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Key Event Title
Increase, Mucin production
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
Key Event Description
Mucins are a family of highly glycosylated proteins produced by epithelial tissues and constitute major macromolecular components of mucus which protects epithelium from chemical and mechanical damage (Dhanisha et al., 2018). Mucin production in healthy airway provides an important role in trapping and removing bacterial and viral pathogens and particulates. Similarly, mucus layer in the intestinal epithelium provides first line of defense against physical and chemical hazards, notably ingested food and bacteria (Kim and Ho, 2010). In airways, major gel-forming secreted mucins MUC5AC and MUC5B are primarily involved in defensive function. MUC2 is the major intestinal mucin but is also expressed in the airway epithelium, and MUC19 is the major mucin in salivary glands (Lillehoj et al., 2013). Specialized mucin-producing goblet cells increase mucin production in respiratory tract in response to various irritants and stressors (Rogers, 2003). Many stressors specifically induce mucin mRNA and protein production through activation of the epidermal growth factor receptor (EGFR) pathway (Nadel, 2013). However, other signaling pathways, not necessarily requiring EGFR activation, via STAT6, FOXA2 and SPDEF have also been implicated in mucin overexpression (Turner and Jones, 2009).
Evidence for Perturbation by Stressor
Various stressors such as cigarette smoke (Shao et al., 2004, Takeyama et al., 2001), reactive oxygen species (Yu et al., 2011, Casalino-Matsuda et al., 2009), phorbol 12-myristate 13-acetate (PMA) (Hewson et al., 2004), 2,3,7,8-tetrachlorodibenzodioxin (TCDD) (Lee et al., 2011), ozone (Wagner et al., 2003), fine particulate matter (Val et al., 2012), allergens such as ovalbumin (Song et al., 2016), as well as bacteria and viruses (Dohrman et al., 1998, Hao et al., 2014, Zhu et al., 2009) increase mucin production in respiratory airways. Wide range of inflammatory cytokines such as interleukin (IL) 1B, IL4, IL6, IL9, IL13, TNF, induce mucin production in different tissues, including respiratory and intestinal epithelium (Linden et al., 2008). Injection of the urban particulate matter into the middle ear cavity of rats increased MUC5AC and MUC5B expression in the middle ear mucosa (Park et al., 2014). Bacterial lipopolysaccharide (LPS) induced mucin expression in human intrahepatic biliary epithelial cells (HIBECs), colon adenocarcinoma cell line HT29, etc (Liu et al., 2013, Smirnova et al., 2003).
How It Is Measured or Detected
In the literature, increased mucin production is frequently equated with increased MUC5AC mRNA and protein expression and less frequently with changes in MUC5B, MUC2 mRNA and protein levels. Due to high molecular weight and extensive glycosylated nature of mucins, conventional polyacrylamide gel-based protein analytic approaches can be challenging for mucin measurements (Kesimer and Sheehan, 2012). Strategies and methods for measuring airway mucins are thoroughly described in a review by Atanasova and Reznikov (Atanasova and Reznikov, 2019). Below we list the methods commonly used for mucin production detection and measurement.
Alterations in mucin genes (MUC5AC, MUC5B) expression in cell and tissue lysates are commonly assessed by RT-PCR or RT-qPCR (Yu et al., 2011, Shao et al., 2004, Lee et al., 2011, Wagner et al., 2003, Hao et al., 2014, Zhu et al., 2009, Val et al., 2012). For absolute quantification of MUC5AC and MUC5B transcript copy numbers droplet digital PCR can be performed (Okuda et al., 2019).
In situ hybridization is used in some studies for mucin (MUC5AC, MUC5B, MUC2) mRNA quantification (Takeyama et al., 2001, Dohrman et al., 1998, Okuda et al., 2019).
For mucin mRNA detection and quantification RNase protection assay (RPA) is also used (Dohrman et al., 1998).
Northern blot of mucin mRNAs can also be applied for mucin gene expression measurement (Chen et al., 2006, Zuhdi Alimam et al., 2000).
In addition, assessment of mucin gene promoter activity by reporter gene expression (e.g. luciferase assay) allows assumptions on mucin expression levels (Chen et al., 2006).
Changes in mucin protein levels can be detected by Western blot in cell and tissue lysates using suitable antibodies (Lee et al., 2011, Okuda et al., 2019, Ramsey et al., 2016).
As a quick alternative to Western blot, dot-blot /slot-blot assay can be performed (Thornton et al., 1989).
Secreted mucin protein levels can be detected and quantified by Enzyme-Linked Immunosorbent Assay (ELISA) (Yu et al., 2011, Shao et al., 2004, Wagner et al., 2003, Dohrman et al., 1998, Song et al., 2016). ELISA method description for detection and quantification of mucin molecules can be found in the article from Steiger and colleagues (Steiger et al., 1994).
Analytical techniques such as immunocyto/histochemistry/immunofluorescence in cytological preparations or histological tissue sections with an appropriate antibody are also common methods of mucin protein level quantification (Zhu et al., 2009, Okuda et al., 2019). For immunofluorescent assays fluorescent dyes such as fluorescein isothiocyanate, Alexa488, Alexa555 are applied with subsequent visualization (e.g. confocal laser scanning or fluorescence microscopy) (Yu et al., 2011, Casalino-Matsuda et al., 2009, Val et al., 2012).
Immunoassay of MUC5AC protein is also used as mucin protein detection method, as described in the study of Takeyama and colleagues (Takeyama et al., 2001).
MUC5AC positive cell number determination through flow cytometry is another method for comparing and quantifying stressor-treated samples to control samples (Val et al., 2012).
For in vivo studies and clinical samples, an experienced pathologist may judge the presence and severity of mucin production on histological tissue sections stained with hematoxylin/eosin and Alcian blue and/or periodic acid Schiff stains (Song et al., 2016, Atanasova and Reznikov, 2019, Okuda et al., 2019).
Mass spectrometric approaches could be utilized for targeted identification of mucins and their quantification in cell and tissue samples (Kesimer and Sheehan, 2012).
Domain of Applicability
Evidence in support of this KE derives from in vitro studies with human cell systems (Casalino-Matsuda et al., 2009, Dohrman et al., 1998, Hao et al., 2014, Hewson et al., 2004, Lee et al., 2011, Val et al., 2012, Zhu et al., 2009), while corroborating in vivo evidence comes from studies in rodents (mouse or rat) (Hao et al., 2014, Song et al., 2016, Takeyama et al., 2001, Wagner et al., 2003).
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