Fibrosis Transmembrane Conductance Regulator Anion Research Articles

A multimodal iPSC platform for cystic fibrosis drug testing

Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants.

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl− and HCO3− across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO3− efflux through CFTR often required the intracellular H+/HCO3− production and/or the Na+-dependent basolateral HCO3− uptake, the intracellular pH (pHi) balance might be disturbed, especially as a consequence of increased endogenous H+ and HCO3− production. However, measurement of pHi by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO3− transport. In addition, although the plasma membrane Na+/K+-ATPase (NKA) is normally essential for basolateral HCO3− uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pHi or NKA abundance on cell membrane.

Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH

Key points Cl− and HCO3 − had similar paracellular permeabilities in human airway epithelia.PCl/PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions.Under basal conditions, calculated paracellular HCO3 − flux was secretory.Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3 − flux.HCO3 − flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3 − secretion. Airway epithelia control the pH of airway surface liquid (ASL), thereby optimizing respiratory defences. Active H+ and HCO3 − secretion by airway epithelial cells produce an ASL that is acidic compared with the interstitial space. The paracellular pathway could provide a route for passive HCO3 − flux that also modifies ASL pH. However, there is limited information about paracellular HCO3 − flux, and it remains uncertain whether an acidic pH produced by loss of cystic fibrosis transmembrane conductance regulator anion channels or proinflammatory cytokines might alter the paracellular pathway function. To investigate paracellular HCO3 − transport, we studied differentiated primary cultures of human cystic fibrosis (CF) and non‐CF airway epithelia. The paracellular pathway was pH‐insensitive at pH 6.0 vs. pH 7.4 and was equally permeable to Cl− and HCO3 −. Under basal conditions at pH ∼6.6, calculated paracellular HCO3 − flux was weakly secretory. Treating epithelia with IL‐17 plus TNFα alkalinized ASL pH to ∼7.0, increased paracellular HCO3 − permeability, and paracellular HCO3 − flux was negligible. Applying IL‐13 increased ASL pH to ∼7.4 without altering paracellular HCO3 − permeability, and calculated paracellular HCO3 − flux was absorptive. These results suggest that HCO3 − flux through the paracellular pathway counterbalances, in part, changes in the ASL pH produced via cellular mechanisms. As the pH of ASL increases towards that of basolateral liquid, paracellular HCO3 − flux becomes absorptive, tempering the alkaline pH generated by transcellular HCO3 − secretion.

Roles of volume-regulatory anion channels, VSOR and Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death, stroke and myocardial infarction.

Two types of anion channels are directly activated by osmotic swelling and are involved in the regulatory volume decrease (RVD) in most types of mammalian cells, and they include the volume-sensitive outwardly rectifying anion channel (VSOR), also called the volume-regulated anion channel (VRAC), and the large-conductance maxi-anion channel (Maxi-Cl). In cardiomyocytes, a splice variant of cystic fibrosis transmembrane conductance regulator anion channel (cardiac CFTR) participates in the RVD mechanism under β-adrenergic stimulation. VSOR and Maxi-Cl are also involved in facilitation of the RVD process by releasing extracellular autocrine/paracrine signals, glutamate and ATP. Apoptotic cell death starts with cell shrinkage, called the apoptotic volume decrease (AVD), which is also caused by activation of VSOR. Since VSOR is implicated not only in the AVD induction but also in the uptake of an anti-cancer drug, cisplatin, downregulation of VSOR activity is causatively involved in acquisition of cisplatin resistance in cancer cells. Necrotic cell death exhibits persistent cell swelling, called the necrotic volume increase (NVI), which is coupled to RVD dysfunction due to impaired VSOR function. Acidotoxic and lactacidosis-induced necrotic cell death is induced both by glutamate release mediated by astroglial VSOR and Maxi-Cl and by exaggerated Cl- influx mediated by neuronal VSOR under prolonged depolarization caused by activation of ionotropic glutamate receptor (iGluR) cation channels. Both VSOR and Maxi-Cl are elaborately involved, in a manner as double-edged swords, in ischemia- and ischemia-reperfusion-induced apoptotic or necrotic cell death in cerebral and myocardial cells by mediating not only Cl- transport but also release of glutamate and/or ATP. Cardiac CFTR exerts a protective action against ischemia(-reperfusion)-induced cardiac injury, called myocardial infarction (MI), which is largely necrotic. Cardiac Maxi-Cl activity may participate in protection against ischemia(-reperfusion) injury by mediating ATP release.

