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

• Deregulation of constitutively Ca2+ influx in cystic fibrosis.
• Expression of PLC-δ1 isoform in CF cells is dramatically decreased in cystic fibrosis.
• Absence of negative regulation of TRPV6 activity in CF cells.

Increase of Ca2+ 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 Ca2+ homeostasis as they are highly selective toward Ca2+ and constitutively activated. Thus, we investigated the involvement of these channels in Ca2+ 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 Ca2+ 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 Ca2+ influx. TRPV6 channel is negatively regulated by the PLC-PIP2 pathway. We measured the Ca2+ influx in the presence of the non-specific PLC inhibitor, U73122, in non-CF human bronchial epithelial cells. Ca2+ 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 Ca2+ 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 Ca2+ homeostasis in CF human bronchial epithelial cells.

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