Franck Brouillard

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.

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.

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.

Cystic Fibrosis Transmembrane Regulator Inhibitors CFTRinh-172 and GlyH-101 Target Mitochondrial Functions, Independently of Chloride Channel Inhibition

Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTRinh-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTRinh-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTRinh-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTRinh-172, but not GlyH-101, induced nuclear translocation of nuclear factor-κB (NF-κB). CFTRinh-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-κB signaling pathway, independently of CFTR inhibition.

NF-κB mediates up-regulation of the CFTR gene expression by interleukin-1β in Calu-3 cells

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.

Concomitant Increase of Histone Acetyltransferase Activity and Degradation of p300 during Retinoic Acid-induced Differentiation of F9 Cells

The p300 and closely related CBP histone acetyltransferases (HAT) function as global transcriptional co-activators that play roles in many cell differentiation and signal transduction pathways. Despite their similarities, p300 and CBP have distinct functions during retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells. F9 cells constitute a well established model system for investigating the first steps of early development and retinoic acid signaling ex vivo. p300, but not CBP, was shown to be essential for F9 differentiation. In this study we have investigated the regulation of p300 during F9 differentiation. We report a dramatic decrease of p300, but not CBP protein levels, after 48 h of retinoic acid treatment. p300 is degraded via the ubiquitin-proteasome pathway. Although the large majority of p300 is degraded, its global HAT activity stays constant during F9 differentiation, which means that its specific HAT activity increases considerably. p300 is strongly phosphorylated in both undifferentiated and differentiated F9 cells; its HAT activity, however, is independent of phosphorylation before differentiation and becomes dependent on phosphorylation during differentiation. Furthermore, we show that protein kinase A affects p300 HAT activity both in vivo and in vitro as well as p300 phosphorylation in differentiated cells. Thus, we show that p300 is differentially phosphorylated in undifferentiated versus differentiated cells and that the changes in phosphorylation affect its HAT activity. Moreover, our study suggests an explanation for the functional switch of p300-mediated repression versus activation during F9 differentiation.

Membrane Cholesterol Content Modulates ClC-2 Gating and Sensitivity to Oxidative Stress

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-β-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.

Contribution of proteomics to the study of the role of cytokeratins in disease and physiopathology.

Cytokeratins (CKs), the most abundant group of cytoskeletal intermediate filaments, and proteomics are strongly connected. On the one hand, proteomics has been extremely useful to uncover new features and functions of CKs, on the other, the highly abundant CKs serve as an exceptional tool to test new technological developments in proteomics. As a result, proteomics has contributed to finding valuable associations of CKs with diseases as diverse as cancer, cystic fibrosis, steatohepatitis, viral and bacterial infection, keratoconus, vitreoretinopathy, preeclampsia or the chronic fatigue syndrome, as well as to characterizing their participation in a number of physiopathological processes, including drug resistance, response to toxicants, inflammation, stem cell differentiation, embryo development, and tissue repair. In some cases, like in cystic fibrosis, CKs have been described as potential therapeutic targets. The development of a specific field of proteomics where CKs become the main subject of research aims and hypotheses is suggested.