A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. While biochemical mechanisms that regulate activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

Download Full-text

Lipid rafts are disrupted in mildly inflamed intestinal microenvironments without overt disruption of the epithelial barrier

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00002.2011 ◽

2012 ◽

Vol 302 (8) ◽

pp. G781-G793 ◽

Cited By ~ 18

Author(s):

Rachel V. Bowie ◽

Simona Donatello ◽

Clíona Lyes ◽

Mark B. Owens ◽

Irina S. Babina ◽

...

Keyword(s):

Lipid Rafts ◽

Intestinal Permeability ◽

Barrier Function ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Intestinal Epithelial Barrier ◽

Epithelial Barrier Function ◽

Barrier Integrity ◽

Ifn Γ

Intestinal epithelial barrier disruption is a feature of inflammatory bowel disease (IBD), but whether barrier disruption precedes or merely accompanies inflammation remains controversial. Tight junction (TJ) adhesion complexes control epithelial barrier integrity. Since some TJ proteins reside in cholesterol-enriched regions of the cell membrane termed lipid rafts, we sought to elucidate the relationship between rafts and intestinal epithelial barrier function. Lipid rafts were isolated from Caco-2 intestinal epithelial cells primed with the proinflammatory cytokine interferon-γ (IFN-γ) or treated with methyl-β-cyclodextrin as a positive control for raft disruption. Rafts were also isolated from the ilea of mice in which colitis had been induced in conjunction with in vivo intestinal permeability measurements, and lastly from intestinal biopsies of ulcerative colitis (UC) patients with predominantly mild or quiescent disease. Raft distribution was analyzed by measuring activity of the raft-associated enzyme alkaline phosphatase and by performing Western blot analysis for flotillin-1. Epithelial barrier integrity was estimated by measuring transepithelial resistance in cytokine-treated cells or in vivo permeability to fluorescent dextran in colitic mice. Raft and nonraft fractions were analyzed by Western blotting for the TJ proteins occludin and zonula occludens-1 (ZO-1). Our results revealed that lipid rafts were disrupted in IFN-γ-treated cells, in the ilea of mice with subclinical colitis, and in UC patients with quiescent inflammation. This was not associated with a clear pattern of occludin or ZO-1 relocalization from raft to nonraft fractions. Significantly, a time-course study in colitic mice revealed that disruption of lipid rafts preceded the onset of increased intestinal permeability. Our data suggest for the first time that lipid raft disruption occurs early in the inflammatory cascade in murine and human colitis and, we speculate, may contribute to subsequent disruption of epithelial barrier function.

Download Full-text

Salmonella Effector SpvB Disrupts Intestinal Epithelial Barrier Integrity for Bacterial Translocation

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.606541 ◽

2020 ◽

Vol 10 ◽

Author(s):

Lanqing Sun ◽

Sidi Yang ◽

Qifeng Deng ◽

Kedi Dong ◽

Yuanyuan Li ◽

...

Keyword(s):

Bacterial Translocation ◽

Junctional Complex ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Barrier Dysfunction ◽

