Abstract
Background
Inflammatory bowel disease (IBD) is due to a combination of factors, including genetics, mucosal barrier dysfunction and dysregulated immune responses. Recently, it has been appreciated that IBD is associated with profound tissue anoxia. Tight junctions (TJs) located at the apical lateral region of adjacent intestinal epithelial cells are largely responsible for regulating the intestinal mucosal barrier. Larazotide acetate (LA, Innovate Biopharmaceuticals, Inc., Raleigh, NC) is a synthetic, eight amino acid peptide that is known to act as a TJ regulator capable of closing ‘leaky’ interepithelial junctions. Presently, LA is being studied in Phase 3 clinical trials for the treatment of celiac disease. Based on prior work in our lab, we hypothesised that LA would protect the TJ barrier in an anoxic injury IBD model associated with upregulation of TJ-associated signalling pathways.
Methods
C2BBe1 (Caco-2 brush border expressing) monolayers were treated apically with LA and were subjected to anoxia for 2 h followed by reoxygenation with 21% O2. Barrier function was assessed by measuring transepithelial electrical resistance (TEER) during anoxic injury and recovery. TJ proteins and cytoskeleton protein F-actin were assessed by western blotting and immunofluorescence microscopy. Then, next-generation RNA sequencing was employed to assess cellular regulatory pathways.
Results
Pre-treatment of anoxic injured C2BBe1cells with 10 mM LA significantly increased TEER as compared with untreated anoxic injured cells. The TJ protein occludin and ZO-1 were disrupted in anoxia-injured monolayer. Alternatively, treatment with 10 mM LA prevented disruption of TJ proteins during anoxic injury. Gene ontology annotation revealed a number of critical signalling pathways that were differentially expressed in cells treated with LA, including biological processes involved in establishment of cell polarity, molecular functions that regulate junctional structures, and cellular components associated with epithelial repair (cell leading edge, ruffle and apical junctional complex). Furthermore, Ras/Rho GTPase binding and protein serine/threonine kinase activity were differentially expressed in cells treated with LA. Additionally, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed enrichment of target genes for ‘cell cycle,’ ‘adherens junction’ and ‘Wnt signalling pathways’.
Conclusion
The results of the present study provide novel insights into the molecular mechanism of action of LA on the protection of TJ integrity in anoxic injury, an IBD microenvironment and the potential for a more broad use in important digestive diseases such as IBD.
DAAM1 stabilizes epithelial junctions by restraining WAVE complex–dependent lateral membrane motility