Structural Changes Fundamental to Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Pore.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial cell anion channel. Potentiator drugs used in the treatment of cystic fibrosis act on the channel to increase overall channel function, by increasing the stability of its open state and/or decreasing the stability of its closed state. The structure of the channel in either the open state or the closed state is not currently known. However, changes in the conformation of the protein as it transitions between these two states have been studied using functional investigation and molecular modeling techniques. This review summarizes our current understanding of the architecture of the transmembrane channel pore that controls the movement of chloride and other small anions, both in the open state and in the closed state. Evidence for different kinds of changes in the conformation of the pore as it transitions between open and closed states is described, as well as the mechanisms by which these conformational changes might be controlled to regulate normal channel gating. The ways that key conformational changes might be targeted by small compounds to influence overall CFTR activity are also discussed. Understanding the changes in pore structure that might be manipulated by such small compounds is key to the development of novel therapeutic strategies for the treatment of cystic fibrosis.

Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.

Inhibition of Heat-Stable Toxin–Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors

Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

Cystic fibrosis: NHLBI Workshop on the Primary Prevention of Chronic Lung Diseases.

Cystic fibrosis (CF) is a life-limiting, monogenic disorder characterized by chronic sinopulmonary and gastrointestinal involvement. Progressive pulmonary disease leads to death in the majority of patients. Despite its well-defined molecular basis related to defects in the cystic fibrosis transmembrane conductance regulator anion transport channel, there are large gaps in our understanding of the origin of CF lung disease. Disease has been shown to be present in infancy, and there is mounting evidence that abnormalities begin in utero. Heterogeneity of clinical presentations and severity suggest that many factors involved in lung disease have yet to be fully elucidated. Although new advances in therapeutic treatments have shown promise in delaying disease progression, the prevention of pulmonary disease at its origin (primary prevention) should be a key goal of CF care. The objective of this workshop was to (1) review our understanding of the origins of CF lung disease, (2) determine gaps in the knowledge base that are most significant and most likely to enable prevention of CF lung disease, and (3) prioritize new research questions that will promote pulmonary health in both CF and other childhood lung diseases. The goal of this report is to provide recommendations for future research that will improve our understanding of pulmonary development in health and disease, improve outcome measures and biomarkers for early lung disease, and determine therapeutic targets and strategies to prevent the development of lung disease in children with CF.

Cystic fibrosis: NHLBI workshop on the primary prevention of chronic lung diseases

Cystic fibrosis (CF) is a life-limiting, monogenic disorder characterized by chronic sinopulmonary and gastrointestinal involvement. Progressive pulmonary disease leads to death in the majority of patients. Despite its well-defined molecular basis related to defects in the cystic fibrosis transmembrane conductance regulator anion transport channel, there are large gaps in our understanding of the origin of CF lung disease. Disease has been shown to be present in infancy, and there is mounting evidence that abnormalities begin in utero. Heterogeneity of clinical presentations and severity suggest that many factors involved in lung disease have yet to be fully elucidated. Although new advances in therapeutic treatments have shown promise in delaying disease progression, the prevention of pulmonary disease at its origin (primary prevention) should be a key goal of CF care. The objective of this workshop was to (1) review our understanding of the origins of CF lung disease, (2) determine gaps in the knowledge base that are most significant and most likely to enable prevention of CF lung disease, and (3) prioritize new research questions that will promote pulmonary health in both CF and other childhood lung diseases. The goal of this report is to provide recommendations for future research that will improve our understanding of pulmonary development in health and disease, improve outcome measures and biomarkers for early lung disease, and determine therapeutic targets and strategies to prevent the development of lung disease in children with CF.

