al., 2011). EGFR blockade also was reported to prevent an increase in goblet cell numbers and cause activation of caspase-3 and loss of ciliated cells, indicating that EGFR is essential for decreased ciliated cell apoptosis (Tyner et al., 2006). However, there is also evidence supporting decreased apoptosis in airway goblet cells in vitro, in a mouse model of asthma, and in rats following intratracheal lipopolysaccharide (LPS) instillation as a result of EGFR activation (Casalino-Matsuda et al., 2006; Song et al., 2016; Tesfaigzi, 2006). Whether the latter only occurs once GCH/GCM is established, as indicated by Harris et al. (2005), or whether additional events are required to maintain GCH/GCM, is currently unclear.

Sp-1 binding sites are required for active MUC5AC gene expression (Hewson et al., 2004), and Sp-1-mediated mucin expression can be blocked by the Sp-1 inhibitor mithramycin A (Lee et al., 2011; Wu et al., 2007). However, since the MUC5AC promoter has multiple transcription factor binding sites, it is likely that alternative pathways might also contribute to increased mucin production, such as activation of HIF-1α or decreased FOXA2 expression (Hao et al., 2014; Kim et al., 2014; Wan et al., 2004).

Mucus hypersecretion is a physiological response to inhalation exposures such as pollutants or infectious agents. As such, it is typically of short duration and does not pose a major problem to normal lung function. However, in the presence of GCH, increased mucus production may decrease airflow. Since this may be accompanied by impaired mucociliary clearance and ineffective cough (Ramos et al., 2014), and owing to the lack of direct evidence, it is currently unclear whether chronic mucus hypersecretion alone is sufficient to affect a decrease in lung function.

Although some KERs may be executed in parallel to and independent of each other, all KEs together contribute to mucus hypersecretion as a result of EGFR activation by oxidative stress.


Weight of Evidence Summary

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Summary Table

File:Mucus hypersecretion empirical support concordance table.pdf (Published studies investigating dose- and time-response of various KEs)

 

 

 

Empirical Support for KERs

 

Defining question: Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses, earlier time points, and higher in incidence than KEdown? Inconsistencies?

High (Strong)

Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. No or few critical data gaps or conflicting data.

 

Moderate

Demonstrated dependent change in both events following exposure to a small number of stressors. Some inconsistencies with expected pattern that can be explained by various factors.

Low (Weak)

Limited or no studies reporting dependent change in both events following exposure to a specific stressor, and/or significant inconsistencies in empirical support across taxa and species.

Oxidative stress directly leading to EGFR activation

Strong

Various sources of ROS, including glucose oxidase, xanthine/xanthine oxidase, acrolein, H2O2, cigarette smoke extract, PMA, TCDD, and supernatant from activated neutrophils or eosinophils cause a measurable, rapid increase in EGFR phosphorylation in human airway epithelial cells and the lungs of F344 rats (Burgel et al., 2001; Casalino-Matsuda et al., 2004; Casalino-Matsuda et al., 2006; Deshmukh et al., 2008; Hewson et al., 2004; Kim et al., 2008; Lee et al., 2011; Qi et al., 2010; Ravid et al., 2002; Takeyama et al., 2000; Takeyama et al., 2001b; Yu et al., 2011; Yu et al., 2015). In some instances, the response was dose-dependent (Hewson et al., 2004; Ravid et al., 2002); in others, it was directly linked to GCH or increased mucin production (Casalino-Matsuda et al., 2006; Takeyama et al., 2001b). Moreover, antioxidant treatment prevented EGFR activation and diminished downstream mucin overexpression (Casalino-Matsuda et al., 2006).

EGFR activation indirectly leading to decreased epithelial cell apoptosis

Moderate

Oxidative stress increased Bcl-2 mRNA and protein levels in human and rat airway goblet cells (Casalino-Matsuda et al., 2006; Foster et al., 2003; Lee et al., 2011; Tesfaigzi et al., 1998; Tesfaigzi et al., 2000). Neutralization of Bcl-2 expression in rat nasal epithelium reduced GCM (Harris et al., 2005), and treatment of OVA-sensitized Balb/c mice with the EGFR inhibitor gefitinib decreased Bcl-2 expression and increased apoptosis (Song et al., 2016).

Decreased epithelial cell apoptosis directly leading to transdifferentiation into goblet cells

Weak

There is no direct evidence linking decreased apoptosis in ciliated cells to their transdifferentiation. Co-localization of EGFR and β-tubulin but not CCSP or MUC5AC expression was observed in Sendai virus-infected mouse airways and in the airways of asthma patients (Takeyama et al., 2001a; Tyner et al., 2006). In addition, ciliated cell tagging studies in vitro indicated that the number of ciliated cells decreases following treatment with IL-13, while the number of goblet cells increases (Turner et al., 2011). Together these studies are supportive of ciliated cells transdifferentiating into goblet cells. 

EGFR activation directly leading to increased epithelial cell proliferation

Moderate

Treatment of human airway epithelial cells with oxidative stressors, EGFR ligands, or IL-13 was shown to increase the number of MUC5AC-positive (i.e. goblet) cells (Casalino-Matsuda et al., 2006; Hirota et al., 2012). Increased proliferation was observed in rat conjunctival goblet cells following treatment with EGFR ligands (Gu et al., 2008; Shatos et al., 2008). In addition, 50% of goblet cells were BrdU-positive in rat airways following LPS instillation, suggesting that they may have been derived from proliferating cells (Tesfaigzi et al., 2004).

