CFTR, mucins, and mucus obstruction in cystic fibrosis

Abstract

Mucus pathology in cystic fibrosis (CF) has been known for as long as the disease has been recognized and is sometimes called mucoviscidosis. The disease is marked by mucus hyperproduction and plugging in many organs, which are usually most fatal in the airways of CF patients, once the problem of meconium ileus at birth is resolved. After the CF gene, CFTR, was cloned and its protein product identified as a cAMP-regulated Cl(-) channel, causal mechanisms underlying the strong mucus phenotype of the disease became obscure. Here we focus on mucin genes and polymeric mucin glycoproteins, examining their regulation and potential relationships to a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR). Detailed examination of CFTR expression in organs and different cell types indicates that changes in CFTR expression do not always correlate with the severity of CF disease or mucus accumulation. Thus, the mucus hyperproduction that typifies CF does not appear to be a direct cause of a defective CFTR but, rather, to be a downstream consequence. In organs like the lung, up-regulation of mucin gene expression by inflammation results from chronic infection; however, in other instances and organs, the inflammation may have a non-infectious origin. The mucus plugging phenotype of the β-subunit of the epithelial Na(+) channel (βENaC)-overexpressing mouse is proving to be an archetypal example of this kind of inflammation, with a dehydrated airway surface/concentrated mucus gel apparently providing the inflammatory stimulus. Data indicate that the luminal HCO(3)(-) deficiency recently described for CF epithelia may also provide such a stimulus, perhaps by causing a mal-maturation of mucins as they are released onto luminal surfaces. In any event, the path between CFTR dysfunction and mucus hyperproduction has proven tortuous, and its unraveling continues to offer its own twists and turns, along with fascinating glimpses into biology.

Figure 1.

Mucin domain maps and mucus. (A) Domain map for a generic, polymeric mucin (after Fig. 1a in Thornton et al. 2007). Indicated are the vWF vB, vC, and D domains, a Cys-rich domain, and the carboxy-terminal cystine knot (CK), in addition to the central glycosylated TR domains. The map most closely represents MUC2; unlike MUC2, MUC5AC and MUC5B have multiple Cys-rich domains within the glycosylated repeat domain (Rose and Voynow 2006). (B) Domain map for a tethered mucin, MUC1 (after Fig. 1A in Hattrup and Gendler 2008). Indicated are the cytoplasmic tail (CT), transmembrane (TM), and SEA domains, as well as the glycosylated TR domains, which for MUC1 can have considerable variation in the number of TRs (Hattrup and Gendler 2008). (C) Mucus harvested from HBE cell cultures, stained with fluorescent wheat germ agglutinin and concentrated to 2.5% or 6.5% solids, from a control of 1.5%. The highest concentration (6.5% solids) is representative of CF sputum. (Panel C is from Matsui et al. 2005; reprinted, with express permission, from the author.)

Figure 2.

Immunolocalization of CFTR, MUC5AC, and MUC5B in normal human airway epithelium. (Left panel) Confocal microscopy overlay image of a human frozen bronchial section from a normal lung donor immunostained with antibodies against MUC5AC (clone 45M1) and MUC5B (VNTR region). MUC5AC expression is restricted to goblet cells of the surface epithelium, whereas MUC5B is expressed in the goblet cells of the surface epithelium and predominantly in submucosal glands both in the ciliated ducts (CD) and acini (GA) in a gradient pattern with the highest levels in the acini. Scale bar, 200 µm. (Inset) Goblet cells of the surface epithelium show that MUC5B is present in the surface epithelium and can be coexpressed with MUC5AC. Scale bar, 20 µm. (Center panel) Confocal microscopy image of a frozen section of human bronchus immunostained with a CFTR antibody. Images represent an overlay of the differential interference contrast (DIC) (gray) and CFTR immunofluorescence (red) confocal channels, and indicate that CFTR protein is mainly localized in the apical membrane of ciliated cells of the surface epithelium and ciliated ducts; CFTR immunostaining is negligible in serous cells of the gland acini. Scale bar, 20 µm. (Only ∼20% of normal bronchial specimens display CFTR immunostaining in glandular serous cells.) (Inset) Confocal image of coimmunostaining of CFTR and MUC5AC in primary cultures of human bronchial epithelium indicating that CFTR is not immunolocalized in goblet cells. Scale bar, 10 µm. (Images from Kreda et al. 2005; reproduced, with permission, from ASCB/Molecular Biology of the Cell.) (Right panel) Confocal microscopy images of CFTR immunolocalization in the gland acini of human nasal epithelium. CFTR is localized in the apical membrane of serous cells in ∼50% of normal nasal specimens (Kreda et al. 2005). Scale bar, 20 µm. SE, surface epithelium; CD, ciliated duct; GA, gland acini.

