Cigarette smoke and calcium conspire to impair CFTR function in airway epithelia

To maintain health and function in response to inhaled environmental irritants and toxins, the lungs and airways depend upon an innate defense system that involves the secretion of mucus (i.e., mucin, salts, and water) by airway epithelium onto the apical surface to trap foreign particles. Airway mucus is then transported in an oral direction via ciliary beating and coughing, which helps to keep the airways clear. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated Cl^- channel in the apical membrane of epithelium that contributes to salt and water secretion onto the luminal surface of airways, thereby ensuring that secreted mucus is sufficiently hydrated for movement along the epithelial surface. Dehydration of airway mucus, as occurs in cystic fibrosis, results in a more viscous, less mobile secretion that compromises the lung’s innate defense system by facilitating a build-up of foreign particles and bacterial growth. Related to this situation is chronic obstructive pulmonary disease (COPD), which is a leading cause of death globally. A major cause of COPD is cigarette smoking, which has been reported to decrease the cellular levels of CFTR in airway epithelia. In their recent article, Rasmussen and coworkers now report that exposure to cigarette smoke elevates cytosolic free Ca²⁺ in airway epithelium, leading to decreased surface localization and cellular expression of CFTR and reduced levels of secreted airway surface liquid. Blocking this increase in cytosolic Ca²⁺ largely prevented CFTR loss in airway epithelium and surprisingly, cellular lysosomes appear to be a major source for smoke-induced Ca²⁺ elevation.     

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.     

Cigarette smoke condensate induces MMP-12 gene expression in airway-like epithelia

Cigarette smoke (CS)-induced emphysema is attributable to matrix metalloproteinase-12 (MMP-12) in mice, however, a relationship between CS and MMP-12 is absent in human emphysema. Here, we show that cigarette smoke condensate (CSC) induces MMP-12 gene expression in airway-like epithelia through a hydrogen peroxide (H(2)O(2))-dependent pathway involving NADPH oxidase, AP-1, and TNF-alpha. Cigarette smoke condensate-induced H(2)O(2) production and MMP-12 gene expression were inhibited by apocynin, a specific inhibitor of NADPH oxidases, while 3-aminobenzamide, an inhibitor of AP-1, attenuated CSC-induced MMP-12 gene expression. Messenger RNAs encoding phagocytic NADPH oxidase components and a homologue of p67phox, p51 (NOXA1), were detected, while mRNA of dual oxidase (Duox)1 was unchanged by CSC. Enbrel, an inhibitor of TNF-alpha function, reduced CSC-induced H(2)O(2) production and MMP-12 expression. These findings provide novel evidence of a direct relationship between CS exposure and MMP-12 in human airway epithelia and suggest several targets for modulation of this potentially pathogenic pathway.     

Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

Background

Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Cigarette smoking is the leading risk factor for the development of COPD and 25% of asthmatics smoke. Smoking asthmatics have worse symptoms and more frequent hospitalizations compared to non-smoking asthmatics. The degree of neutrophil recruitment to the airways correlates with disease severity in COPD and during viral exacerbations of asthma. We have previously shown that HRV and cigarette smoke, in the form of cigarette smoke extract (CSE), each induce expression of the neutrophil chemoattractant and activator, CXCL8, in human airway epithelial cells. Additionally, we demonstrated that the combination of HRV and CSE induces expression of levels of CXCL8 that are at least additive relative to induction by each stimulus alone, and that enhancement of CXCL8 expression by HRV+CSE is regulated, at least in part, via mRNA stabilization. Here we further investigate the mechanisms by which HRV+CSE enhances CXCL8 expression.

Methods

Primary human bronchial epithelial cells were cultured and treated with CSE alone, HRV alone or the combination of the two stimuli. Stabilizing/destabilizing proteins adenine/uridine-rich factor-1 (AUF-1), KH-type splicing regulatory protein (KHSRP) and human antigen R (HuR) were measured in cell lysates to determine expression levels following treatment. siRNA knockdown of each protein was used to assess their contribution to the induction of CXCL8 expression following treatment of cells with HRV and CSE.

Results

We show that total expression of stabilizing/de-stabilizing proteins linked to CXCL8 regulation, including AUF-1, KHSRP and HuR, are not altered by CSE, HRV or the combination of the two stimuli. Importantly, however, siRNA-mediated knock-down of HuR, but not AUF-1 or KHSRP, abolishes the enhancement of CXCL8 by HRV+CSE. Data were analyzed using one-way ANOVA with student Newman-Keuls post hoc analysis and values of p≤ 0.05 were considered significant.

