CDCA Activation of CFTR-Dependent Cl- Secretion Requires a Complex Network of Signaling Cascades

Bile acids are synthesized in the liver and secreted into the small intestine to aid in lipid digestion and absorption. The majority of bile acids are reabsorbed and recycle back to the liver, while the remaining 5% enter the colon and may be reabsorbed or excreted. Bile acid malabsorption greatly increases colonic bile acid concentrations leading to excess fluid secretion and bile acid-associated diarrhea, however the molecular mechanisms underlying bile acid action have yet to be fully elucidated. Our laboratory established that chenodeoxycholic acid (CDCA) activates Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) in human colonic cells, and was partially dependent on protein kinase A (PKA) activation (Ao et al., AJP:305:C447-C456, 2013). It was hypothesized that CDCA utilizes a membrane receptor-mediated activation of multiple intracellular signaling cascades to stimulate CFTR-dependent Cl- secretion. We tested this hypothesis in the human colonic T84 cell line, HEK-293 cells and in mouse intestinal organoids. We found that in T84 cells, CDCA does not act through the nuclear farnesoid X receptor, transmembrane G-protein coupled receptor 5, or via muscarinic receptors. However, CDCA transactivated the epidermal growth factor receptor (EGFR), increasing its phosphorylation. Intracellularly, CDCA action involved intricate crosstalk between cAMP, Ca2+, and EGFR signaling cascades to activate CFTR. CDCA activated cAMP signaling, leading to activation of PKA and the exchange protein directly activated by cAMP (EPAC). While PKA phosphorylated CFTR, CDCA also activated Rap2 via EPAC, which modulated changes in intracellular Ca2+. Furthermore, cAMP signaling mediated CDCA-induced EGFR activation. In a stable CFTR-expressing HEK-293 cell line, CDCA stimulated Cl- transport independently of CFTR, suggesting a cell type specific activation of Cl- transport by CDCA. Furthermore, CDCA responsiveness was examined in mouse intestinal organoids derived from crypt stem cells. CDCA increased organoid swelling, especially in colon-derived organoids, which was partially dependent on EPAC activation, paralleling our findings in T84 cells. The data presented here reveal the complex network of signaling cascades initiated by bile acids, underlying the secretory processes that contribute to bile acid-associated diarrhea, and lay the foundation for identification of new therapeutic targets to alleviate diarrheal symptoms.