Interleukin-1 β-induced decrease of NHE3 interacting protein PDZK1, as well as sh-RNA-mediated PDZK1 knockdown, is associated with NHE3 dysfunction in the colonic epithelial cell line Caco-2bbe.

Background: Inflammatory bowel disease (IBD) is a major gastrointestinal disease with diarrhea being a leading symptom. The pathophysiology of inflammatory diarrhea is multifacto-rial and still ill understood. We have previously reported a dysfunction of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, in inflamed colonocytes of ulcer-ative colitis patients and mouse models of colitis as well as a downregulation of the NHE3-interacting PDZ-protein PDZK1 (NHERF-3) in chronically inflamed murine colonic mucosa. Aim: The present study investigates whether the inflammation induced reduction of PDZK1 expression is causally related to the defective function of its binding partner, NHE3. Methods: We used cytokine-treated Caco-2bbe cells as an in vitro model of intestinal inflammation. Real-time PCR and western blots were done to measure PDZK1 and NHE3 mRNA and protein expression. pH-fluorometry using the pH sensitive dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein(BCECF), was performed to measure acid activated NHE3 activity. Con-focal microscopy was done to analyze NHE3 localization in interleukin-1β (IL-1 β) treated Caco-2bbe cells stably expressing human NHE3 (C2N3cells). Results: Treatment of Caco-2bbe cells with Th1 pro-inflammatory cytokines revealed, IL-1 β as the main cytokine with a significant inhibitory effect on PDZK1 expression (50% of decrease in both mRNA and protein compared to control). Treatment of C2N3 cells with IL-1 β lead to a significant decrease in PDZK1 protein to approx. 50% of control value, but no change in NHE3 protein expression and membrane localization. However, acid activated NHE3 transport rates were significantly decreased after IL-1 β treatment in these cells (22% less than control). When PDZK1 expression was decreased by lentiviral shRNA knockdown in NHE3-transfected Caco2bbe cells to approx 80% of control value, and NHE3 transport activity assessed, an even stronger decrease in acid-activated NHE3 activity (49% less than control) was observed than in IL-1 β treated cells. This demonstrates a causal relationship of PDZK1 downregulation and NHE3 dysfunction, independent of inflammation. Conclusion: A marked decrease in the PDZ-adaptor protein, PDZK1, was observed in the IL-1 β treated Caco-2bbe cells. This corresponded to a decrease in NHE3 transport function but no change in NHE3 expression and membrane localization. We therefore conclude that during intestinal inflammation, cytokine mediated PDZK1 down regulation is likely an important causative factor for inflammation-associated NHE3 dysfunction and diarrhea. [ABSTRACT FROM AUTHOR]