EGFR activation directly leading to Sp-1 activation

Weak

Treatment of H292 cells with PMA dose-dependently increased MUC5AC mRNA and protein production, shown to be dependent on ligand-dependent EGFR phosphorylation and subsequent Sp-1-mediated transactivation of the MUC5AC promoter (Hewson et al., 2004). Moreover, Sp-1 phosphorylation and MUC5AC promoter activity increased in TCDD-treated NHBECs, and increased promoter activity was suppressed in the presence of the EGFR inhibitor AG1478 (Lee et al., 2011).

EGFR activation indirectly leads to increased mucin production

Strong

Oxidative stress increased EGFR phosphorylation and MUC5AC gene and protein expression in human lung and nasal epithelial cells, as well as in the airways of mice and rats (Casalino-Matsuda et al., 2006; Deshmukh et al., 2008; Hao et al., 2014; Hegab et al., 2007; Kim et al., 2010; Val et al., 2012). Pre-treatment with catalase, glutathione, AG1478, erlotinib, gefitinib, or a neutralizing antibody preventing EGFR ligand binding markedly reduced mucin production.

Increased epithelial cell proliferation directly leading to GCH

Weak

Inferred: The term ‘hyperplasia’ refers to an increase in a tissue or organ that is linked to an increase in cell number or cell size. Therefore, increased proliferation can be considered a root cause of GCH.

Transdifferentiation into goblet cells directly leading to GCM

Weak

Inferred: Following injury, airway epithelial repair is accomplished by (transient) remodeling processes. In the absence of cell proliferation, this remodeling is thought to be facilitated by transdifferentiation, i.e. the generation of specialized cell types, such as goblet cells, from other specialized cells, such as ciliated and club cells (Evans et al., 2004; Tesfaigzi, 2006).

Sp-1 activation directly leading to increased mucin production

Moderate

Treatment of A549 cells with cigarette smoke extract increased MUC5AC promoter activity, which was accompanied by an increase in Sp-1 protein expression, nuclear translocation, and Sp-1-DNA-binding (Di et al., 2012). Similarly, increased MUC5AC gene and protein expression in H292 cells infected with IVA was shown to be linked to activation of Sp-1 (Barbier et al., 2012).

Increased mucin production directly leading to mucus hypersecretion

Weak

Inferred: Increased mucin production is a requirement in states of mucus hypersecretion to restore depleted mucin stores (Rose and Voynow, 2006). Mucus hypersecretion is a cardinal feature of chronic lung diseases and has been linked to both increased intraluminal mucus volume (Aikawa et al., 1989), a measure of mucus hypersecretion, and increased mucin production.

GCH/GCM directly leading to mucus hypersecretion

Weak

Inferred:  According to Rose et al., “Secretory cell hyperplasia is a prerequisite for sustained mucus hypersecretion/mucin overproduction” (Rose and Voynow, 2006).

Chronic mucus hypersecretion directly leads to decreased lung function

Moderate

GCH and MUC5AC expression was increased in the airways of COPD patients compared with non-COPD patients (with normal lung function) (Ma et al., 2005). The volume of epithelial mucin stores was larger in bronchial biopsies from smokers compared with those without airflow obstruction, and correlated with the FEV1/FVC ratio (Innes et al., 2006).  MUC5AC expression in bronchial epithelium was inversely correlated with FEV1 (% predicted) (Caramori et al., 2009). Epidemiological data indicate that self-reported symptoms, including chronic sputum production and/or chronic cough, in middle-aged current smokers increased the likelihood of airflow limitation at later stages of life, and that lung function declines more rapidly the longer chronic mucus hypersecretion persists, at least in this middle-aged group (Allinson et al., 2015; Pistelli et al., 2003; Vestbo et al., 1996).

 

 


Quantitative Considerations

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Summary Table

There is good quantitative understanding of how oxidative stress affects EGFR signaling and influences mucus production, epithelial cell proliferation, apoptosis, and transdifferentiation, individually assayed. In addition, in the majority of these studies, the summary evidence indicates dose-response relationships, time-response relationships, and causality for oxidative stress-induced EGFR activation leading to increased cell proliferation, lending strong support for these KERs. However, quantitative knowledge is lacking with respect to the identity of airway epithelial cells undergoing proliferation and apoptosis, which makes empirical support for these KERs weak. Furthermore, while cause-effect relationships can be derived from studies investigating Sp-1 activation, dose-response relationships are difficult to derive. Moreover, data for increased mucin production and mucus hypersecretion at the organism level are mainly derived from surrogate measures, and while those may not adequately reflect quantitative mucus production, they are accepted in the clinical community as an indicator of chronic bronchitis. Taken together, the quantitative evidence for the KEs and KERs on the tissue and organism level are moderate at best.


Considerations for Potential Applications of the AOP (optional)

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The future application of this AOP lies in its potential for predicting decreased lung function in humans exposed to potentially harmful inhaled substances. This becomes especially pertinent as impaired lung function carries a significant risk of morbidity and mortality. Owing to the long latency period between exposure and detectable decreases in lung function, together with the fact that lung function tests alone may not be sufficiently sensitive to account for early lung damage that remains asymptomatic (Celli et al., 2003), means for early identification of potentially hazardous exposures are critical for the development of appropriate public health interventions.


References

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Booth, B.W., Adler, K.B., Bonner, J.C., Tournier, F., and Martin, L.D. (2001). 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.

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