Activating AMPK to Restore Tight Junction Assembly in Intestinal Epithelium and to Attenuate Experimental Colitis by Metformin.
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ABSTRACT:
SUBMITTER: Chen L
PROVIDER: S-EPMC6054982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK ?1/?2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.
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Project description:In the intestinal epithelium, the aberrant regulation of cell/cell junctions leads to intestinal barrier defects, which may promote the onset and enhance the severity of inflammatory bowel disease (IBD). However, it remains unclear how the coordinated behaviour of cytoskeletal network may contribute to cell junctional dynamics. In this report, we identified ACF7, a crosslinker of microtubules and F-actin, as an essential player in this process. Loss of ACF7 leads to aberrant microtubule organization, tight junction stabilization and impaired wound closure in vitro. With the mouse genetics approach, we show that ablation of ACF7 inhibits intestinal wound healing and greatly increases susceptibility to experimental colitis in mice. ACF7 level is also correlated with development and progression of ulcerative colitis (UC) in human patients. Together, our results reveal an important molecular mechanism whereby coordinated cytoskeletal dynamics contributes to cell adhesion regulation during intestinal wound repair and the development of IBD.
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Project description:In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.
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Project description:The intestinal epithelium is a single-cell layer on the mucosal surface that absorbs food-derived nutrients and functions as a barrier that protects mucosal integrity. Hesperidin (hesperetin-7-rhamnoglucoside) is a flavanone glycoside composed of the flavanone hesperetin and the disaccharide rutinose, which has various physiological benefits, including antioxidative, anti-inflammatory, and antiallergic effects. Here, we used human intestinal Caco-2 cell monolayers to examine the effect of hesperidin on intestinal barrier function. Hesperidin-treated Caco-2 cell monolayers displayed enhanced intestinal barrier integrity, as indicated by an increase in transepithelial electrical resistance (TEER) and a decreased apparent permeability (Papp ) for fluorescein. Hesperidin elevated the mRNA and protein levels of occludin, MarvelD3, JAM-1, claudin-1, and claudin-4, which are encoded by tight junction (TJ)-related genes. Moreover, hesperidin significantly increased the phosphorylation of AMP-activated protein kinase (AMPK), indicating improved intestinal barrier function. Thus, our results suggest that hesperidin enhances intestinal barrier function by increasing the expression of TJ-related occludin, MarvelD3, JAM-1, and claudin-1 via AMPK activation in human intestinal Caco-2 cells.
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Project description:Background and aimsDysfunction of the vitamin D receptor [VDR] contributes to the aetiology of IBD by regulating autophagy, immune response, and mucosal permeability. VDR directly controls the paracellular tight junction protein Claudin-2. Claudin-2 and Claudin-15 are unique in maintaining paracellular permeability. Interestingly, claudin-15 mRNA was downregulated in patients with ulcerative colitis. However, the exact mechanism of Claudin-15 regulation in colitis is still unknown. Here, we investigated the protective role of VDR against intestinal inflammation via upregulating Claudin-15.MethodsWe analysed the correlation of Claudin-15 with the reduction of VDR in human colitis. We generated intestinal epithelial overexpression of VDR [O-VDR] mice to study the gain of function of VDR in colitis. Intestinal epithelial VDR knockout [VDR∆IEC] mice were used for the loss of function study. Colonoids and SKCO15 cells were used as in vitro models.ResultsReduced Claudin-15 was significantly correlated with decreased VDR along the colonic epithelium of human IBD. O-VDR mice showed decreased susceptibility to chemically and bacterially induced colitis and marked increased Claudin-15 expression [both mRNA and protein] in the colon. Correspondingly, colonic Claudin-15 was reduced in VDR∆IEC mice, which were susceptible to colitis. Overexpression of intestinal epithelial VDR and vitamin D treatment resulted in a significantly increased Claudin-15. ChIP assays identified the direct binding of VDR to the claudin-15 promoter, suggesting that claudin-15 is a target gene of VDR.ConclusionWe demonstrated the mechanism of VDR upregulation of Claudin-15 to protect against colitis. This might enlighten the mechanism of barrier dysfunction in IBD and potential therapeutic strategies to inhibit inflammation.
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Project description:Superoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase, is an enzyme that scavenges reactive oxygen species (ROS). It has been reported that SOD3 exerts anti-inflammatory abilities in several immune disorders. However, the effect of SOD3 and the underlying mechanism in inflammatory bowel disease (IBD) have not been uncovered. Therefore, in the present study, we investigated whether SOD3 can protect intestinal cells or organoids from inflammation-mediated epithelial damage. Cells or mice were treated with SOD3 protein or SOD3-transduced mesenchymal stem cells (MSCs). Caco-2 cells or intestinal organoids stimulated with pro-inflammatory cytokines were used to evaluate the protective effect of SOD3 on epithelial junctional integrity. Dextran sulfate sodium (DSS)-induced colitis mice received SOD3 or SOD3-transduced MSCs (SOD3-MSCs), and were assessed for severity of disease and junctional protein expression. The activation of the mitogen-activated protein kinase (MAPK) pathway and elevated expression of cytokine-encoding genes decreased in TNF-α-treated Caco-2 cells or DSS-induced colitis mice when treated with SOD3 or SOD3-MSCs. Moreover, the SOD3 supply preserved the expression of tight junction (ZO-1, occludin) or adherence junction (E-cadherin) proteins when inflammation was induced. SOD3 also exerted a protective effect against cytokine- or ROS-mediated damage to intestinal organoids. These results indicate that SOD3 can effectively alleviate enteritis symptoms by maintaining the integrity of epithelial junctions and regulating inflammatory- and oxidative stress.
