A Role for p38 in Bile Acid (BA)‐Mediated Tight Junction (TJ) Dysfunction in Human Colon T84 Cells
Authors: Nair Tanushree , Sirajuddin Hafsa , Ao Mei , Alendry Sharyl , Haery Ladan , Domingue Jada , Rao Meena , Sarathy Jayashree 
Published: November 2023

BA‐associated diarrhea commonly occurs in patients with inflammatory bowel diseases where the epithelial barrier is compromised. We have previously shown in T84 cells that chenodeoxycholic acid (CDCA, 500 μM) caused a time‐dependent increase in p38 phosphorylation, but SB203580 (SB, p38 antagonist, 10μM) had no effect on CDCA‐induced increase in ISC(AJP ′16; 311: C777). We also demonstrated that BAs play a role in the regulation of intestinal barrier function, with CDCA disrupting TJs and lithocholic acid (LCA, 50 μM) reversing these effects (FASEB J 2016 30:1223.29). Furthermore, p38 signaling has been linked to inflammation‐related barrier disruption. Hence, using confluent T84 cells (transepithelial resistance, TER >1000Ω.cm2), we further studied the role of p38 by examining the effects of BAs±SB (apical addition, 0.5 – 18Hr) on: i. apoptosis (Annexin‐V, flow cytometry); ii. intracellular adhesion strength (IAS, Dispase‐II assay); iii. paracellular permeability [TER, dilution potential (Dψ), and fluorescein isothiocyanate‐10 kDa dextran flux (DFlux)]; and iv. ROS production (CellRox, flow cytometry).

We found that p38 inhibition did not alter CDCA‐induced apoptosis; however, it reversed LCA's inhibition of CDCA's action (Annexin V+ cells [in %]: DMSO: 7.5±0.4; CDCA: 18±1; LCA: 8±0.2; CDCA+LCA: 11±0.3; SB: 7±0.1; CDCA+SB: 19±0.4; LCA+SB: 10±0.2; CDCA+LCA+SB: 20±1, p<0.05 vs CDCA+LCA; n=3), suggesting that LCA's anti‐apoptotic effect is p38‐mediated. Next, we examined the role of p38 in BA‐regulated IAS by using dispase to disrupt cell‐matrix adhesion and visualizing monolayer fragmentation. While CDCA caused a time‐dependent increase in fragmentation, neither LCA nor SB had any impact on IAS. Examining the effect of p38 on TJs revealed that while it played no role in BA‐mediated changes in DFlux, it decreased CDCA±LCA‐induced drop in TER [0.5 Hr in Ω.cm2; DMSO:1140±267; CDCA: 287±62; LCA: 3151±241; CDCA+LCA: 344±31; SB: 1366±180; CDCA+SB: 774±110, p<0.05 vs CDCA; LCA+SB:1921±284; CDCA+LCA+SB: 868±71, p<0.05 vs CDCA+LCA; n=4]. SB also caused a significant reduction in TER in the presence of LCA (p<0.05). While p38 inhibition did not reverse CDCA‐induced charge selectivity, it decreased the overall DΨ of the pore (25 min, in mV; DMSO: 7.5 ± 0.5, CDCA: −6.5 ± 2.0, LCA: 8.0 ± 1; CDCA+LCA: −5 ± 2, SB: 2.4±0.5, CDCA+SB: −1.1±0.8, LCA+SB: 2.8±1.5, CDCA+LCA+SB: −2.6±1; p<0.05 for all but CDCA+LCA vs CDCA+LCA+SB; n≥3). These studies establish p38's involvement in BA regulation of TJs and the pore function of the barrier.

In a concurrent study, we found that CDCA‐induced TJ disruption was due to ROS production and hence we investigated if p38 played a role in this pathway. Inhibition of p38 decreased BA‐induced oxidative stress (CellRox+ cells, 18 Hr, [in %]; DMSO: 8±1; CDCA: 24±3; LCA: 11±1; CDCA+LCA: 25±3; SB: 15±0.2%; CDCA+SB: 15±1% (p<0.05); LCA+SB: 6±1% (p<0.05); CDCA+LCA+SB: 13.5±1% (p<0.05); n=3). In summary, our study highlights a role for p38 in BA signaling, specifically in BA regulation of barrier function and ROS production. Further delineation of BA‐>p38‐>ROS‐>TJ pathway will help identify key players in BA‐associated diarrhea.

Support or Funding Information

This study was supported by NSF‐MRI: DBI‐1427937 and Institutional Funds: BU to JS, UIC to MR, APS‐UGSRF to HS.