Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) is a major cause of cystic fibrosis (CF), one of the most common inherited childhood diseases. ΔF508 CFTR is a trafficking mutant that is retained in the endoplasmic reticulum (ER) and unable to reach the plasma membrane. Efforts to enhance exit of ΔF508 CFTR from the ER and improve its trafficking are of utmost importance for the development of treatment strategies. Using protein interaction profiling and global bioinformatics analysis we revealed mammalian target of rapamycin (mTOR) signalling components to be associated with ∆F508 CFTR. Our results demonstrated upregulated mTOR activity in ΔF508 CF bronchial epithelial (CFBE41o-) cells. Inhibition of the Phosphatidylinositol 3-kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) pathway with 6 different inhibitors demonstrated an increase in CFTR stability and expression. Mechanistically, we discovered the most effective inhibitor, MK-2206 exerted a rescue effect by restoring autophagy in ΔF508 CFBE41o- cells. We identified Bcl-2-associated athanogene 3 (BAG3), a regulator of autophagy and aggresome clearance to be a potential mechanistic target of MK-2206. These data further link the CFTR defect to autophagy deficiency and demonstrate the potential of the PI3K/Akt/mTOR pathway for therapeutic targeting in CF.

Bile acids (BA) are becoming increasingly appreciated as enteric hormones that regulate many aspects of intestinal physiology. The BA receptor, TGR5, has been recently shown to be expressed on enteric nerves and enterochromaffin cells (ECs), where its activation regulates small intestinal and colonic motility. Here, we show that TGR5 is also expressed on colonic epithelial cells and that its activation decreases basal secretory tone and inhibits cholinergic-induced secretory responses. Our data demonstrate a new role for TGR5 in regulating colonic fluid and electrolyte transport and suggest that the receptor represents a good therapeutic target for intestinal transport disorders.

These data reveal a novel antisecretory role for the FXR in colonic epithelial cells and suggest that FXR agonists have excellent potential for development as a new class of antidiarrheal drugs.

Key points• Cl − secretion, the predominant driving force for fluid secretion in the intestine, can be dysregulated in conditions of disease, such as cystic fibrosis.• We have previously shown that acute exposure to epidermal growth factor (EGF) chronically upregulates the capacity of colonic epithelial cells to secrete Cl − .• Here, we show that the effects of EGF are mediated by upregulation of the Ca 2+ -dependent Cl − channel, transmembrane protein 16A (TMEM16A), in the apical membrane of colonic epithelial cells.• EGF-induced TMEM16A expression is mediated by sequential activation of phosphatidylinositol 3-kinase and PKCδ.• These findings are among the first to elucidate molecular mechanisms that regulate TMEM16A expression in epithelial cells and suggest the channel represents a good target for development of new therapeutics for intestinal transport disorders.Abstract Dysregulated epithelial fluid and electrolyte transport is a common feature of many intestinal disorders. However, molecular mechanisms that regulate epithelial transport processes are still poorly understood, thereby limiting development of new therapeutics. Previously, we showed that epidermal growth factor (EGF) chronically enhances intestinal epithelial secretory function. Here, we investigated a potential role for altered expression or activity of apical Cl − channels in mediating the effects of EGF. Cl − secretion across monolayers of T 84 colonic epithelia was measured as changes in short-circuit current. Protein expression/phosphorylation was measured by RT-PCR and Western blotting. Under conditions that specifically isolate apical Ca 2+ -activated Cl − channel (CaCC) currents, EGF pretreatment (100 ng ml −1 for 15 min) potentiated carbachol (CCh)-induced responses to 173 ± 25% of those in control cells, when measured 24 h later (n = 26; P < 0.01). EGF-induced increases in CaCC currents were abolished by the transmembrane protein 16A (TMEM16A) inhibitor, T16A inh -A01 (10 μM). Furthermore, TMEM16A mRNA and protein expression was increased by EGF to 256 ± 38% (n = 7; P < 0.01) and 297 ± 46% (n = 9, P < 0.001) of control levels, respectively. In contrast, EGF did not alter CFTR expression or activity. EGF-induced increases in Cl − secretion, CaCC currents and TMEM16A expression were attenuated by a PKCδ inhibitor, rottlerin (20 μM), and a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042 (25 μM). Finally, LY290042 inhibited EGF-induced phosphorylation of PKCδ. We conclude that EGF chronically upregulates Ca 2+ -dependent Cl − conductances and TMEM16A expression in intestinal epithelia by a mechanism involving sequential activation of PI3K and PKCδ. Therapeutic targeting of EGF