Chenodeoxycholic Acid (CDCA) Initiates Distinct Signaling Mechanisms to Stimulate Cl− Transport in Intestinal and Non‐Intestinal Epithelial Cells
Authors: Domingue Jada , George A , Ao M , Bijvelds M , de Jonge H , Sarathy J , Rao MC 
Published: November 2023

We had reported earlier that CDCA stimulates Cl− transport in human colonic T84 cells via cAMP‐dependent protein kinase (PKA)‐mediated activation of the cystic fibrosis transmembrane conductance regulator (CFTR) (Am J Physiol 305:C447–56, 2013). More recently we showed that CDCA‐stimulated Cl− secretion also involves Ca2+ and EGF Receptor (EGFR)‐PI3 kinase signaling, but does not involve TGR5, FXR, EGF, p38, ERK1/2, or AKT (FASEB 29:855.1, 2015). We hypothesize that CDCA signaling requires crosstalk between cAMP and Ca2+ cascades and we used mammalian cell and organoid models to further elucidate the mechanisms of CDCA action. In T84 cells, we established a central role for EGFR by demonstrating that EGFR siRNA but not scrambled siRNA reduced EGFR protein expression by 67% and anion transport (n=5), as measured by iodide efflux. Next, we determined if the exchange protein directly activated by cAMP (EPAC), which is known to trigger [Ca2+]i signaling, plays a role in CDCA action. The EPAC inhibitor, ESI09 (10μM), significantly reduced the CDCA‐induced Cl− secretory response across T84 monolayers by 70% (short circuit current: ΔIsc, μA/cm2; CDCA: 17±2; CDCA+ESI09: 5±1; n=6; p=0.0004). Combined addition of the PKA inhibitor H89 (30μM) and ESI09 (10μM) reduced the CDCA response by 97% (n=5). In addition, preliminary results indicate that H89 and ESI09 alter CDCA‐induced EGFR phosphorylation (n=2), suggesting that cAMP signaling may mediate CDCA's transactivation of EGFR. Along with its effects on Ca2+ signaling, EPAC, via Ras‐related protein 1A (Rap1A) and Rho‐associated protein kinase (ROCK), activates K+ channels to increase the driving force for Cl− secretion. However, neither Rap1A nor ROCK inhibitors altered the CDCA response (n≥4). In contrast to T84 cells, in human embryonic kidney cells (HEK293), which do not express CFTR, we had shown that CDCA‐stimulated Cl− transport is Ca2+ ‐dependent (FASEB J 29:LB764, ‘15). Here we tested the hypothesis that the Ca2+‐activated Cl− channel TMEM16a is involved in CDCA action. The TMEM16a inhibitor, T16Ainh (20μM) caused a significant 45% (n=3) reduction in CDCA‐induced iodide efflux in HEK293 cells. Although T84 cells express TMEM16a, T16Ainh had no effect on the CDCA response (n=5), but increased baseline Isc. We speculate that the effect on basal Isc may be due to inhibition of a basolateral TMEM16a. Finally to study CDCA's effect on Cl− transport in a more physiological model, we cultured mouse intestinal organoids and examined organoid swelling (as an index of fluid transport), in response to CDCA (500μM) and forskolin (10μM). In preliminary results, organoids from ileum and proximal colon exhibited CDCA‐ and forskolin‐induced swelling, suggestive of Cl− secretion. In summary, CDCA increases Cl− transport in intestinal and non‐intestinal epithelial cells. In T84 cells, CDCA elicits CFTR activation through EGFR, Ca2+ and cAMP (PKA and EPAC) signaling. Further studies will determine if these signaling routes are involved in the CDCA‐induced secretory response of mouse organoids.

Support or Funding Information

This study was supported by UIC institutional funds and the APS Porter Physiology Development Fellowship.