Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux.

BACKGROUND AND AIMS: Functional loss of the gut epithelium's paracellular tight junction [TJ] barrier and defective autophagy are factors potentiating inflammatory bowel disease [IBD]. Previously, we showed the role of autophagy in enhancing the intestinal TJ barrier via pore-forming claudin-2 degradation. How autophagy regulates the TJ barrier-forming proteins remains unknown. Here, we investigated the role of autophagy in the regulation of occludin, a principal TJ component involved in TJ barrier enhancement. RESULTS: Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in intestinal epithelial cells, mouse colonocytes and human colonoids. Autophagy induction enriched membrane occludin levels and reduced paracellular permeability of macromolecules. Autophagy-mediated TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier-enhancing effect against macromolecular flux. Autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss. Autophagy enhanced the phosphorylation of ERK-1/2 and inhibition of these kinases in Caco-2 cells and human colonic mucosa prevented the macromolecular barrier-enhancing effects of autophagy. In vivo, autophagy induction by rapamycin enhanced occludin levels in wild-type mouse intestines and protected against lipopolysaccharide- and tumour necrosis factor-α-induced TJ barrier loss. Disruption of autophagy with acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ permeability and exacerbating the effect of experimental colitis. CONCLUSION: Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 mitogen-activated protein kinase-dependent mechanism.

AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability.

The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXФ) (67-70 and 148-151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation.Abbreviations: Amil: amiloride; AP2: adaptor protein complex 2; AP2A1: adaptor related protein complex 2 subunit alpha 1; AP2M1: adaptor related protein complex 2 subunit mu 1; ATG7: autophagy related 7; CAL: calcitriol; Cas9: CRISPR-associated protein 9; Con: control; CPZ: chlorpromazine; DSS: dextran sodium sulfate; EBSS: Earle's balanced salt solution; IBD: inflammatory bowel disease; TER: trans-epithelial resistance; KD: knockdown; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MßCD: Methyl-ß-cyclodextrin; MET: metformin; MG132: carbobenzoxy-Leu-Leu-leucinal; MTOR: mechanistic target of rapamycin kinase; NT: non target; RAPA: rapamycin; RES: resveratrol; SMER: small-molecule enhancer 28; SQSTM1: sequestosome 1; ST: starvation; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Matrix Metalloproteinase MMP-12 Promotes Macrophage Transmigration Across Intestinal Epithelial Tight Junctions and Increases Severity of Experimental Colitis.

BACKGROUND AND AIMS: Matrix metalloproteinases [MMPs] play an important role in extracellular matrix regulation during cell growth and wound healing. Increased expression of MMP-12 [human macrophage elastase] has been reported in inflammatory bowel disease [IBD] which is characterised by the loss of epithelial tight junction [TJ] barrier function and an excessive inflammatory response. The aim of this study was to investigate the role of MMP-12 in intestinal TJ barrier function and inflammation. METHODS: Wild type [WT] and MMP-12-/- mice were subjected to experimental acute or chronic dextran sodium sulphate [DSS] colitis. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon and ex vivo by Ussing chamber studies. RESULTS: DSS administration increased colonic permeability through modulation of TJ proteins and also increased MMP-12 expression in the colonic mucosa of WT mice. The acute as well as chronic DSS-induced increase in colonic TJ permeability and the severity of DSS colitis was found to be markedly attenuated in MMP-12-/- mice. The resistance of MMP-12-/- mice to DSS colitis was characterised by reduced macrophage infiltration and transmigration, and reduced basement membrane laminin degradation. Further in vitro and in vivo studies show that macrophage transmigration across the epithelial layer is MMP-12 dependent and the epithelial TJ barrier is compromised during macrophage transmigration. Conclusions: Together, these data demonstrate that MMP-12 mediated degradation of basement membrane laminin, macrophage transmigration, and associated loss of intestinal TJ barrier are key pathogenic factors for intestinal inflammation.

Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1.

A defective tight junction (TJ) barrier is a key pathogenic factor for inflammatory bowel disease. Previously, we have shown that autophagy, a cell survival mechanism, enhances intestinal epithelial TJ barrier function. Autophagy-related protein-6 (ATG6/beclin 1), a key protein in the autophagy pathway, also plays a role in the endocytic pathway. The constitutive role of beclin 1 in the intestinal TJ barrier is not known. In Caco-2 cells, beclin 1 was found to be coimmunoprecipitated with the TJ protein occludin and colocalized with occludin on the membrane. Treatment of Caco-2 cells with beclin 1 peptide [transactivating regulatory protein (Tat)-beclin 1] reduced TJ barrier function. Activation of beclin 1 increased occludin endocytosis and reduced total occludin protein level. In contrast, beclin 1 siRNA transfection enhanced Caco-2 TJ barrier function. In pharmacologic and genetic autophagy inhibition studies, the constitutive function of beclin 1 in the TJ barrier was found to be autophagy independent. However, de novo induction of autophagy with starvation or rapamycin prevented Tat-beclin 1-induced increase in TJ permeability and reduction in occludin level. Induction of autophagy also resulted in reduced beclin 1-occludin association. In mouse colon, beclin 1 colocalized with occludin on the epithelial membrane. Perfusion of mouse colon with beclin 1 peptide caused an increase in colonic TJ permeability that was prevented by in vivo induction of autophagy. These findings show that beclin 1 plays a constitutive, autophagy-independent role in the regulation of intestinal TJ barrier function via endocytosis of occludin. Autophagy terminates constitutive beclin 1 function in the TJ barrier and enhances the TJ barrier.