Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

Abstract

Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers.

Short-Chain Fatty Acids Manifest Stimulative and Protective Effects on Intestinal Barrier Function Through the Inhibition of NLRP3 Inflammasome and Autophagy

“…Remarkably, the activation of autophagy could impair the barrier function of Caco-2 monolayers through down-regulating the expression of ZO-1, occludin and claudin-1, up-regulating claudin-2 expression, and destroying the morphology of both ZO-1 and occludin. Similarly, inhibition of autophagic degradation process was reported to contribute to epithelial tight junction dysfunction in TNF-α treated cell monolayers [48]. However, autophagy was demonstrated to enhance tight junction through down- [19].…”

New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD

“…In 2015, autophagy was reported for the first time to regulate intestinal barrier function via inducing lysosomal degradation of the tight junction protein CLDN2 (claudin 2), thus decreasing epithelial permeability [10]. Increased CLDN2 level and tight junction defects in intestinal epithelial Caco-2 monolayers treated with the pro-inflammatory cytokine TNF/TNFA (tumor necrosis factor) partly arises from the inhibition of autophagy-mediated CLDN2 degradation [11] ( Figure 1A). It was recently shown that defects in mitochondria and ER functions induce intestinal permeability, promoting E. coli internalization and transcytosis across the epithelium, and these are counteracted by selective autophagy-mediated elimination of intracellular bacteria, which is so-called xenophagy [12].…”

“…Atg16l1 ΔIEC mice infected with murine norovirus and then treated with dextran sulfate sodium (DSS) exhibit exacerbated pathological score and increased nonapoptotic epithelial cell death compared to control mice [15]. Moreover, the viability in response to TNF treatment of intestinal organoids carrying the CD-associated ATG16L1 T300A variant is [10,11]. (B) Defective autophagy leads to intestinal dysbiosis and increased IgA-coated bacterial amount [26,27,32].…”

Suppressing glucose metabolism with epigallocatechin-3-gallate (EGCG) reduces breast cancer cell growth in preclinical models

“…Next, to monitor the effect of EGCG on autophagy in live cells, we transfected 4T1 cells with an adenovirus expressing the m-Cherry-GFP-LC3B fusion protein (Ad-mCherry-GFP-LC3B). 20 Under normal conditions, the mCherry-GFP-LC3B protein disperses in the cytoplasm and displays homogeneous yellow fluorescence. When the autophagic process is triggered, the mCherry-GFP-LC3B will gather around at the membrane of the autophagosome, and yellow dots appear.…”