Aleksander Edelman

In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study.

BackgroundCystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.g. gentamicin) suppress nonsense mutations located in CFTR permitting translation to continue to the natural termination codon. Pharmacologic suppression of stop mutations within the CFTR may be of benefit to a significant number of patients. Our pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits.MethodsA dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. We investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment.ResultsAfter in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations.ConclusionSuppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.

NH3Is Involved in the Transport Induced by the Functional Expression of the Human Rh C Glycoprotein

Renal ammonium (NH3 + \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document}) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document} transmembrane translocation without high specificity \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document}, but it is still debated whether NH3, and more generally, gas, may be transported by transmembrane proteins. The human Rh glycoproteins have been proposed to mediate ammonium transport. Transport of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document} and/or NH3 by the epithelial Rh C glycoprotein (RhCG) may be of physiological importance in renal ammonium excretion because RhCG is mainly expressed in the distal nephron. However, RhCG function is not yet established. In the present study, we search for ammonium transport by RhCG. RhCG function was investigated by electrophysiological approaches in RhCG-expressing Xenopus laevis oocytes. In the submillimolar concentration range, NH4Cl exposure induced inward currents (IAM) in voltage-clamped RhCG-expressing cells, but not in control cells. At physiological extracellular pH (pHo) = 7.5, the amplitude of IAM increased with NH4Cl concentration and membrane hyperpolarization. The amplitude of IAM was independent of external Na+ or K+ concentrations but was enhanced by alkaline pHo and decreased by acid pHo. The apparent affinity of RhCG for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document} was affected by NH3 concentration and by changing pHo, whereas the apparent affinity for NH3 was unchanged by pHo, consistent with direct NH3 involvement in RhCG function. The enhancement of methylammonium-induced current by NH3 further supported this conclusion. Exposure to 500 μm NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document} enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH3 and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document} transport.

Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftrtm1Unc, cftr−/−) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 6–11, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr−/− mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr−/− mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A2. More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.

Rescue of ΔF508-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) by Curcumin: Involvement of the Keratin 18 Network

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, ΔF508, causes retention of ΔF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl– channels in the plasma membrane. ΔF508-CFTR retains some Cl– channel activity so increased expression of ΔF508-CFTR in the plasma membrane can restore Cl– secretion deficiency. Recently, curcumin was shown to rescue ΔF508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in ΔF508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional ΔF508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of ΔF508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or ΔF508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epithelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in ΔF508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated ΔF508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic ΔF508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments. With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

SummaryTo assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl− activity (αiCl) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (VBL) and Cl-selective electrode PD (VBLCl) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate αiCl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl− transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased αiCl, whereas addition of the drug in luminal fluid slightly increased αiCl; both results are consistent with basolateral membrane uphill Cl− transport from interstitium to the cell. TMA+ for Na+ substitutions in either luminal or peritubular perfusate had no effect on αiCl. Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO3− outflux), resulted in an increase of αiCl, presumably related to enhanced Cl− cell influx: we infer that Cl− is exchanged against HCO3− at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl− concentration above equilibrium values: intracellular CO2 hydration gives rise to cell HCO3− concentrations above equilibrium. The passive exit of HCO3− at the basolateral membrane energizes an uphill entry of Cl− into the cell. The resulting increase of αiCl, above equilibrium, generates downhill Cl− diffusion from cell to lumen. As a result, luminal Cl− concentration also increases.

Neutrophil Elastase Degrades Cystic Fibrosis Transmembrane Conductance Regulator via Calpains and Disables Channel Function In Vitro and In Vivo

RATIONALE Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel regulating fluid homeostasis at epithelial surfaces. Its loss of function induces hypohydration, mucus accumulation, and bacterial infections in CF and potentially other lung chronic diseases. OBJECTIVES To test whether neutrophil elastase (NE) and neutrophil-mediated inflammation negatively impact CFTR structure and function, in vitro and in vivo. METHODS Using an adenovirus-CFTR overexpression approach, we showed that NE degrades wild-type (WT)- and ΔF508-CFTR in vitro and WT-CFTR in mice through a new pathway involving the activation of intracellular calpains. MEASUREMENTS AND MAIN RESULTS CFTR degradation triggered a loss of function, as measured in vitro by channel patch-clamp and in vivo by nasal potential recording in mice. Importantly, this mechanism was also shown to be operative in a Pseudomonas aeruginosa lung infection murine model, and was NE-dependent, because CFTR integrity was significantly protected in NE(-/-) mice compared with WT mice. CONCLUSIONS These data provide a new mechanism and show for the first time a link between NE-calpains activation and CFTR loss of function in bacterial lung infections relevant to CF and to other chronic inflammatory lung conditions.

