title: The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity.
abstract: Increase 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.
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires ERL 7368 CNRS, Université de Poitiers, 86073 Poitiers, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Vachel
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires ERL 7368 CNRS, Université de Poitiers, 86073 Poitiers, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Norez
authors.affiliation: Service de Chirurgie Cardiothoracique, CHU Poitiers, Poitiers, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Jayle
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires ERL 7368 CNRS, Université de Poitiers, 86073 Poitiers, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Becq
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires ERL 7368 CNRS, Université de Poitiers, 86073 Poitiers, France. Electronic address: clarisse.vandebrouck@univ-poitiers.fr.
authors.forename: Clarisse
authors.initials: C
authors.lastname: Vandebrouck
mesh_terms: D000328:Adult; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008297:Male; D008875:Middle Aged; D009154:Mutation; D054803:Phospholipase C delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D004963:Estrenes; D020033:Protein Isoforms; D011760:Pyrrolidinones; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C120776:TRPV6 channel; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C518024:PLCD1 protein, human; D054803:Phospholipase C delta; D002118:Calcium
keywords: Calcium; Cystic fibrosis; Human bronchial epithelial cells; TRPV6