Structural Variations and Functional Ramifications of Bile Acids (BA) on Tight Junctions (TJ) in Human Colon T84 Cells

Abstract

BA‐induced diarrhea affects ⅓ of patients with chronic intestinal inflammation, but the underlying mechanisms remain to be elucidated. We previously reported a yin/yang in BA action, with dihydroxy, chenodeoxycholic acid (CDCA), disrupting TJs and its monohydroxy derivative, lithocholic acid (LCA) attenuating it. We hypothesize that structural differences account for the varied actions, namely the presence of the 7‐OH in CDCA, which can serve as a hydrogen bond donor. Recently, we used a fluorescein amine‐tagged CDCA (CDCA‐FA), to show that apical CDCA‐FA travels paracellularly to the basolateral surface (BLS), and like CDCA alters reactive oxygen species‐dependent TJ permeability (FASEB J’ 18:32, 747.19). We predict that, in contrast, LCA‐FA will not alter TJ or access BLS. We studied the structural basis for the yin/yang in BA action on TJ function, by synthesizing: 1. a fluorescein amine‐tagged LCA and 2. a 7, methoxy CDCA (CDCA‐Me).CDCA‐FA and LCA‐FA were synthesized by protecting their alcohol(s), converting‐COOH to an acid chloride, adding the fluorescein amine tag to form an amide, and removing the protecting groups. They were purified by column chromatography and the structure confirmed by NMR and mass spectrometry. We have synthesized CDCA‐Me, structurally similar to LCA, by protecting ‐ COOH of CDCA as a methyl ester, protecting 3‐OH as a silyl ether, converting 7‐OH to a–OCH3 using methyl triflate, and removing the protecting groups. The yield of pure CDCA‐Me is being optimized so its biological effects can be tested.Confluent T84 cells (TransEpithelial Resistance, TER >1000Ωcm2), were treated apically with DMSO (CTRL), 500μM CDCA‐FA, 50μM LCA‐FA ± 500μM CDCA/CDCA‐FA for 0.5–18 H. Cell viability was measured by propidium iodide staining, fluorescence microscopy and Image J analysis. TJ function was assessed by examining: a. pore function measured as TER; b. leak function measured as apparent permeability of CDCA‐FA (Papp CDCA‐FA) vs. LCA‐FA (Papp LCA‐FA) or FITC‐10kDa dextran flux across the monolayer.Exposure (18H) to 50μM LCA‐FA, like LCA, did not alter cell viability nor change CDCA‐FA's effect on cell death (% cell death, CTRL: 7±3 vs LCA‐FA: 9±4; CDCA‐FA: 17±5 vs. CDCA‐FA+ LCA‐FA: 18±6, n=5). Also similar to LCA, LCA‐FA alone or ± CDCA‐FA did not alter pore function (TER) over time (Ωcm2; t=18H; CTRL: 731 ± 10; LCA‐FA: 761 ± 157, CDCA‐FA: 96 ± 12*; CDCA‐FA+LCA‐FA: 118±24*, n=4; *p<0.05 vs CTRL). However, similar to LCA, LCA‐FA attenuated CDCA‐FA's permeability (18 H, Papp LCA‐FA cm/sec, LCA‐FA: 6±3; Papp CDCA‐FA, CDCA‐FA: 57±2; CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA‐FA: 21±5; p<0.05, n>3). Thus, addition of the fluorescent tag did not alter the function of BAs. In summary, CDCA, but not LCA, moves paracellularly to the BLS whereas LCA limits CDCA movement to protect barrier integrity. We postulate that the 7‐OH group in CDCA as a hydrogen bond donor is critical in its role in disrupting barrier function and triggering inflammation and our structure/function studies will guide new therapeutic strategies.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR, APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.