Targeting the PI3K/Akt/mTOR signalling pathway in Cystic Fibrosis.

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.

The bile acid receptor, TGR5, regulates basal and cholinergic-induced secretory responses in rat colon.

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.

Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

BACKGROUND: Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function. METHODS: Cl(-) secretion was measured as changes in short-circuit current across voltage-clamped T(84) colonic epithelial cells. Protein expression was measured by western blotting and intracellular Ca(2+) levels by Fura-2 fluorescence. KEY RESULTS: While acute (15 min) treatment of T(84) cells with a cholinergic G(q) PCR agonist, carbachol (CCh), rapidly stimulated Cl(-) secretion, subsequent CCh-induced responses were attenuated in a biphasic manner. The first phase was transient and resolved within 6 h but this was followed by a chronic phase of attenuated responsiveness that was sustained up to 48 h. CCh-pretreatment did not chronically alter responses to another G(q)PCR agonist, histamine, or to thapsigargin or forskolin which elevate intracellular Ca(2+) and cAMP, respectively. This chronically acting antisecretory mechanism is not shared by neurotransmitters that activate G(s)PCRs. Conditioned medium from CCh-pretreated cells mimicked its chronic antisecretory actions, suggesting involvement of an epithelial-derived soluble factor but further experimentation ruled out the involvement of epidermal growth factor receptor ligands. Acute CCh exposure did not chronically alter surface expression of muscarinic M(3) receptors but inhibited intracellular Ca(2+) mobilization upon subsequent agonist challenge. CONCLUSIONS & INFERENCES: These data reveal a novel, chronically acting, antisecretory mechanism that downregulates epithelial secretory capacity upon repeated G(q)PCR agonist exposure. This mechanism involves release of a soluble factor that uncouples receptor activation from downstream prosecretory signals.