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. 2018 Mar;66:93-109.
doi: 10.1016/j.matbio.2017.09.003. Epub 2017 Oct 1.

Layilin Is Critical for Mediating Hyaluronan 35kDa-induced Intestinal Epithelial Tight Junction Protein ZO-1 in Vitro and in Vivo

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Layilin Is Critical for Mediating Hyaluronan 35kDa-induced Intestinal Epithelial Tight Junction Protein ZO-1 in Vitro and in Vivo

Yeojung Kim et al. Matrix Biol. .
Free PMC article

Abstract

Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.

Keywords: Epithelial barrier; Hyaluronan; Layilin; Mouse intestinal organoids; Tight junction protein.

Figures

Figure 1
Figure 1
HA35, but not HA2000 treatment increases ZO-1 expression and HA35 but not HA2000 is internalized in mouse intestinal organoids in vitro. A. Sections of mouse intestinal organoids treated with no HA, HA35 (350µg/ml) or HA2000 (350µg/ml) for 30h were fixed and stained for ZO-1 or E-cadherin (red), HA (green), and nuclei (blue). Z-stack images were obtained using confocal microscopy. B. Quantification of stained images from 8 organoids per condition. ZO-1 staining intensity was measured as described in “Methods” and normalized to the number of epithelial cells by counting the number of nuclei. The unpaired t-test was used to test the statistical significance of differences between no HA and the HA35-treated groups (*, p<0.05.) Error bars = S.E.M.
Figure 2
Figure 2
HA35-mediated ZO-1 induction is TLR-4 independent and murine distal colon epithelium does not express detectable levels of CD44. A. Distal colons from wild type or tlr4−/− mice gavaged with HA35 (300µg per mouse daily) or water for 5 days were immunostained for ZO-1 (red) (n=5/group). Images were taken using fluorescence microscopy of a single plane. B. The quantification of ZO-1 intensity was performed on 2 stained sections per mouse, 5 mice per group and normalized to the number of epithelial cells. The statistical significance was tested with the unpaired t-test. (**, p<0.01, ***, p<0.0001). Error bars=S.E.M. C. Sections of proximal and distal colons from wild type mice were stained for CD44 (green) and nuclei (blue). Nonspecific (NS) indicates the secondary antibody staining control. D. Western blot analysis of CD44 protein in mouse colon tissue lysates from wild type mice and mouse colonic epithelial organoids cultured in 50% L-WRN conditioned media. Villin-1, an epithelial cell marker, and GAPDH were used as loading controls.
Figure 3
Figure 3
Layilin mediates HA35-induced ZO-1 increase in vitro. A. Sections of no HA and HA35 treated (350µg/ml for 30h) wild type and Layn−/− mouse intestinal organoids were stained for ZO-1 (red), HA (green) and nuclei (blue). Z-stacks images were obtained using confocal microscopy. B. ZO-1 intensity was quantified using 9 organoids per condition and normalized to the number of epithelial cells. The statistical analysis was done using the one-tailed unpaired t-test. (**, p<0.01). C. Intracellular HA intensity was measured using 9 organoids per condition and normalized to the number of epithelial cells. The one-tailed unpaired test was used to test the statistical significance. (*, p<0.05.) Error bars = S.E.M.
Figure 4
Figure 4
Layilin mediates HA35 induced ZO-1 induction in healthy and DSS-treated mice. A. Sections of distal colon from wild type and Layn−/− mice gavaged once daily with water or HA35 (300µg per mouse) for five days were immunostained for ZO-1 (red). Single plane images were taken with a confocal microscope. B. ZO-1 staining intensity was quantified using 2 stained sections per mouse and 6 mice per group. Quantified ZO-1 intensity was normalized to the number of epithelial cells. C. Groups of wild type and Layn−/− mice were orally gavaged with water or HA35 (300µg per mouse, once daily) for 5 days before, as well as 3 days during 2.5% DSS treatment. After euthanasia, colons were collected for fixation and histologic examination. Transverse and distal colons were immunostained for ZO-1 (red). Images were taken using confocal microscopy of a single plane. D. The intensity of ZO-1 staining was quantified using 2 stained sections per mouse, with 7 mice per group. ZO-1 intensity was normalized to the number of epithelial cells and the statistical significance of difference between water-treated groups and HA35-treated groups was tested using unpaired t-test. (*, p<0.05, **, p<0.01) Error bars = S.E.M.
Figure 5
Figure 5
The inhibition of ROCK kinase decreases HA35-induced ZO-1 levels and affects HA internalization in mouse intestinal organoids. A. Sections of mouse intestinal organoids treated with no HA or HA35 (350µg/ml, for 30h) in the presence or absence of ROCK inhibitor (Y-27632,10µM) were fixed and stained for ZO-1 (red), HA (green), and nuclei (blue). Confocal microscopy was performed to visualize and obtain Z-stack images. B. Quantification of fluorescence from images for multiple organoids (n=13) per condition. ZO-1 intensity was measured as described in “Methods” section and are normalized to the number of epithelial nuclei by manually counting each image. One-tailed unpaired t-test was used for statistical analysis between no HA and the HA35-treated groups (*, p<0.05). C. The intensity of intracellular HA was measured using 16 organoids per condition and normalized to the number of epithelial nuclei. The one-tailed unpaired test was used to test the statistical significance (*, p<0.05). Error bars = S.E.M.
Figure 6
Figure 6
ZO-1 expression is decreased, yet layilin expression is similar, in the intestinal epithelium of IBD patients as compared to non-IBD controls. A. Western blot analysis of ZO-1 protein in epithelial cell lysates from non-IBD, ulcerative colitis (UC), and Crohn’s disease (CD) patients. The same amount of proteins was loaded per lane and the levels of ZO-1 were normalized to the levels of villin-1 (an epithelial cell marker). Representative blots show two different patient samples per group (1 and 2). The relative expression level of ZO-1 in epithelium of non-IBD, UC, and CD patients was calculated using the densitometric quantification (n=11 (non-IBD), n=8 (UC), n=7 (CD)). Unpaired t-test was used to test the statistical significance between groups. (*, p<0.05, ***, p<0.0001) Error bars = S.E.M. B. Sections of colon from non-IBD, UC, and CD patients were immunostained for ZO-1 (red) and nuclei were stained blue (DAPI). Images were obtained using confocal microscopy of a single plane C. Western blot analysis of layilin protein in epithelial cell lysates from non-IBD, UC, and CD patients was performed by loading the same amount of protein. The densitometric quantification of layilin and vilin-1 protein bands was used to calculate the relative expression level of layilin.

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