CFTR and Ca2+ Signaling in Cystic Fibrosis

Among the diverse physiological functions exerted by calcium signaling in living cells, its role in the regulation of protein biogenesis and trafficking remains incompletely understood. In cystic fibrosis (CF) disease the most common CF transmembrane conductance regulator (CFTR) mutation, F508del-CFTR generates a misprocessed protein that is abnormally retained in the endoplasmic reticulum (ER) compartment, rapidly degraded by the ubiquitin/proteasome pathway and hence absent at the plasma membrane of CF epithelial cells. Recent studies have demonstrated that intracellular calcium signals consequent to activation of apical G-protein-coupled receptors by different agonists are increased in CF airway epithelia. Moreover, the regulation of various intracellular calcium storage compartments, such as ER is also abnormal in CF cells. Although the molecular mechanism at the origin of this increase remains puzzling in epithelial cells, the F508del-CFTR mutation is proposed to be the onset of abnormal Ca2+ influx linking the calcium signaling to CFTR pathobiology. This article reviews the relationships between CFTR and calcium signaling in the context of the genetic disease CF

Effets des acides gras saturés sur la voie de sécrétion. Relation avec la mucoviscidose

Les acides gras saturés (AGS) altèrent la fonctionnalité des organites dans de nombreux types cellulaires. Il a été proposé que ce processus, également nommé lipointoxication, puisse être responsable de plusieurs pathologies humaines telles que le diabète de Type 2.Au niveau cellulaire, l'accumulation d'AGS est associée à une augmentation du taux de saturation des phospholipides (PL) membranaires, les composants majoritaires des membranes des organites, mais également du taux de céramides, impliqués dans l'induction de l'apoptose.Dans une première partie de ce travail, nous avons étudié, chez le modèle cellulaire simple Saccharomyces cerevisiae, la contribution relative des PL saturés et des céramides à la cytotoxicité des AGS. Nous avons pu démontrer que les céramides agissaient à des étapes précoces de la voie de sécrétion, alors que les PL saturés impactaient des étapes plus tardives en altérant en particulier la formation de vésicules de sécrétion.Parallèlement, nous avons également constaté que le taux d'AGS était significativement augmenté dans les PL membranaires des patients atteints d'une maladie génétique, la mucoviscidose. La mutation la plus fréquente responsable de cette maladie, résulte en la rétention de la protéine correspondante dans le réticulum endoplasmique. Des molécules pharmacologiques, capables de corriger le trafic de la protéine à sa destination finale ont été isolées in vitro, mais des limitations importantes ont pu être observées lors des tests cliniques. Nous proposons dans le présent manuscrit que la lipointoxication liée aux AGS pourrait être un écueil important à l'utilisation des correcteurs actuels pour le traitement de la mucoviscidose.Saturated fatty acids (SFA) have been reported to alter organelle integrity in many cell types. This process, also known as lipotoxicity, has been proposed to be responsible for several human pathologies such as type 2 diabetes.At the cellular level, SFA accumulation is associated with an increase of the saturation rate of membrane phospholipids (PL), the major components of organelle membranes, and an increase of ceramides levels, implicated in apoptosis induction.In the first part of this work, we took advantage of a simple yeast-based model to study the relative contributions of saturated PL and ceramides to SFA cytotoxicity. We demonstrated that ceramides act early in the secretory pathway, while saturated PL impact the later steps, and particularly the formation of secretory vesicles.In parallel, we observed that SFA amounts were significantly increased in the membrane PL of cystic fibrosis (CF) patient cells. The most common mutation responsible for this genetic disease results in the retention of the corresponding protein in the endoplasmic reticulum. Pharmacological agents, which correct the mistrafficking of the protein, have been isolated in vitro, but they did not show significant improvements in clinical trials. We propose in the present manuscript, that SFA-related lipointoxication could be an important bottleneck for the use of these pharmacological agents in clinical trials.POITIERS-SCD-Bib. électronique (861949901) / SudocSudocFranceF

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers

Phospholipase C controls chloride-dependent short-circuit current in human bronchial epithelial cells

