THU0048 Soluble uric acid increased the expression of ABCG2 and PDZK1 in human intestinal cell lines through the TLR4/NLRP3/CASPASE-1 and PI3K/AKT signaling pathways

M Chen, X Lu, H Wu
2017 Poster Presentations   unpublished
Scientific Abstracts Thursday, 15 June 2017 217 and intracellular level. This inhibition was more pronounced after polyphenol cell pretreatment. In this case cytokine production was completely suppressed both in control and in stimulated cells. Cell pretreatment was extremely effective also in reducing mRNA expression of IL-1 after crystal stimulation while NLRP3 expression was to some extent affected by RES. RES and PD had a slight inhibitory effect on crystal phagocytosis when added along
more » ... the stimulus, while pretreated cells did not show any difference in the phagocytosis index. ROS production induced by MSU crystals was more pronounced (4 fold increase) with respect to CPP crystals (2 fold increase). RES was more effective than PD in inhibiting ROS production (p<0.05 crystals vs crystals+RES). The pretreatment showed a more marked decrease of ROS than the simultaneous treatment and the effect reached significance for both PD and RES. Similar inhibitory effects have been obtained when NO production was considered. Conclusions: Our results demonstrated that RES and PD are effective in inhibiting crystal-induced inflammation affecting IL-1 production and NLRP3 expression and activation. Data obtained after cell pretreatment allow us to hypothesize that these polyphenols act on specific signaling pathway preventing inflammation, and that this action is independent from crystal phagocytosis. Background: Hyperuricemia is the key pathophysiological basis of gout [1] . The most common reason generating hyperuricemia is verified the obstacle of urate excretion by current researches [2] . The intestine is known as the most important organ involved the excretion of uric acid besides the kidney [1] . ABCG2 has an outsized genetic contribution to extra-renal underexcretion and causes a compensatory increase in urinary urate output [3] . PDZK1 is a kind of an important structural protein regulated ABCG2 function, by potential molecular interaction with ABCG2 [4] . Objectives: The present study was undertaken to explore the effect and its related mechanisms of soluble uric acid on the urate excretion PDZK1 and ABCG2 in HT-29 and Caco-2 cell lines Methods: HT-29 and Caco-2 cell lines were used as a well-established model of human intestinal epithelial cells. Cells were pretreated with or without inhibitors and then stimulated with soluble uric acid. siRNA transfection was used to assess the interaction between ABCG2 and PDZK1. qRT-PCR and western blotting were used to measure mRNA and protein levels, respectively. Subcellular fractionation methods and immunofluorescence were used to examine the proteins in different subcellular compartments. Flow cytometry experiments examined the function of ABCG2. Results: Soluble uric acid significantly up regulated the expression of PDZK1 and ABCG2 in human intestinal cell lines. It also activated TLR4/NLRP3/caspase-1 inflammasome and PI3K/AKT signalling pathway through the phosphorylation of the ser473 pAkt. The increased expression of PDZK1 and ABCG2 was suppressed by a block of TLR4 (TAK-242) and caspase-1 inhibitor (acetyl-YVADchloromethylketone) and partly reduced by wortmanning, a specific inhibitior of PI3K. Additionally, lipopolysaccharide (LPS), the potent inducer of inflammatory responses mediated through TLR4, activated TLR4/NLRP3/caspase-1 inflammasome and up regulated the expression of ABCG2 and PDZK1. Besides, the stimulation of soluble uric acid facilitated the translocation of ABCG2 from intracellular compartment to plasma membrane and increased the transport activity. Furthermore, PDZK1 knockdown via siRNA significantly inhibited the expression and transport activity of ABCG2 regardless of the activation by soluble uric acid. Conclusions: The PI3K/AKT and TLR4/NLRP3/caspase-1 signaling pathways modulate ABCG2 and PDZK1 expression stimulated by soluble uric acid in human intestinal cells. PDZK1 plays a pivotal role in the regulation of ABCG2.
doi:10.1136/annrheumdis-2017-eular.5673 fatcat:5nihabxrybbmjb3uq2bu73xko4