The strong IL-1beta-induced downregulation of the multifunctional PDZ adaptor PDZK1 in inflamed enterocytes is attenuated by ERKI/II inhibition and RXR-alpha stimulation

Background: The PDZ adaptor protein PDZK1 is an important scaffolding protein that modulates membrane expression of a number of important epithelial ion transporters. PDZK1 expression is strongly decreased as well as mistargeted in inflamed intestinal mucosa of mice and IBD patients, which is in part responsible for the disturbed fluid absorption in inflamed intestinal mucosa (1,2,)

Aim and methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by exposing intestinal epithelial Caco2bbe cells to tumor necrosis factor alpha (TNF-α), interferon-γ and interleukin-1β (IL-1β) alone and in combination, and measuring the effect on PDZK1 mRNA expression by quantitative polymerase chair reaction and on PDZK1 protein expression by Western analysis.. PDZK1 promotor constructs were expressed in Caco2bbe cells and promotor activity was assayed using the luciferase system.

Results: IL-1β significantly decreased PDZK1 mRNA and protein as well as PDZK1 promotor activity in Caco2bbe cells in a time and concentration-dependent manner, and this effect was seen at an earlier time point in the early than the late differentiation stage, but was evident both in proliferating and in differentiated Caco2bbe cells. Deletion of base pairs -4689 to -3995 strongly diminished basal activity of the PDZK1 promotor and abolished IL-1β mediated inhibition. This region of the promotor harbours putative binding sites for transcription factors NF-κB, AP-1, SP-1 and retinoid X receptor alpha (RXR-α). RXR-α expression was found to be decreased in biopsies from ulcerative colitis patients and was downregulated by IL-1β in Caco2 cells. 9-cis retinoic acid (RA) 9-cis retinoic acid (RA), a ligand for RXR-α, stimulated PDZK1 mRNA expression and partially reversed IL-1β-mediated PDZK1 downregulation. Since IL-1β may affect downstream targets via several signal pathways, we studied the effect of coadministration of different concentrations of IL1β with inhibitors for the NF-κB, the p38 MAP kinase, the Jun kinase and the ERK1/2 pathways. A significant attenuation of IL-1β-induced decrease in PDZK1 protein expression in Caco-2BBe cells was seen with ERK1/2 inhibition.

Conclusions: The results suggest that the cytokine IL-1β strongly decreases PDZK1 expression, in part via a decreased expression of RXR-α. This suggests that the strong decrease in PDZK1 during intestinal inflammation is, at least in part, a direct consequence of the increased tissue levels of IL-1β and may be amenable to pharmacological therapy.

1. Lenzen et al.(2012) PlosOne 7(7): e40657

2. Yeruva et al. (2015) Pflugers Arch. 467(8): 1795-807