Figure 3.

Mucin secretion in human bronchial epithelial cell cultures from healthy (normal) and CF individuals (A), and from Calu-3 cells exposed to CFTR_inh-172 (B). Mucin secretion, assessed as described in Abdullah et al. (2012) and Kreda et al. (2007), was not affected in either case. The data are expressed as the mean ± S.E.: HBE cell culture data (LH Abdullah and CW Davis, unpubl.) (n = 6); Calu-3 cell data (S Kreda, unpubl.) (n = 3). a.u., arbitrary units.

Figure 4.

MUC5AC gene expression is transcriptionally regulated by various mediators and pathways. The figure depicts mediators, receptors, and cis-sites reported since earlier reviews on regulation of MUC5AC gene expression (Rose and Voynow 2006; Thai et al. 2008; Voynow and Rubin 2009). Each reference is color-coded by arrows. (Red) IL1β and IL17A induce MUC5AC expression by activation of the NFκB cis-site at −3594/−3582 bp. Direct binding of the NFκB p50 subunit was observed in HBE1 cells (Fujisawa et al. 2009). (Blue) PGF_2α signals through the prostaglandin F receptor (FP), which is coupled to the Gq protein to activate PKC, which then signals through an MEK, ERK, RSK pathway. RSK translocates to the nucleus after phosphorylation and activates CREB to bind to the CRE cis-site (−878/−871) to up-regulate MUC5AC expression in differentiated HBE cells and NCI-H292 cells. A model summarizing the mechanisms whereby prostaglandins and IL1β induce MUC5AC overexpression has been reported (Chung et al. 2009). (Pink) CREB binds maximally to the CRE cis-site in the MUC5AC promoter 2 h after IL1β exposure to A549 or differentiated HBE cells (YA Chen, AM Watson, LM Garvin, et al., unpubl.). (Orange) PMA activates the PKC isoforms δ and θ, resulting in activation of matrix metalloproteinase (MMP) and secretion of TGFα, which binds to its cognate receptor EGFR. EGFR activates the MEK/ERK1/2 pathway to activate Sp1, which binds to three Sp1 sites (−192/−63) in the MUC5AC promoter. Similar results were obtained in H292 cells (Hewson et al. 2004) and in differentiated HBE and HBE1 cells (Yuan-Chen et al. 2007). (Green) ATP exposure to H292 cells induces MUC5AC expression by activating PLCβ3, which, in turn, activates Akt to signal through ERK or p38 to activate RSK, thereby activating CREB and c-Ets1 binding to the CRE (−889/−869) and c-Ets1 (−938/−930) sites in the MUC5AC promoter (Song et al. 2008). (Purple) Neuregulin 1β1 (NRG1β1) signals through the ERB2/ERB3 heterodimer receptor to activate three different pathways: p38, ERK1/2, and PI3K, which signals through Akt to up-regulate MUC5AC gene expression in differentiated HBE cells (Kettle et al. 2010).

Figure 5.

MUC5B gene expression is transcriptionally regulated by various mediators and pathways. (Red) IL1β and IL17A induce MUC5B gene expression by activation of the NFκB cis-site at −2921/−2909 bp in the MUC5B promoter. Direct binding of the NFκB p50 subunit was observed by ChIP in HBE1 cells (Fujisawa et al. 2011). (Orange) PMA activates PKCδ, which signals through Ras to activate MEKK1, which then signals through either a p38 pathway or the JNK pathway to phosphorylate Sp1, which binds to the Sp1 site at −184/−196. The Sp1 cis-site (−144/−122) functions to maintain basal regulation of MUC5B in differentiated HBE cells and in HBE1 cells (Wu et al. 2007a). (Purple) NRG1β1 increases MUC5B gene expression in differentiated HBE cells by signaling through the ErbB2/ErbB3 heterodimer receptor, which then signals through three different pathways including p38, ERK1/2, and PI3k. PI3k goes on to activate Akt (Kettle et al. 2010).