Zinc ions regulate opening of tight junction favouring efflux of macromolecules via the GSK3β/snail-mediated pathway

Abstract

Zinc is an essential trace element presenting in particularly high concentration in the brain. In some regions, e.g. lateral amygdala, subiculum and hippocampus, rapidly-exchangeable zinc may transiently reach even up to 600 μM. To explore the possible roles of high-concentration Zn²⁺ in regulating the blood–brain barrier (BBB), we investigated the effects of Zn²⁺ on the functions and structures of the tight junction (TJ) with an in vitro model of a Madin-Darby canine kidney (MDCK) cell monolayer. The experimental results indicated that high concentrations (>200 μM) of Zn²⁺ can affect the TJ integrity in a polarized manner. Basolateral addition of Zn²⁺ led to reversible TJ opening with pore paths of r ∼ 2 nm or more depending on Zn²⁺ concentration. The efflux/influx ratios of different sized probes were found to be ∼4.6 for FD4 (M_W 4000) and ∼1.8 for Eu–DTPA (M_W 560), suggesting that the Zn²⁺-induced paracelluar channels favour efflux especially for macromolecules. Further mechanistic studies revealed that the elevated intracellular Zn²⁺ taken from the basolateral side can increase phosphorylation of glycogen synthase kinase (GSK) 3β, primarily due to the inhibition of calcineurin (CaN), thus resulting in the elevation of the snail transcriptional repressors. Subsequently, Zn²⁺ can cause the down-regulation of claudin-1, breakage of occludin and ZO-1 rings, and collapse of basolateral F-actin structures. These overall factors result in the formation of a trumpet-like paracellular channel, which allows asymmetric solute permeation. The ERK1/2 and JNK1/2 pathways may also be involved in the Zn²⁺-induced TJ opening process, while the activation of matrix metalloproteinase was not observed. Our results may suggest a potential role of zinc in regulation of BBB permeability associated with brain clearance of metabolites through the glymphatic system.