IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro

AbstractRecent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6–48 h causes a marked inhibition of the amiloride-sensitive Na+ channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca2+-dependent Cl− channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Rα. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the γ amiloride-sensitive Na+ channel mRNA, one of the subunits of the amiloride-sensitive Na+ channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid

High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva.

open12noopenCappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello E.; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R.Cappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R

TITLE PAGE α-AMINOAZAHETEROCYCLIC-METHYLGLYOXAL ADDUCTS DO NOT INHIBIT CFTR CHLORIDE CHANNEL ACTIVITY

ABSTRACT Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. Recently, several highly polar α-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (Routaboul et al. J. Pharmacol. Exp. Ther. 322:1023-1035, more than 10,000-fold better than that of thiazolidionone and glycine hydrazide CFTR inhibitors identified previously by highthroughout screening. Here, we resynthesized and evaluated the α-aminoazaheterocyclicmethylglyoxal adducts of Routaboul et al. reported to have high CFTR inhibition potency (compounds 5, 7 and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 µM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp and YFPfluorescence quenching. As positive controls, near 100% CFTR inhibition was found by thiazolidionone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by α-aminoazaheterocyclic-methylglyoxal adducts

Cystic Fibrosis: A New Target for 4-Imidazo[2,1-b]thiazole-1,4-dihydropyridines

The pharmacology of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel has attracted significant interest in recent years with the aim to search for rational new therapies for diseases caused by CFTR malfunction. Mutations that abolish the function of CFTR cause the life-threatening genetic disease cystic fibrosis (CF). The most common cause of CF is the deletion of phenylalanine 508 (ΔF508) in the CFTR chloride channel. Felodipine, nifedipine, and other antihypertensive 1,4-dihydropyridines (1,4-DHPs) that block L-type Ca(2+) channels are also effective potentiators of CFTR gating, able to correct the defective activity of ΔF508 and other CFTR mutants ( Mol. Pharmacol. 2005 , 68 , 1736 ). For this purpose, we evaluated the ability of the previously and newly synthesized 4-imidazo[2,1-b]thiazoles-1,4-dihydropyridines without vascular activity and inotropic and/or chronotropic cardiac effects ( J. Med. Chem. 2008 , 51 , 1592 ) to enhance the activity of ΔF508-CFTR. Our studies indicate compounds 17, 18, 20, 21, 38, and 39 as 1,4-DHPs with an interesting profile of activity

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector in vivo and in vitro only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR

Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins

Managing the underlying cause of cystic fibrosis: A future role for potentiators and correctors

Cystic fibrosis (CF), a severe genetic disease, is caused by mutations that alter the structure and function of CFTR, a plasma membrane channel permeable to chloride and bicarbonate. Defective anion transport in CF irreversibly damages the lungs, pancreas, liver, and other organs. CF mutations cause loss of CFTR function in multiple ways. In particular, class 3 mutations such as p.Gly551Asp strongly decrease the time spent by CFTR in the open state (gating defect). Instead, class 2 mutations impair the maturation of CFTR protein and its transport from the endoplasmic reticulum to the plasma membrane (trafficking defect). The deletion of phenylalanine 508 (p.Phe508del), the most frequent mutation among CF patients (70-90 %), destabilizes the CFTR protein, thus causing both a trafficking and a gating defect. These two defects can be overcome with drug-like molecules generically called correctors and potentiators, respectively. The potentiator Kalydeco™ (also known as Ivacaftor or VX-770), developed by Vertex Pharmaceuticals, has been recently approved by the US FDA and the European Medicines Agency (EMA) for the treatment of CF patients carrying at least one CFTR allele with the p.Gly551Asp mutation (2-5 % of all patients). In contrast, the corrector VX-809, which significantly improves p.Phe508del-CFTR trafficking in vitro, is still under study in clinical trials. Because of multiple defects caused by the p.Phe508del mutation, it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action. This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect

TMEM16 Proteins: Membrane Channels with Unusual Pores

no abstract availabl

Structure and function of tmem16 proteins (anoctamins)

TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion transport, phospholipid scrambling, and regulation of other membrane proteins. The first two members of the family, TMEM16A (anoctamin-1, ANO1) and TMEM16B (anoctamin-2, ANO2), function as Ca2+-activated Cl- channels (CaCCs), a type of ion channel that plays important functions such as transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, and control of neuronal excitability. Genetic ablation of TMEM16A in mice causes impairment of epithelial Cl- secretion, tracheal abnormalities, and block of gastrointestinal peristalsis. TMEM16A is directly regulated by cytosolic Ca2+ as well as indirectly by its interaction with calmodulin. Other members of the anoctamin family, such as TMEM16C, TMEM16D, TMEM16F, TMEM16G, and TMEM16J, may work as phospholipid scramblases and/or ion channels. In particular, TMEM16F (ANO6) is a major contributor to the process of phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane. Intriguingly, TMEM16F is also associated with the appearance of anion/cation channels activated by very high Ca2+ concentrations. Furthermore, a TMEM16 protein expressed in Aspergillus fumigatus displays both ion channel and lipid scramblase activity. This finding suggests that dual function is an ancestral characteristic of TMEM16 proteins and that some members, such as TMEM16A and TMEM16B, have evolved to a pure channel function. Mutations in anoctamin genes (ANO3, ANO5, ANO6, and ANO10) cause various genetic diseases. These diseases suggest the involvement of anoctamins in a variety of cell functions whose link with ion transport and/or lipid scrambling needs to be clarified

Pharmacological correctors of mutant CFTR mistrafficking

The lack of phenylalanine 508 (ΔF508 mutation) in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel represents the most frequent cause of CF, a genetic disease affecting multiple organs such as lung, pancreas, and liver. ΔF508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g., histone deacetylase inhibitors) or a single CFTR-interacting protein. The presence of multiple defects of the CFTR protein caused by the ΔF508 mutation and the redundancy of quality control mechanisms detecting ΔF508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector) may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects