VIP-dependent increase in F508del-CFTR membrane localization is mediated by PKCε

Abstract

The most common cystic fibrosis causing mutation F508del induces early degradation and reduced trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. In the human nasal epithelial cells JME/CF15, we previously reported that vasoactive intestinal peptide (VIP) exposure corrects trafficking and membrane insertion of functional F508del-CFTR channels at 37°C. Correction of trafficking was PKA dependent, whereas enhanced membrane localization involved PKC. In the present study, we have identified PKCε as the isoform involved in VIP-dependent F508del-CFTR membrane insertion. Iodide effluxes were used to monitor the presence of VIP-rescued functional F508del-CFTR channels at the surface of JME/CF15 cells maintained at 37°C. Iodide efflux peaks measured in response to stimulation with forskolin were insensitive to PKC α, β, γ, δ, ζ inhibitors. In contrast, efflux peaks were completely inhibited by pretreatment with the PKCε inhibitor peptide EAVSLKPT with an IC(50) of 4.9 μM or by PKCε small interfering RNA (siRNA). Immunostaining and confocal microscopy confirmed that membrane localization of F508del-CFTR induced by VIP was abolished in the presence of EAVSLKPT but not with other isoform inhibitors. In recombinant baby hamster kidney cells, endogenously expressing PKCε but no VIP receptor, wild-type, and F508del-CFTR sensitivity to cpt-cAMP stimulation was increased by PMA treatment. Biotinylation assays and immunoblots confirmed that PMA (0.5-2 h) induced a greater than threefold increase in membrane CFTR, whereas forskolin had no effect. The PMA effect was abolished by specifically inhibiting PKCε (EAVSLKPT IC(50) = 5.7 μM) but not other PKC isoforms. Taken together, these results indicate that stimulating PKCε by VIP or PMA increases membrane insertion and activity of WT- and F508del-CFTR.

VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner

“…In Calu-3 cells, the most widely used model for human airway submucosal glands, stimulation of the G␣ s -PKA signaling cascade activates CFTR function by direct phosphorylation (21,22), whereas the G␣ q -PKC signaling pathway mainly regulates CFTR membrane density, also enhancing PKA-dependent activation by phosphorylation (11). In the present study, we first confirmed the role of PKCε in VIP-mediated increase in CFTR membrane density in Calu-3 cells with a specific inhibitor peptide, showing good correlation with our previous work on human airways and recombinant cells (1,11,46). Actin network and its associated proteins actively regulate the surface stability of many membrane proteins including CFTR (8,41).…”

“…We previously reported that PKCε was involved in VIPdependent increase in F508del-CFTR membrane stability in the human nasal epithelial cells JME/CF15 (1,46). Here we used the cell-permeant PKCε inhibitor peptide EAVSLKPT to demonstrate the role of this isoform in VIP signaling cascade responsible for increase in CFTR membrane expression.…”

“…VIP is released from nerves surrounding the airways where it performs several functions, including the control of inflammation, relaxation of smooth muscle, and regulation of CFTR chloride channels. Although initially considered as a cAMP-mediated secretagogue, VIP regulates CFTR through both PKA-and PKC-dependent signaling pathways (1,31). VIP plays a significant role in apical CFTR insertion in several in vivo preparations and in cell lines, implicating either PKA or PKC signaling depending on the kind of epithelium considered (3,4,33).…”

Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice

“…In human nasal epithelial cells (JME/CF15), derived from a F508del-CFTR homozygous patient, VIP treatment (300 nM) for 1 or 2 h induced the maturation and membrane insertion of functional F508del-CFTR proteins by stimulation of the VPAC1 receptor. Both the PKA and PKC⑀-dependent signaling cascade were found to be involved (1,30). The receptors are widely distributed in the body with some variation in cell type expressing VPAC1 and VPAC2 receptors.…”

Vasoactive intestinal peptide inhibits the activation of murine fibroblasts and expression of interleukin 17 receptor C

“…Cunha‐Reis et al () found that activation of VPAC1 receptors inhibited and activation of VPAC2 receptors increased gamma‐aminobutyric acid release, and both effects depend on the PKC mechanism. It has been reported that F508del‐cystic fibrosis trans‐membrane potential conductance regulator (CFTMP) membrane localization by VIP is mediated by PKC13ε (Alcolado et al, ). Our study suggests that both PKA and PKC pathways are involved in inhibiting the expression of IL‐17RC in fibroblasts.…”