Ca2+ influx in human bronchial epithelial cells : implication of TRP channels

Les canaux TRP (Transient Receptor Potential) sont des acteurs clés de l'homéostasie calcique. Plusieurs de ces canaux interviennent dans l'influx calcique des cellules épithéliales bronchiques, notamment TRPC6, qui est impliqué dans un couplage fonctionnel avec le canal Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Les mutations du CFTR (F508del et G551D) sont à l'origine de la mucoviscidose (Cystic Fibrosis (CF)), qui aboutit à l'augmentation de l'influx calcique dans les cellules CF. L'objectif de ce travail a été d'étudier l'implication des canaux TRP dans la dérégulation de l'influx calcique des cellules épithéliales bronchiques CF. Nous avons mis en évidence que CFTR régulait négativement l'activité de TRPC6, tandis que l'influx calcique via TRPC6 permettait de potentialiser l'activité du canal muté CFTR-G551D, activé au préalable par le VX-770. Nous proposons donc une nouvelle stratégie thérapeutique, combinant un potentiateur de CFTR et un activateur spécifique de TRPC6. Nous nous sommes ensuite intéressés au rôle des canaux TRPV, en particulier TRPV5 et TRPV6, dans l'influx calcique des cellules épithéliales bronchiques. Nous avons observé que l'influx Ca2+ constitutif, attribuable à ces deux canaux, était doublé dans les cellules CF, dû à une augmentation de l'activité de TRPV6. En effet, l'expression de la PLC-δ1, une enzyme régulant négativement TRPV6, est dramatiquement réduite dans les cellules CF. La correction de l'adressage du F508del-CFTR a permis de normaliser l'activité de TRPV6 sans restaurer l'expression de la PLC-δ1 dans les cellules CF, suggérant un contrôle plus complexe de TRPV6 dans les cellules épithéliales bronchiques.TRP (Transient Receptor Potential) channels are keys actors of Ca2+ homeostasis. Several of these channels are involved in the Ca2+ influx of bronchial epithelial cells, including TRPC6 which is implicated in a functional coupling with the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel. CFTR mutation leads Cystic Fibrosis (CF) disease and causes abnormal Ca2+ homeostasis trought an increased of Ca2+ influx in CF bronchial epithelial cells. Our objective is to investigate the implication of TRP channels in abnormal Ca2+ influx of CF bronchial epithelial cells.We showed that CFTR down regulates TRPC6 activity whereas Ca2+ influx through TRPC6 potentiates G551D-CFTR, activated by VX-770. We propose a new therapeutic strategy that combines a CFTR potentiator and a specific activator of TRPC6. Then, we focused on the role of TRPV channels, particularly TRPV5 and TRPV6, in Ca2+ influx of bronchial epithelial cells. We observed that constitutive Ca2+ influx, related to TRPV5/TRPV6 activity, was twice higher in CF cells due to the increase of TRPV6 activity. The expression of PLC-δ1, an enzyme that negatively regulates TRPV6 activity, is dramatically decreased in CF cells. The correction of F508del-CFTR trafficking allows TRPV6 activity normalization but do not restore PLC-δ1 expression level in CF cells, suggesting a more complex control of TRPV6 in bronchial epithelial cells

The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity.

International audienceIncrease of Ca(2+) influx in Cystic Fibrosis (CF) cells has been reported to be related to Transient Receptor Potential Canonical (TRPC6) channel, which is implicated in a functional coupling with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Several members of the Transient Receptor Potential Vanilloid (TRPV) channels family have already been described as emerging target for respiratory diseases. Two specific isoforms, TRPV5 and TRPV6 are of particular interest in the context of CF Ca(2+) homeostasis as they are highly selective toward Ca(2+) and constitutively activated. Thus, we investigated the involvement of these channels in Ca(2+) influx in CF and non-CF human bronchial epithelial cell lines. 16HBE14o-, CFBE41o- cell lines, primary human airway epithelial cells (hAEC) and freshly isolated human airway epithelial cells from CF and non-CF individuals were used. We showed that both channels are expressed in CF and non-CF cells and constitutive Ca(2+) influx was significantly higher (85%) in cells from CF individuals compared to cells from non-CF ones. Using the selective inhibitor of TRPV6 channel SOR-C27 and a siRNA strategy, our results revealed that TRPV6 was mostly involved in the increase of Ca(2+) influx. TRPV6 channel is negatively regulated by the PLC-PIP2 pathway. We measured the Ca(2+) influx in the presence of the non-specific PLC inhibitor, U73122, in non-CF human bronchial epithelial cells. Ca(2+) influx was increased by 33% with U73122 and this increase was largely reduced in the presence of SOR-C27. PLC inhibition in CF cells by U73122 had no effect on Ca(2+) influx. These results showed that PLC-PIP2 pathway is dysregulated in CF cells and leads to the increase of TRPV6 activity. The regulation of TRPV6 by PLC-PIP2 pathway implicates the specific PLC isoform, PLC-δ1. Immunoblot experiments revealed that expression of PLC-δ1 was decreased by 70% in CF cells. TRPV6 activity was normalized but not the level of expression of PLC-δ1 protein after F508del-CFTR rescue by low temperature for 48 h or treated for 24 h by 10 μM VX-809 in CF cells. This study revealed TRPV6 and PLC-δ1 as critical actor of Ca(2+) homeostasis in CF human bronchial epithelial cells

Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca2+ signaling complexes at the ER/PM compartment

Communication and material transfer between membranes and organelles take place at membrane contact sites (MCSs). MCSs between the ER and PM, the ER/PM junctions, are the sites where the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1 cluster. MCSs are formed by tether proteins that bridge the opposing membranes, but the identity and role of these tethers in receptor-evoked Ca2+ signaling is not well understood. Here, we identified Anoctamin 8 (ANO8) as a key tether in the formation of the ER/PM junctions that is essential for STIM1-STIM1 interaction and STIM1-Orai1 interaction and channel activation at a ER/PM PI(4,5)P2-rich compartment. Moreover, ANO8 assembles all core Ca2+ signaling proteins: Orai1, PMCA, STIM1, IP3 receptors, and SERCA2 at the ER/PM junctions to mediate a novel form of Orai1 channel inactivation by markedly facilitating SERCA2-mediated Ca2+ influx into the ER. This controls the efficiency of receptor-stimulated Ca2+ signaling, Ca2+ oscillations, and duration of Orai1 activity to prevent Ca2+ toxicity. These findings reveal the central role of MCSs in determining efficiency and fidelity of cell signaling. © 2019 The Author

A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells

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