NF-kappa B mediates up-regulation of CFTR gene expression in Calu-3 cells by interleukin-1beta.

Inflammation of the airways is a major feature of the inherited disease cystic fibrosis. Previous studies have shown that the pro-inflammatory cytokines tumor necrosis factor α and interferon γ reduce the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR) in HT-29 and T84 cells by acting post-transcriptionally. We have investigated the effect of the pro-inflammatory peptide interleukin 1β (IL-1β) on the expression of the CFTR in Calu-3 cells. IL-1β increased the production of CFTR mRNA in a dose- and time-dependent manner. Its action was inhibited by inhibitors of the NF-κB pathway, includingN-acetyl-l-cysteine, pyrrolidine dithiocarbamate, and a synthetic cell-permeable peptide containing the NF-κB nuclear localization signal sequence. Gel shift analysis showed that IL-1β activated NF-κB in Calu-3 cells, and transfection experiments using p50 and RelA expressing vectors showed that exogenous transfected NF-κB subunits increased the concentration of CFTR mRNA. Gel shift analysis with antibody supershifting also showed that IL-1β caused the binding of NF-κB to a κB-like response element at position −1103 to −1093 in the CFTR5′-flanking region. Transfection experiments using −2150 to +52CFTR reporter gene constructs showed that the activity of the CFTR promoter is enhanced by exogenous transfected NF-κB and IL-1β and that this enhancement is due, at least in part, to the −1103 to −1093 κB site. We conclude that the intracellular signaling that leads to increased CFTR mRNA in response to IL-1β in Calu-3 cells includes the binding of NF-κB to the −1103 κB element and a subsequent increase in CFTR promoter activity.

Expression of the human erythroid Rh glycoprotein (RhAG) enhances both NH3 and NH4+ transport in HeLa cells

The erythroid Rh-associated glycoprotein (RhAG) is strictly required for the expression of the Rh blood group antigens carried by Rh (D,CE) proteins. A biological function for RhAG in ammonium transport has been suggested by its ability to improve survival of an ammonium-uptake-deficient yeast. We investigated the function of RhAG by studying the entry of NH3/NH4+ in HeLa cells transiently expressing the green fluorescent protein (GFP)-RhAG fusion protein and using a fluorescent proton probe to measure intracellular pH (pHi). Under experimental conditions that reduce the intrinsic Na/H exchanger activity, exposure of control cells to a 10 mM NH4Cl-containing solution induces the classic pHi response profile of cells having a high permeability to NH3 (PNH3) but relatively low permeability to NH4+ (PNH4). In contrast, under the same conditions, the pHi profile of cells expressing RhAG clearly indicated an increased PNH4, as evidenced by secondary reacidification during NH4Cl exposure and a pHi undershoot below the initial resting value upon its removal. Measurements of pHi during methylammonium exposure showed that RhAG expression enhances the influx of both the unprotonated and ionic forms of methylammonium. Using a mathematical model to adjust passive permeabilities for a fit to the pHi profiles, we found that RhAG expression resulted in a threefold increase of PNH4 and a twofold increase of PNH3. Our results are the first evidence that the human erythroid RhAG increases the transport of both NH3 and NH4+.

Coronin-1 Is Associated with Neutrophil Survival and Is Cleaved during Apoptosis: Potential Implication in Neutrophils from Cystic Fibrosis Patients

Because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. Using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. Coronin-1 is an actin-binding protein that can associate with phagosomes and NADPH oxidase, but its involvement in apoptosis was currently unknown. In coronin-1-transfected PLB985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by CD11b labeling. Concerning apoptosis, increased coronin-1 expression in dimethylformamide-differentiated PLB985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. Likewise, coronin-1 significantly decreased TRAIL-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or Bid truncation suggesting that coronin-1 interfered with mitochondria-related events. To validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (CF) patients were studied. Circulating neutrophils from CF patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. This was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. In addition, inflammatory neutrophils from CF patients lungs showed an intense coronin-1 immunolabeling. We concluded that coronin-1 could constitute a potential target in resolving inflammation.

Blue Native/SDS-PAGE Analysis Reveals Reduced Expression of the mClCA3 Protein in Cystic Fibrosis Knock-out Mice

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr−/−). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr−/− mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr−/− mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 μm) was impaired in the distal colon of cftr−/− mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.