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 characterized 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 sulfate (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 characterized by reduced macrophage infiltration and transmigration, and reduced basement membrane laminin degradation. Further in vitro and in vivo studies show that macrophage transmigration across epithelial layer is MMP-12 dependent and the epithelial TJ barrier is compromised during macrophage transmigration. Together, this data demonstrates 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.

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.

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Background and aim 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 remain 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 WT mouse intestines and protected against LPS and TNF-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 MAPK-dependent mechanism.