Expression of ENaC subunits as an investigation tool of the interaction between CFTR and ENaC and therapeutic approaches by epigenetic manipulation and activity reduction.

Background ENaC (Epithelial Na+ channel) has been shown to be involved in CF and CF-like diseases. Although in last years our knowledge has greatly improved, the exact role and molecular mechanisms involved deserve further studies. The wild-type but deregulated ENaC may be a better therapeutic target than the mutated CFTR. Proteolytic activation and epigenetics could play a role both as regulatory mechanisms and as therapeutic targets. Aims The functional characterization of mutations in ENaC genes, identified in CF patients, requires a cellular model that recapitulates the physiological interactions between ENaC and CFTR channels. To this end, we transfected FRT (Fisher Rat Thyroid) cells, which do not express endogenous ENaC and CFTR genes, with tagged human ENaC genes. Moreover, we investigated whether epigenetic mechanisms (DNA methylation and chromatin compaction), known to control several biological functions, are involved in the physiologic and pathologic regulation of ENaC genes and whether inhibition of ENaC by S-adenosyl methionine (SAM), curcumin and camostat may be synergistic or additive. Methods Mammalian expression vectors carrying the three tagged ENaC genes were achieved for each wild-type human ENaC subunit and used to transfect FRT cells transiently. To study ENaC activity, we measured fluid absorption from the apical side in polarized cells in the absence or in the presence of a low-molecular weight trypsin-like protease inhibitor camostat mesylate at different doses (0.3-30 µM). Moreover we measured fluid absorption in 16HBE, CFBE and primary bronchial epithelial cells both wild-type and mutated homozygous F508del / F508del after treatment with with 50 µM camostat, 100 µM SAM or 6 µM curcumin separately or in combination. Results FRT transfected with ENaC subunits in a ratio of alpha, beta and gamma of 2:1:1 showed a significantly higher fluid absorption than non-transfected cells. Nevertheless, camostat reduced significantly the rate of fluid reabsorption in transfected cells in a dose-dependent way. Concerning the epigenetic approach, in both 16HBE and CFBE all three compounds lowered the fluid absorption rate, with the strongest effect provided by curcumin in 16HBE and by SAM and camostat in CFBE. Both in 16HBE and CFBE no either synergic or additive effect was detected. In WT primary cells a significant effect was obtained by SAM and camostat and when all the three drugs were used in combination. In mutated homozygous F508del / F508del primary cells, SAM lowered fluid absorption both alone or in combination with the other compounds. A significant reduction of fluid absorption was also detected when camostat alone was used and when all the three drugs were used in combination. Conclusions We have found optimal doses of camostat as inhibitor of ENaC function in a heterologous expression system by a fluid absorption assay. The goal of the following work will be to analyze transient and stable ENaC-FRT clones with CFTR expression. Moreover, our results suggest that the function of ENaC, as evaluated by fluid absorption assay, may be downregulated by epigenetic modulation acting on both DNA methylation and chromatin, as well as by inhibiting peptidase. Acknowledgments This work was supported by the Italian Cystic Fibrosis Research Foundation (FFC#03/2012).