Aim: 

We have previously demonstrated that NHERF1 overexpression increases the wild type (wt) Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) functional expression on the apical membrane in human airway cells and rescues F508del-CFTR functional expression in airway CF cells derived from a subject homozygous for the F508del mutation. The aim of this study was to explore the hypothesis that NHERF1 overexpression in CF cells may increase the redistribution of F508del-CFTR from the cytoplasm to the plasma membrane via cytoskeleton re-organization and the formation of the multi-protein complex, NHERF1-ezrin-actin.

Methods: 

All the experiments were performed in human airway cells CFBE41o- (CFBE) homozygous for the F508del allele, CFBE cells stably overexpressing wt NHERF1 and the control clone CFBE (stably transfected with empty pcDNA3 vector).

Results: 

We observed that CFBE cell monolayers displayed substantial disassembly of actin filaments and that overexpression of wt NHERF1 caused an increase in F-actin content and a re-distribution of F-actin towards the apical membrane. Transfection with NHERF1 lacking the ezrin binding domain, NHERF1-DERM, reversed the wt NHERF1 overexpression-induced increase in both F actin content and CFTR-dependent chloride secretion. Moreover, the overexpression of wt NHERF1 in CFBE cells greatly enhanced the interaction between NHERF1 and ezrin as well as between ezrin and actin. The overexpression of wt NHERF1, but not NHERF1-DERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Overexpression of wt NHERF1 also induced RhoA activation and constitutively active RhoA, per-se, was sufficient to induce the redistribution of phospho-ezrin to the membrane fraction and increase both the F actin content and the CFTR-dependent chloride efflux. In addition, specific ROCK inhibition reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, of F-actin content and of CFTR-dependent secretion.

Conclusions: 

We conclude that NHERF1 rescues F508-del CFTR-dependent chloride secretion via the re-organization of the actin cytoskeleton network induced by the formation of the multiprotein complex NHERF1-RhoA-ROCK-ezrin-actin. NHERF1 overexpression activates RhoA and leads to ROCK-mediated ezrin phosphorylation and stabilization in its open, active conformation which could further regulate F508del-CFTR stability in the apical membrane by tethering it to the actin cytoskeleton overexpression.