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Key Event Title
Cystic Fibrosis Transmembrane Regulator Function, Decreased
|Level of Biological Organization|
Key Event Components
|chloride channel activity||decreased|
Key Event Overview
AOPs Including This Key Event
|All life stages||High|
Key Event Description
The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain membrane protein that belongs to the large family of adenine nucleotide binding cassette transporters consisting of two transmembrane domains, two nucleotide binding domains (NBDs) and a unique regulatory domain (Riordan, 2008). It is an integral membrane glycoprotein that functions as cAMP-activated and phosphorylation-regulated Cl– channel at the apical membrane of epithelial cells (Farinha et al., 2013). In respiratory epithelia, CFTR is the major Cl– channel that mediates fluid and electrolyte transport, and CFTR function is critical to normal ASL homeostasis. Exposure to inhaled oxidants, such as ozone and cigarette smoke, leads to decreased CFTR gene and protein expression as well as CFTR internalization, thereby reducing or abolishing short-circuit currents (Qu et al., 2009; Cantin et al., 2006a; Cantin et al., 2006b; Clunes et al., 2012; Sloane et al., 2012; Rasmussen et al., 2014). Reduced CFTR gene transcription rates were mechanistically linked to mobilization of intracellular Ca2+, resulting in decreased mRNA and protein expression, presumably in a protein kinase-dependent manner (Bargon et al., 1992a; Bargon et al., 1992b). Cigarette smoke exposure of primary human bronchial epithelial cells at the air-liquid interface was shown to rapidly increase intracellular Ca2+, followed by a decrease in cell surface CFTR expression (Rasmussen et al., 2014). Of note, this decrease by CFTR internalization was subsequently linked to decreased active Cl– transport and a reduction in ASL height/volume (Clunes et al., 2012). Similarly, treatment with pyocyanin, a redox-active virulence factor secreted by Pseudomonas aeruginosa which commonly infects the airways of cystic fibrosis patients, increased hydrogen peroxide levels in CFBE41o- bronchial epithelial cells in a dose- and time-dependent manner, leading to oxidation of the cytosol and inhibited forskolin-stimulated ion transport (Schwarzer et al., 2008). Other possible mechanisms of acquired CFTR dysfunction include direct covalent modification of the protein by cigarette smoke and acrolein (Raju et al., 2013; Raju et al., 2016a) or modulation of channel open probability (Zhang et al., 2013; Woodworth, 2015).
How It Is Measured or Detected
In cystic fibrosis patients, who carry a defect in the CFTR gene, the determination of the residual levels of normal, full-length CFTR transcripts may have some clinical utility in estimating CFTR function (Amaral et al., 2004). Moreover, decreased CFTR mRNA and protein expression were previously shown to result in reduced CFTR-mediated Cl− transport (Cantin et al., 2006a; Cantin et al., 2006b; Clunes et al., 2012; Sloane et al., 2012; Rasmussen et al., 2014). Therefore, measuring decreased CFTR function could be achieved by a combination of multiple techniques. For example, decreased expression of CFTR mRNA and protein in cells and tissues can be directly assessed using RT-PCR, Northern blot and Western blot or immunocyto-/histochemical methods, respectively. Of note, CFTR gene expression is generally low as is protein abundance, and protein detection methods in general perform more robustly in cultured cells than in native tissues (Farinha et al., 2004). Other, less frequently used methods include cell surface biotinylation, enabling a distinction between intracellular and cell surface forms of the protein if one wishes to study plasma membrane-expressed CFTR. In vitro or ex vivo, CFTR channel function can be assessed in real-time using patch-clamping of whole (single) cells or cell patches. In the whole-cell patch-clamp approach, current flow through CFTR can be assessed by voltage-clamp, whereas current-clamping provides insights into the effects of CFTR currents on membrane voltage (Sheppard et al., 2004). Measuring the efflux of radiolabeled tracers is another means of studying CFTR channel function, permitting a higher throughput than patch-clamping (Norez et al., 2004). The most commonly used method to study CFTR ion transport, however, utilizes the Ussing chamber to measure transepithelial voltage or “active transport potential” and short-circuit current (Li et al., 2004). In vivo, CFTR dysfunction is demonstrated by the chloride sweat test, the gold standard diagnostic tool for cystic fibrosis. The sweat test should be performed according to clinical guidelines using the Gibson and Cooke technique (also known as quantitative pilocarpine iontophoresis sweat test) (Farrell et al., 2017; Smyth et al., 2014). As a complementary diagnostic measure, nasal potential difference (NPD) can be assessed to gauge net transepithelial active ion transport and epithelial ion conductance (Schüler et al., 2004). An entire issue of the Journal of Cystic Fibrosis dedicated to the Virtual Repository of the CFTR Working Group, including the description of consensus research methods, selected principles, techniques and reagents for the assessment of CFTR expression and function is available here: https://www.sciencedirect.com/journal/journal-of-cystic-fibrosis/vol/3/suppl/S2
Domain of Applicability
Phylogenetic analysis of CFTR DNA sequences across multiple species suggests a close evolutionary relationship between human and primate CFTR, followed by rabbit, guinea pig, equine, ovine, and bovine CFTR, whereas rodent CFTR DNA largely diverges from the human DNA (Chen et al., 2001). Of note, CFTR ion permeability differs from species to species (Higgins, 1992). For example, murine CFTR displays reduced channel activity compared with its human counterpart, while ovine CFTR exhibits higher ATP sensitivity, greater single-channel conductance and larger open probability than human CFTR. Moreover, sensitivity to pharmacological agents able to potentiate or block CFTR gating varies greatly from species to species (Bose et al., 2015). Therefore, results from animal studies are not easily and directly transferable to human.