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here, we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over-expressing wild type (WT)- or F508del-CFTR. Western blot analysis shows expression of the three endogenous phospholipase C (PLC) isoforms, namely, PLCδ1, PLCγ1, and PLCβ3 in 16HBE14o- cells. In 16HBE14o- cells, we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCβ3 and PLCγ1 in CFTR-dependent short-circuit current activated by forskolin, but not of PLCδ1. In CFBE-WT CFTR and corrected CFBE-F508del CFTR cells, PLCβ3 silencing also inhibits CFTR-dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR-dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells

Homéostasie calcique dans les cellules épithéliales mucoviscidosiques (F508del-CFTR)

Le calcium est un messager intracellulaire secondaire impliqué dans le contrôle de nombreux processus biologiques. Un mauvais contrôle de sa concentration peut conduire à la mort cellulaire. Pour ce faire, le Ca2 + est stocké dans les compartiments intracellulaires tel que le réticulum endoplasmique (RE). La mucoviscidose (CF) est une maladie génétique résultant de la mutation du gène codant pour la protéine CFTR (Cystic fibrosis transmembrane conductance regulator). Chez une majorité de patients (92%), il est retrouvé une délétion d'un résidu phénylanine en position 508. La protéine ainsi délétée (F508del-CFTR) se retrouve retenue dans le RE par des protéines chaperonnes dépendantes du Ca2 + Dans un premier temps, nous avons caractérisé et comparé l'homéostasie calcique des cellules épithéliales CF et non FC, précisément au niveau : du ER, de la membrane plasmique et pour finir au niveau des mitochondries. Puis, nous avons regarder les conséquences sur l'homéostasie calcique de la correction du défaut de l'adressage défectueux de la protéine F508del-CFTR (correction pharmacologique ( miglustat) ou par une incubation 24 heures à 27 C) . Nous avons mis en évidence l'implication de 3 isoformes de recepteurs à l'IP3 (RIP3),des canaux calciques membranaires TRPC1 et TRPC6, ainsi qu'une participation des mitochondries. Dans la mucoviscidose, les RIP3s, les canaux TRPC6 apparaissent suractivés. Il semble que le contenu en cholestérol de la membrane plasmique joue un rôle primordial dans la régulation du TRPC6. tandis que le pouvoir tampon calcique des mitochondries apparaît diminué dans les cellules CF. Le contrôle du signal calcique des cellules mucovisidosiques apparaît fortement perturbé. Ces perturbations du signal calcique pourraient avoir de nombreuses implications dans la physiopathologie de la mucoviscidose, notamment au niveau de l'état inflammatoire des cellules mucoviscidosiques.Calcium is a secondary intracellular messenger implicated in numerous biological processes, nevertheless calcium induce cell death. For this, Ca2+ is stored in intracellular compartments as endoplasmic reticulum (ER). In the genetic disease cystic fibrosis (CF), the most common mutation F508del results in endoplasmic reticulum retention of misfolded CF proteins (CFTR). The endoplasmic reticulum (ER) F508del-CFTR proteins retention is dependent upon chaperone proteins, many of which require Ca2+ for optimal activity. In first time, we characterized and compared calcium homeostasis in CF and non CF epithelial cells, at the ER, plasma membrane and mitochondrial levels. Secondly, we studied the consequence of rescue functional F508del-CFTR in CF cells by treatment with the pharmacological corrector miglustat or low temperature incubation (24h at 27C) on the Ca2+ homeostasis. In this present work, we show that the Ca2+ homeostasis is due to the presence of 3 IP3R isoforms on the ER membrane, the plasma membrane TRPC1 and TRPC6 are expressed, and that the mitochondria participated to Ca2+ homeostasis. In CF epithelial cells, IP3Rs and TRPC6 appear up regulated. The plasma membrane content plays a key role in TRPC6 regulation, whereas the mitochondria Ca2+ up-take is weaker in epithelial CF cells. These results show that the Ca2+ signalling is highly perturbed in human airway epithelial cells. These disturbances may have profound implications in CF physiopathology, notably in inflammatory state of CF cells.POITIERS-BU Sciences (861942102) / SudocSudocFranceF