Conclusions

Induction of CXCL8 by the combination of HRV and CSE is regulated by mRNA stabilization involving HuR. Thus, targeting the HuR pathway may be an effective method of dampening CXCL8 production during HRV-induced exacerbations of lower airway disease, particularly in COPD patients and asthmatic patients who smoke.     

Molecular mechanisms controlling CFTR gene expression in the airway

The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3. We identify regions of open chromatin that appear selective for primary airway epithelial cells and show that several of these are enriched for a histone modification (H3K4me1) that is characteristic of enhancers. Consistent with these observations, three of these sites encompass elements that have cooperative enhancer function in reporter gene assays in 16HBE14o- cells. Finally, we use chromosome conformation capture (3C) to examine the three-dimensional structure of nearly 800 kb of chromosome 7 encompassing CFTR and observe long-range interactions between the CFTR promoter and regions far outside the locus in cell types that express high levels of CFTR.     

Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.     

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.     

Cigarette smoke alters IL-33 expression and release in airway epithelial cells

Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored.Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.     

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

Mitochondria-induced oxidative stress and flawed autophagy are common features of neurodegenerative and lysosomal storage diseases (LSDs). Although defective autophagy is particularly prominent in Pompe disease, mitochondrial function has escaped examination in this typical LSD. We have found multiple mitochondrial defects in mouse and human models of Pompe disease, a life-threatening cardiac and skeletal muscle myopathy: a profound dysregulation of Ca²⁺ homeostasis, mitochondrial Ca²⁺ overload, an increase in reactive oxygen species, a decrease in mitochondrial membrane potential, an increase in caspase-independent apoptosis, as well as a decreased oxygen consumption and ATP production of mitochondria. In addition, gene expression studies revealed a striking upregulation of the β 1 subunit of L-type Ca²⁺ channel in Pompe muscle cells. This study provides strong evidence that disturbance of Ca²⁺ homeostasis and mitochondrial abnormalities in Pompe disease represent early changes in a complex pathogenetic cascade leading from a deficiency of a single lysosomal enzyme to severe and hard-to-treat autophagic myopathy. Remarkably, L-type Ca²⁺channel blockers, commonly used to treat other maladies, reversed these defects, indicating that a similar approach can be beneficial to the plethora of lysosomal and neurodegenerative disorders.     

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.     

Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia

Ambroxol is often used as a mucolytic agent in various lung diseases. However, it is unclear how ambroxol acts on bronchial epithelial cells. To clarify the action of ambroxol, we studied the effects of ambroxol on the ion transport in human Calu-3 cells, a human submucosal serous cell line, measuring the transepithelial short-circuit current and conductance across monolayers of Calu-3 cells. Ambroxol of 100 microM diminished the terbutaline (a beta2-adrenergic agonist)-stimulated Cl-/HCO3(-)-dependent secretion without any decreases in the conductance of cystic fibrosis transmembrane conductance regulator (CFTR) channel locating on the apical membrane. On the other hand, under the basal (unstimulated) condition ambroxol increased the Cl(-)-dependent secretion with no significant change in the apical CFTR channel conductance and decreased the HCO3- secretion associated with a decrease in the apical CFTR channel conductance. Ambroxol had no major action on the epithelial Na+ channel (ENaC) or the ENaC-mediated Na+ absorption. These results indicate that in Calu-3 cells: (1) under the basal (unstimulated) condition ambroxol increases Cl- secretion by stimulating the entry step of Cl- and decreases HCO3- secretion by diminishing the activity of the CFTR channel and/or the Na+/HCO3(-)-dependent cotransporter, (2) under the adrenergic agonist-stimulated condition, ambroxol decreases Cl- secretion by acting on the Cl-/HCO3- exchanger, and (3) ambroxol has a more powerful action than the adrenergic agonist on the Cl-/HCO3- exchanger, leading fluid secretion to a moderately stimulated level from a hyper-stimulated level.     

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.     

Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function

Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiology on allergic asthma phenotypes are currently unknown. The mitochondrial Ca²⁺ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca²⁺ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca²⁺ uptake and reactive oxygen species (ROS) production, preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL-13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU-/- mice after exposure to IL-13. In the ovalbumin-model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO-1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca²⁺ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.     

Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin

Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.     

Small airway dysfunction is associated to excessive bronchoconstriction in asthmatic patients

Background

We investigated whether a relationship between small airways dysfunction and bronchial hyperresponsiveness (BHR), expressed both in terms of ease of airway narrowing and of excessive bronchoconstriction, could be demonstrated in asthma.

Methods

63 (36 F; mean age 42 yr ± 14) stable, mild-to-moderate asthmatic patients (FEV₁ 92% pred ±14; FEV₁/FVC 75% ± 8) underwent the methacholine challenge test (MCT). The degree of BHR was expressed as PD₂₀ (in μg) and as ∆FVC%. Peripheral airway resistance was measured pre- and post-MCT by impulse oscillometry system (IOS) and expressed as R5-R20 (in kPa sL⁻¹).

Results

All patients showed BHR to methacholine (PD₂₀ < 1600 μg) with a PD₂₀ geometric (95% CI) mean value of 181(132–249) μg and a ∆FVC% mean value of 13.6% ± 5.1, ranging 2.5 to 29.5%. 30 out of 63 patients had R5-R20 > 0.03 kPa sL⁻¹ (>upper normal limit) and showed ∆FVC%, but not PD₂₀ values significantly different from the 33 patients who had R5-R20 ≤ 0.03 kPa sL⁻¹ (15.8% ± 4.6 vs 11.5% ± 4.8, p < 0.01 and 156(96–254) μg vs 207 (134–322) μg, p = 0.382). In addition, ∆FVC% values were significantly related to the corresponding pre- (r = 0.451, p < 0.001) and post-MCT (r = 0.376, p < 0.01) R5-R20 values.

Conclusions

Our results show that in asthmatic patients, small airway dysfunction, as assessed by IOS, is strictly associated to BHR, expressed as excessive bronchoconstriction, but not as ease of airway narrowing.     

Intercellular calcium signalling in cultured renal epithelia: a theoretical study of synchronization mode and pacemaker activity

We investigate a two-dimensional lattice model representation of intercellular Ca²⁺ signalling in a population of epithelial cells coupled by gap junctions. The model is based on and compared with Ca²⁺ imaging data from globally bradykinin-stimulated MDCK-I (Madin-Darby canine kidney)-I cell layers. We study large-scale synchronization of relevance to our laboratory experiments. The system is found to express a wealth of dynamics, including quasiperiodic, chaotic and multiply-periodic behaviour for intermediate couplings. We take a particular interest in understanding the role of pacemaker cells in the synchronization process. It has been hypothesized that a few highly hormone-sensitive cells control the collective frequency of oscillation, which is close to the natural frequencies (without coupling) of these cells. The model behaviour is consistent with the conjectures of the pacemaker cell hypothesis near the critical coupling where the cells lock onto a single frequency. However, the simulations predict that the frequency in globally connected systems decreases with increasing coupling. It is found that a pacemaker is not defined by its natural frequency alone, but that other intrinsic or local factors must be considered. Inclusion of partly sensitized cells that do not oscillate autonomously in the cell layer increases the coupling necessary for global synchronization. For not excessively high coupling, these cells oscillate irregularly and with distinctive lower frequencies. In summary, the present study shows that the frequency of synchronized oscillations is not dictated by one or few fast-responding cells. The collective frequency is the result of a two-way communication between the phase-advanced pacemaker and its environment.     

Mitochondria attached to desmosomes in the ciliary epithelia of human,monkey, and rabbit eyes

In the normal ciliary epithelia of the rhesus monkey, owl monkey, albino rabbit, and human eye, a previously unreported relationship exists between mitochondria and certain desmosomes. At these sites, two mitochondria appear like "sentinels" attached to the cytoplasmic surfaces of their respective sides of a desmosome. In other instances, only one side of the junction may be afforded an associated mitochondrion. In each case the cytoplasmic filaments of the desmosome are seen to blend with the outer membrane of the mitochondrion. The relationship between desmosomes and mitochondria in the ciliary epithelium is unique among ocular tissues. A survey of ocular epithelia in the various species examined, failed to give any evidence of similar junctional/organelle complexes. Various functional roles for this relationship are discussed including the possibility that the mitochondria could control the cytoplasmic calcium ion concentration in the microenvironment of their associated desmosomal junctions.