Glucagon-like peptide 2 attenuates intestinal mucosal barrier injury through the MLCK/pMLC signaling pathway in a piglet model.

Glucagon-like peptide-2 (GLP-2), an intestinotrophic hormone, has drawn considerable attention worldwide due to its potential to promote intestinal development. We investigated the effects and mechanisms of GLP-2 against lipopolysaccharide (LPS)-induced intestinal inflammation and injury both in vitro and in vivo. Forty healthy piglets weaned at the age of 28 days with similar body weight (BW) were assigned to four in vivo treatments with ten piglets each: (i) nonchallenged control; (ii) LPS-challenged control; (iii) LPS + low dose GLP-2; and (iv) LPS + high dose GLP-2. Piglets were subcutaneously injected with phosphate-buffered saline supplemented with GLP-2 at doses of 0, 0, 2, and 10 nmol/kg BW per day for seven consecutive days. The piglets were challenged with an intraperitoneal injection with 100 µg/kg LPS on day 14 to induce intestinal damage. After that, the gene and protein expression levels of representative tight junction proteins and myosin light-chain kinase (MLCK)/phosphorylated myosin light chain (pMLC), as well as proinflammatory cytokine levels were determined using quantitative reverse transcription polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay methods. A high dose of GLP-2 pretreatment increased intestinal permeability by downregulating and redistributing tight junction proteins (p < .05), for example, zona occluden-1 (ZO-1) and occludin. GLP-2 decreased the transcription of proinflammatory cytokines genes including interleukin-1ß (IL-1ß), IL-6, IL-8, and tumor necrosis factor-α in small intestines (p < .05). GLP-2 prevented the LPS-induced increase in the expression of MLCK dose-dependently and the increase in pMLC levels in the duodenum, jejunum, and ileum. To assess further the protective effect of GLP-2 on LPS-induced intestinal barrier injury after weaning and its possible mechanism, an in vitro intestinal epithelial barrier model was established with IPEC-J2 monolayers and treated with 100 µg/ml LPS with or without 1 × 10-8 mol/L GLP-2 pretreatment. The in vitro analysis included control, LPS, and GLP-2 + LPS treatments. GLP-2 treatment alleviated the destructive effect of LPS on barrier permeability by restoring the expression and ultrastructure of ZO-1 and occludin (p < .05). In addition, GLP-2 reversed the LPS-induced MLCK hyperexpression and pMLC hyperphosphorylation (p < .05). Taken together, our findings revealed a mechanism by which GLP-2 alleviated LPS-challenged intestinal barrier injury and inflammation in weaned piglets and IPEC-J2 cells via the MLCK/pMLC signaling pathway.

Structural Features of Tight-Junction Proteins.

Tight junctions are complex supramolecular entities composed of integral membrane proteins, membrane-associated and soluble cytoplasmic proteins engaging in an intricate and dynamic system of protein-protein interactions. Three-dimensional structures of several tight-junction proteins or their isolated domains have been determined by X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These structures provide direct insight into molecular interactions that contribute to the formation, integrity, or function of tight junctions. In addition, the known experimental structures have allowed the modeling of ligand-binding events involving tight-junction proteins. Here, we review the published structures of tight-junction proteins. We show that these proteins are composed of a limited set of structural motifs and highlight common types of interactions between tight-junction proteins and their ligands involving these motifs.

Immunolocalization of junctional proteins in human hairs indicates that the membrane complex stabilizes the inner root sheath while desmosomes contact the companion layer through specific keratins.

The inner root sheath (IRS) sustains and addresses the hair shaft outside the follicle. Ultrastructural analysis of immunolabeling for beta-catenin, plakophilin-1, desmoglein-4 and keratin-17 in human hairs has indicated that adherens junctions and desmosomes initially connect cells in mature IRS and the companion layer. Beta-catenin immunolabeling for adherens junctions is only seen in sparse regions of differentiating Huxley cells, Flugelzellen cells and Henle cells, but disappears in cornified cells of the IRS. Desmoglein-4 and plakophilin-1 immunolabeling are observed in differentiating and cornified desmosomes of the Huxley and Henle layers and in the membrane complex joining these cells. Desmoglein-4 and plakophilin-1 are more frequently immunolocalized in the intracellular side of the junctions, but some labeling is also present in the delta-layer of the membrane complex. The labeling indicates a prevalent intracellular redistribution of desmoglein-4 and plakophilin-1 when the final cornification of the IRS occurs. Intense keratin-17 immunolabeling is observed in tonofilaments of the companion layer joining the plakophilin-1 rich desmosomes of the Henle layer. This suggests that this elastic type of keratin is present at desmosome junctions during the movements of the companion layer along the slippage plane of the hair shaft.

Diverse types of junctions containing tight junction proteins in stratified mammalian epithelia.

Molecular compositions and functions of tight junctions (TJs), that is, continuous, cell-cell-connecting zonulae occludentes serving as barrier structures for the paracellular transport of molecules and particles, have hitherto been determined for simple epithelia and for endothelia. In 2002, special TJ structures with barrier functions were identified in the stratum granulosum of mammalian epidermis. In addition, using biochemical and immunocytochemical methods, various types of TJ-type junctions have also been described that also contain claudins and/or occludin as well as typical TJ plaque proteins, in cell layers of all stratified squamous epithelia (e.g., various types of epidermis, gingiva, lingual, and other kinds of oral mucosa, pharynx, esophagus, trachea, vagina, and exocervix), including tissues without a lumen, such as the reticulum and Hassall corpuscles of the thymus, and tumors derived from such epithelia, notably squamous cell carcinomas. Biological and pathological aspects of TJ-related structures in such tissues are discussed.