Mice heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator anion channel display attenuated cardiopulmonary dysfunction and lung injury after influenza H1N1 infection (406.4)

Influenza A viruses cause a highly‐contagious respiratory disease associated with significant morbidity and mortality. The purpose of this study was to utilize F508del CFTR‐heterozygous mice to determine the contribution of CFTR‐mediated Cl‐ secretion to influenza pathogenesis and to evaluate CFTR as a therapeutic target.F508del CFTR‐heterozygous progeny (HETs) and wild‐type (WT) littermate controls were infected intranasally with 50 μl virus diluent (mock) or with 10,000 FFU/mouse influenza A/WSN/33 (H1N1) for 1‐6 days. Body weight, lung mechanics, cardiopulmonary function and levels of pulmonary inflammatory mediators were determined over the time of disease.Relative to WT littermate controls, infection‐induced hypoxemia and bradycardia were attenuated in HETs at 2‐6 days post‐infection (d.p.i.), although weight loss and viral replication did not differ. At 2 and 6 d.p.i., alterations in lung function in infected WT mice were absent in HETs at both 2 and 6 d.p.i. Bronchoalveolar lavage fluid from influenza‐infected HETs contained approximately 3‐fold greater numbers of alveolar macrophages and more than 10‐fold higher IL‐6 levels at 6 d.p.i.Reduced CFTR expression resulting from F508del CFTR heterozygosity significantly alters influenza pathogenesis. CFTR‐mediated Cl‐ secretion is therefore a potential therapeutic target to decrease lung injury after influenza H1N1 infection.Grant Funding Source: Supported by C. Glenn Barber Fund and NHLBI R01‐HL102469

Differential activation of the HCO3‐ conductance through the cystic fibrosis transmembrane conductance regulator anion channel by genistein and forskolin in murine duodenum

Many cystic fibrosis (CF)-associated mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels affect CFTR-activated HCO(3)(-) transport more than Cl(-) transport. Targeting the CFTR HCO(3)(-) conductance, if possible, may therefore be of major therapeutic benefit. In the present study, we examined the effects of genistein and forskolin on duodenal mucosal HCO(3)(-) and Cl(-) secretion. Murine duodenal mucosal HCO(3)(-) and Cl(-) secretions were examined in vitro in Ussing chambers by the pH stat and short circuit current (I(sc)) techniques. Genistein markedly stimulated duodenal HCO(3)(-) secretion and I(sc) in a dose-dependent manner in CFTR wild-type mice, but not in CFTR null mice. CFTR(inh)-172, a highly specific CFTR inhibitor, inhibited genistein-stimulated duodenal HCO(3)(-) secretion and I(sc) in wild-type mice. Genistein induced 59% net HCO(3)(-) increase and 123% net I(sc) increase over basal value, whereas forskolin, an activator of adenylate cyclase, induced 94% net HCO(3)(-) increase and 507% net I(sc) increase, indicating that, compared with forskolin, genistein induced a relatively high HCO(3)(-)/I(sc) ratio. Further data showed that CFTR HCO(3)(-)/Cl(-) conductance ratio was 1.05 after genistein stimulation, whereas after forskolin stimulation, the CFTR HCO(3)(-)/Cl(-) conductance ratio was 0.27. Genistein stimulates duodenal HCO(3)(-) and Cl(-) secretion through CFTR, and has a relatively high selectivity for the CFTR HCO(3)(-) conductance, compared with forskolin. This may indicate the feasibility of selective targeting of the HCO(3)(-) conductance of the CFTR channels.