CFTR dysfunction as a consequence of inherited CFTR gene defects is studied in pediatric as well as adult cystic fibrosis patients. Acquired CFTR dysfunction following inhalation exposures (e.g. to cigarette smoke) may also apply to both pediatric and adult populations, depending on the setting and type of exposure. To our knowledge, the role of gender has not been systematically evaluated in acquired CFTR dysfunction. It is thought that the observed suppression of CFTR expression and impairment of CFTR function in cigarette smokers is a contributing factor to the pathogenesis of chronic obstructive pulmonary disease (COPD) (Dransfield et al., 2013; Raju et al., 2016b). The main risk factor for COPD is cigarette smoking, and COPD is more common in men than in women, which may be directly related to the higher prevalence of smoking in men, although this gender gap is closing (Hitchman and Fong, 2011; Ntritsos et al., 2018; Syamlal et al., 2014). Nevertheless, the available clinical evidence in support of this AOP suggests that there is no remarkable gender difference.
Evidence for Perturbation by Stressor
Acrolein exhibited a complex dose-dependent response with respect to CFTR-mediated Cl− transport in primary murine nasal septal epithelia: At 100 µM acrolein, Cl− currents increased, whereas 300 µM acrolein reduced forskolin-induced total apical Cl− secretion and 300 µM acrolein abolished all Cl− transport. These effects were independent of cAMP, suggesting that channel activation was not PKA/cAMP phosphorylation-dependent (Alexander et al., 2012). Acrolein decreased cAMP-mediated CFTR ion transport in human bronchial epithelial cells grown in monolayers and in human Calu-3 lung cancer cells, where the response was dose-dependent. Repeated, low-level exposure of human bronchial epithelial cells to acrolein (2.5 – 10 ng/mL for 7 days) had a similar effect on CFTR function and was shown to be unrelated to modulation of CFTR expression. Pretreatment with the antioxidant N-acetylcysteine could prevent acrolein-induced CFTR inhibition (Raju et al., 2013). Similar effects on CFTR function (as measured by nasal and intestinal transepithelial potential difference) were elicited by subcutaneous administration of 1 mg/kg acrolein for 4 weeks, and these could also be counteracted by co-treatment with NAC (Raju et al., 2013).
Tracheas of Wistar rats exposed to 1.5 ppm ozone for 1 h/day for 3 days exhibited reduced CFTR protein expression. Similarly, at 4 hours following a 30-min exposure to ozone, CFTR mRNA and protein were down-regulated in 16HBE14o- cells. At 24 hours post-exposure, a reduction in forskolin-stimulated CFTR Cl− conductance was observed (Qu et al., 2009).
CFTR transcript and protein levels were reduced in human Calu-3 lung cancer cells exposed to the gas phase of cigarette smoke (Cantin et al., 2006b), human immortalized bronchial epithelial 16HBE14o- cells treated with 10% cigarette smoke extract (Hassan et al., 2014; Rasmussen et al., 2014; Xu et al., 2015), differentiated primary human bronchial epithelial cells exposed to whole cigarette smoke (Sloane et al., 2012; Hassan et al., 2014), and in airways of smokers compared to non-smokers (Dransfield et al., 2013). Following exposure to cigarette smoke, Cl− conductance (i.e., CFTR-mediated Cl− transport) decreased in primary human bronchial epithelial cells grown in monolayers (Lambert et al., 2014), differentiated primary human bronchial epithelial cells (Schmid et al., 2015; Chinnapaiyan et al., 2018), and nasal respiratory and intestinal epithelia of A/J mice (Raju et al., 2013; Raju et al., 2017). In the lower airways, healthy smokers and smokers with chronic obstructive pulmonary disease (COPD) showed reduced CFTR-dependent Cl− transport, whereas COPD former smokers showed an intermediate response to chloride-free isoproterenol solution compared to non-smokers. Similarly, amiloride-sensitive lower airway potential difference was also lower in healthy smokers and COPD smokers than in healthy non-smokers. This was linked to reduced CFTR protein levels in the airways of smokers compared to non-smokers, although there were no significant differences between healthy and COPD subjects (Dransfield et al., 2013). CFTR-dependent Cl− conductance as measured by nasal potential difference was also significantly reduced in healthy and COPD smokers compared to healthy non-smokers or to former smokers with COPD (Sloane et al., 2012). In addition, healthy never-smokers had higher mean sweat chloride concentrations than COPD smokers and COPD former smokers (Raju et al., 2013; Courville et al., 2014).
Cadmium (Cd) decreased CFTR protein expression in Calu-3 cells in a dose- and time-dependent manner. CFTR transcript levels, however, appeared to only be transiently affected. Reduced CFTR expression at the plasma membrane was associated with a reduction in CFTR Cl− conductance. Treatment of cells with NAC did not rescue CFTR expression in Cd-treated cells. In contrast, co-treatment with α-tocopherol prevented CFTR inhibition, and this effect was linked to α-tocopherol suppressing the accumulation of ubiquitinated CFTR (Rennolds et al., 2010).
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