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

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Key points• Although diarrhoeal diseases represent a significant health and economic burden to society, therapeutic options remain limited.• While several bile acids are known to stimulate epithelial Cl − secretion, the major driving force for fluid secretion in the intestine, the effects of ursodeoxycholic acid (UDCA) on epithelial transport function are not well described.• We report that in contrast to other bile acids, UDCA exerts anti-secretory actions on colonic epithelial cells in vitro.• In contrast, in vivo administration of UDCA enhances epithelial secretory function, an affect we ascribe to being due to its bacterial metabolism to lithocholic acid. In keeping with this hypothesis, in vivo administration of a metabolically stable analogue of UDCA, 6α-methyl-UDCA, was anti-secretory.• Our findings reveal novel anti-secretory actions of UDCA and suggest that metabolically stable analogues of bile acid may be useful for development as a new class of anti-diarrhoeal drug.Abstract Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl − secretion was measured across voltage-clamped monolayers of T 84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T 84 cells with bilateral UDCA attenuated Cl − secretory responses to the Ca 2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na + /K + -ATPase activity and basolateral K + channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg −1 ) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA ( agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

In patients with bile acid malabsorption, high concentrations of bile acids enter the colon and stimulate Cl− and fluid secretion, thereby causing diarrhoea. However, deoxycholic acid (DCA), the predominant colonic bile acid, is normally present at lower concentrations where its role in regulating transport is unclear. Thus, the current study set out to investigate the effects of physiologically relevant DCA concentrations on colonic epithelial secretory function. Cl− secretion was measured as changes in short‐circuit current across voltage‐clamped T84 cell monolayers. At high concentrations (0.5–1 mM), DCA acutely stimulated Cl− secretion but this effect was associated with cell injury, as evidenced by decreased transepithelial resistance (TER) and increased lactate dehydrogenase (LDH) release. In contrast, chronic (24 hrs) exposure to lower DCA concentrations (10–200 μM) inhibited responses to Ca2+ and cAMP‐dependent secretagogues without altering TER, LDH release, or secretagogue‐induced increases in intracellular second messengers. Other bile acids – taurodeoxycholic acid, chenodeoxycholic acid and cholic acid – had similar antisecretory effects. DCA (50 μM) rapidly stimulated phosphorylation of the epidermal growth factor receptor (EGFr) and both ERK and p38 MAPKs (mitogen‐activated protein kinases). The EGFr inhibitor, AG1478, and the protein synthesis inhibitor, cycloheximide, reversed the antisecretory effects of DCA, while the MAPK inhibitors, PD98059 and SB203580, did not. In summary, our studies suggest that, in contrast to its acute prosecretory effects at pathophysiological concentrations, lower, physiologically relevant, levels of DCA chronically down‐regulate colonic epithelial secretory function. On the basis of these data, we propose a novel role for bile acids as physiological regulators of colonic secretory capacity.

The intestinal epithelium constitutes an innate barrier which, upon injury, undergoes self-repair processes known as restitution. Although bile acids are known as important regulators of epithelial function in health and disease, their effects on wound healing processes are not yet clear. Here we set out to investigate the effects of the colonic bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on epithelial restitution. Wound healing in T cell monolayers grown on transparent, permeable supports was assessed over 48 h with or without bile acids. Cell migration was measured in Boyden chambers. mRNA and protein expression were measured by RT-PCR and Western blotting. DCA (50-150 µM) significantly inhibited wound closure in cultured epithelial monolayers and attenuated cell migration in Boyden chamber assays. DCA also induced nuclear accumulation of the farnesoid X receptor (FXR), whereas an FXR agonist, GW4064 (10 µM), inhibited wound closure. Both DCA and GW4064 attenuated the expression of CFTR Cl channels, whereas inhibition of CFTR activity with either CFTR--172 (10 µM) or GlyH-101 (25 µM) also prevented wound healing. Promoter/reporter assays revealed that FXR-induced downregulation of CFTR is mediated at the transcriptional level. In contrast, UDCA (50-150 µM) enhanced wound healing in vitro and prevented the effects of DCA. Finally, DCA inhibited and UDCA promoted mucosal healing in an in vivo mouse model. In conclusion, these studies suggest bile acids are important regulators of epithelial wound healing and are therefore good targets for development of new drugs to modulate intestinal barrier function in disease treatment. NEW & NOTEWORTHY The secondary bile acid, deoxycholic acid, inhibits colonic epithelial wound healing, an effect which appears to be mediated by activation of the nuclear bile acid receptor, FXR, with subsequent downregulation of CFTR expression and activity. In contrast, ursodeoxycholic acid promotes wound healing, suggesting it may provide an alternative approach to prevent the losses of barrier function that are associated with mucosal inflammation in IBD patients.