Intestinal Epithelial Barrier ◽

Barrier Integrity ◽

Epithelial Barrier Dysfunction

Salmonella are common enteric bacterial pathogens that infect both humans and animals. Intestinal epithelial barrier, formed by a single layer of epithelial cells and apical junctional complex (AJC), plays a crucial role in host defense against enteric pathogens to prevent bacterial translocation. However, the underlying mechanisms of intestinal epithelial barrier dysfunction caused by Salmonella are poorly understood. It is found that a locus termed Salmonella plasmid virulence (spv) gene exists extensively in clinically important Salmonella serovars. SpvB is a key effector encoded within this locus, and closely related to Salmonella pathogenicity such as interfering with autophagy and iron homeostasis. To investigate the interaction between SpvB and intestinal epithelial barrier and elucidate the underlying molecular mechanism, we used the typical foodborne disease agent Salmonella enterica serovar Typhimurium (Salmonella typhimurium) carrying spvB or not to construct infection models in vivo and in vitro. C57BL/6 mice were orally challenged with S. typhimurium wild-type strain SL1344 or spvB-deficient mutant strain SL1344-ΔspvB. Caco-2 cell monolayer model, as a widely used model to mimic the human intestinal epithelium in vitro, was infected with SL1344, SL1344-ΔspvB, or spvB complementary strain SL1344-c-ΔspvB, respectively. The results showed that SpvB enhanced bacterial pathogenicity during S. typhimurium infection in vivo, and contributed to intestinal epithelial barrier dysfunction in both infection systems. This SpvB-mediated barrier dysfunction was attributed to the cellular redistribution of Claudin-1, Occludin, and E-cadherin junctional proteins. Moreover, by using pharmacological inhibitors, we found that F-actin rearrangement and suppression of protein kinase C (PKC) signaling pathway were involved in SpvB-mediated barrier dysfunction. In conclusion, the study reveals the contribution of Salmonella effector SpvB to the dysfunction of intestinal epithelial barrier integrity, which facilitates bacterial translocation via the paracellular route to promote Salmonella systemic dissemination. Our findings broaden the understanding of host–pathogen interactions in salmonellosis, and provide new strategies for the therapy in limiting bacterial dissemination during infection.

Download Full-text

Butyrate-producing bacteria supplemented in vitro to Crohn’s disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity

Scientific Reports ◽

10.1038/s41598-017-11734-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 102

Author(s):

Annelies Geirnaert ◽

Marta Calatayud ◽

Charlotte Grootaert ◽

Debby Laukens ◽

Sarah Devriese ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Disease Patient ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Intestinal Epithelial Barrier ◽

Barrier Integrity ◽

Butyrate Production

Download Full-text

Plumericin Protects against Experimental Inflammatory Bowel Disease by Restoring Intestinal Barrier Function and Reducing Apoptosis

Biomedicines ◽

10.3390/biomedicines9010067 ◽

2021 ◽

Vol 9 (1) ◽

pp. 67 ◽

Cited By ~ 1

Author(s):

Shara Francesca Rapa ◽

Rosanna Di Paola ◽

Marika Cordaro ◽

Rosalba Siracusa ◽

Ramona D’Amico ◽

...

Keyword(s):

Barrier Function ◽

Structural Integrity ◽

Intestinal Barrier ◽

Intestinal Barrier Function ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Intestinal Epithelial Barrier ◽

Bowel Diseases ◽

Inflammatory Bowel

Intestinal epithelial barrier impairment plays a key pathogenic role in inflammatory bowel diseases (IBDs). In particular, together with oxidative stress, intestinal epithelial barrier alteration is considered as upstream event in ulcerative colitis (UC). In order to identify new products of natural origin with a potential activity for UC treatment, this study evaluated the effects of plumericin, a spirolactone iridoid, present as one of the main bioactive components in the bark of Himatanthus sucuuba (Woodson). Plumericin was evaluated for its ability to improve barrier function and to reduce apoptotic parameters during inflammation, both in intestinal epithelial cells (IEC-6), and in an animal experimental model of 2, 4, 6-dinitrobenzene sulfonic acid (DNBS)-induced colitis. Our results indicated that plumericin increased the expression of adhesion molecules, enhanced IEC-6 cells actin cytoskeleton rearrangement, and promoted their motility. Moreover, plumericin reduced apoptotic parameters in IEC-6. These results were confirmed in vivo. Plumericin reduced the activity of myeloperoxidase, inhibited the expression of ICAM-1, P-selectin, and the formation of PAR, and reduced apoptosis parameters in mice colitis induced by DNBS. These results support a pharmacological potential of plumericin in the treatment of UC, due to its ability to improve the structural integrity of the intestinal epithelium and its barrier function.

Download Full-text