scholarly journals Detection of Tight Junction Barrier Function In Vivo by Biotin

2011 ◽  
pp. 91-100 ◽  
Author(s):  
Lei Ding ◽  
Yuguo Zhang ◽  
Rodney Tatum ◽  
Yan-Hua Chen
Keyword(s):  
Tight Junction ◽  
Barrier Function

scholarly journals HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity

2015 ◽  
Vol 26 (12) ◽  
pp. 2252-2262 ◽  
Author(s):  
Bejan J. Saeedi ◽  
Daniel J. Kao ◽  
David A. Kitzenberg ◽  
Evgenia Dobrinskikh ◽  
Kayla D. Schwisow ◽  
...  
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
Experimental Colitis ◽  
Site Directed Mutagenesis ◽  
Intestinal Epithelial ◽  
Low Oxygen ◽  
T84 Cells ◽  
Transcriptional Responses ◽  
Epithelial Tight Junction

Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA–mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.

scholarly journals Enteropathogenic E. coli disrupts tight junction barrier function and structure in vivo

2005 ◽  
Vol 85 (10) ◽  
pp. 1308-1324 ◽  
Author(s):  
Donnie E Shifflett ◽  
Daniel R Clayburgh ◽  
Athanasia Koutsouris ◽  
Jerrold R Turner ◽  
Gail A Hecht
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
E Coli

scholarly journals Colon epithelial cell TGFβ signaling modulates the expression of tight junction proteins and barrier function in mice

2021 ◽  
Author(s):  
Paula Marincola Smith ◽  
Yash A Choksi ◽  
Nicholas O Markham ◽  
David N Hanna ◽  
Jinghuan Zi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colon Cancer ◽  
Tight Junction ◽  
Barrier Function ◽  
Protein Translation ◽  
Tight Junction Proteins ◽  
Tgfβ Signaling ◽  
Mouse Colon ◽  
Junction Proteins

Background: Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). While TGFβ signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFβ and intestinal barrier function. Here, we examine the role of TGFβ signaling via SMAD4 in modulation of colon barrier function. Methods: The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4kD FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and trans-epithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. Results: Mice lacking intestinal expression of Smad4 demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in Cldn2/Claudin 2 and Cldn8/Claudin 8, and decreases in Cldn3, Cldn4, and Cldn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFb1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and Cldn8 are regulated by canonical TGFβ signaling, and TGFβ-dependent regulation of these genes is dependent on nascent RNA transcription (Cldn2, Cldn4, Cldn8) but not nascent protein translation (Cldn4, Cldn8). Human IBD/colon cancer specimens demonstrate decreased SMAD4, CLDN4, CLDN7, and CLDN8 and increased CLDN2 compared to healthy controls. Conclusion: Canonical TGFβ signaling modulates the expression of tight junction proteins and barrier function in mouse colon.

scholarly journals Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

2013 ◽  
Vol 305 (5) ◽  
pp. F714-F726 ◽  
Author(s):  
Jialing Bao ◽  
Renee E. Yura ◽  
Gail L. Matters ◽  
S. Gaylen Bradley ◽  
Pan Shi ◽  
...  
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
Fluorescein Isothiocyanate ◽  
Tight Junction Protein ◽  
Sodium Fluorescein ◽  
Monocyte Migration ◽  
Junction Protein ◽  
Epithelial Barriers

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.

scholarly journals COOH Terminus of Occludin Is Required for Tight Junction Barrier Function in Early Xenopus Embryos

1997 ◽  
Vol 138 (4) ◽  
pp. 891-899 ◽  
Author(s):  
Yan-hua Chen ◽  
Christa Merzdorf ◽  
David L. Paul ◽  
Daniel A. Goodenough
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Barrier Function ◽  
Integral Membrane Protein ◽  
Full Length ◽  
Intercellular Spaces ◽  
Zonula Occludens ◽  
Zonula Occludens 1 ◽  
First Time

Occludin is the only known integral membrane protein localized at the points of membrane– membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero–dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.

Rho GTPase overactivation by E. coli CNF-1 disrupts epithelial tight junction structure and barrier function

2001 ◽  
Vol 120 (5) ◽  
pp. A110-A110
Author(s):  
A HOPKINS ◽  
S WALS ◽  
P VERKADE ◽  
P BOQUET ◽  
A NUSRAT
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
Rho Gtpase ◽  
E Coli ◽  
Epithelial Tight Junction ◽  
Junction Structure

Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle

2017 ◽  
Vol 95 (3) ◽  
pp. 1313 ◽  
Author(s):  
L. Zhang ◽  
L. F. Schütz ◽  
C. L. Robinson ◽  
M. L. Totty ◽  
L. J. Spicer
Keyword(s):  
Gene Expression ◽  
Tight Junction ◽  
Tight Junction Proteins ◽  
Junction Proteins
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