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Project description:Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKC?. We recently reported that Eya1 phosphatase functions through aPKC?-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKC? phosphorylation levels, aPKC?-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKC? in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1(-/-) lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKC?-Notch1-Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.
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Project description:Kidney cysts in autosomal dominant polycystic kidney disease (ADPKD) undergo progressive enlargement together with luminal fluid secretion. This involves active, uphill transcellular Cl(-) transport which drives passive Na(+) and water secretion. Implicit in this mechanism is the assumption that the paracellular permeability of the cyst epithelium to Cl(-) must be very low. Claudins are tight junction (TJ) transmembrane proteins that determine the ion selectivity of paracellular barriers. The aim of this study was to determine the expression and localization of claudins within renal cysts in a mouse hypomorphic model of ADPKD and in human patients. We found that the majority of cysts were of collecting duct origin. Claudins normally expressed in collecting duct (3, 4, 7, 8, and 10) were found in small cysts. However, only claudin-7 persisted at substantive levels in the dedifferentiated epithelium of large, presumably late-stage cysts, where it was localized both at the TJ and basolaterally. The constitutively expressed TJ proteins, ZO-1 and occludin, were also abundantly expressed and correctly localized, suggesting that the basic infrastructure of the TJ is preserved. A previous study suggested that claudin-7 may function as a paracellular Cl(-) barrier. We postulate that the role of claudin-7 in ADPKD is to seal the paracellular route in Cl(-)-secreting cyst epithelium, preventing backleak of Cl(-), and that it thereby plays a permissive role in fluid secretion and cyst growth.
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Project description:Sepsis induces both intestinal hyperpermeability and epithelial apoptosis. While each has been implicated in mediating sepsis mortality, the relationship between these two processes is unclear. We hypothesized that preventing intestinal apoptosis would prevent gut barrier dysfunction. To test this hypothesis, transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in the gut epithelium (Fabpl-Bcl-2 mice) and wild-type (WT) mice were subjected to sham laparotomy or cecal ligation and puncture and orally gavaged with fluorescein isothiocyanate conjugated-dextran (FD-4) 5 h before sacrifice. Serum FD-4 concentration was assayed to measure intestinal permeability, and jejunal tight junctions were assayed for mRNA and protein expression. Baseline FD-4 concentration was similar between WT and Fabpl-Bcl-2 mice. Intestinal permeability increased 6, 12, 24, and 48 h following sepsis in WT mice; however, FD-4 concentration was significantly lower at each timepoint in Fabpl-Bcl-2 mice. In addition, there were no statistically significant changes in permeability between septic and sham transgenic mice. Intestinal mRNA expression of claudin 3, claudin 5, and occludin was lower in septic Fabpl-Bcl-2 mice, while claudin 4 mRNA levels were higher in Fabpl-Bcl-2 mice. In contrast, no differences were detected in claudins 2, 7, 15, JAM-A, or ZO-1. Protein levels followed the same trend for all tight junction mediators different between WT and Fabpl-Bcl-2 mice except occludin was significantly higher in transgenic mice. Together these results demonstrate that decreasing intestinal epithelial apoptosis prevents hyperpermeability following sepsis via tight junction alterations which may be at least partially responsible for improved survival conferred by Bcl-2 overexpression.
| S-EPMC7156322 | biostudies-literature
Project description:BACKGROUND & AIMS:Inflammatory bowel diseases (IBD) increase risk for colorectal cancer. Mutations in the Mediterranean fever gene (MEFV or pyrin) are associated with hereditary autoinflammatory disease and severe IBD. Expression of MEFV, a sensor protein that the initiates assembly of the inflammasome complex, is increased in colon biopsies from patients with IBD. We investigated the role of pyrin in intestinal homeostasis in mice. METHODS:Mefv-/- mice and C57/BL6 mice (controls) were given azoxymethane followed by multiple rounds of dextran sodium sulfate (DSS) to induce colitis and tumorigenesis. In some experiments, Mefv-/- mice were given injections of recombinant interleukin 18 (rIL18) or saline (control) during DSS administration. Colon tissues were collected at different time points during colitis development and analyzed by histology, immunohistochemistry, immunoblots, or ELISAs (to measure cytokines). Spleen and mesenteric lymph node were collected, processed, and analyzed by flow cytometry. Colon epithelial permeability was measured in mice with colitis by gavage of fluorescent dextran and quantification of serum levels. RESULTS:MEFV was expressed in colons of control mice and expression increased during chronic and acute inflammation; high levels were detected in colon tumor and adjacent non-tumor tissues. Mefv-/- mice developed more severe colitis than control mice, with a greater extent of epithelial hyperplasia and a larger tumor burden. Levels of inflammatory cytokines (IL6) and chemokines were significantly higher in colons of Mefv-/- mice than control mice following colitis induction, whereas the level IL18, which depends on the inflammasome for maturation and release, was significantly lower in colons of Mefv-/- mice. Mefv-/- mice had increased epithelial permeability following administration of DSS than control mice, and loss of the tight junction proteins occludin and claudin-2 from intercellular junctions. STAT3 was activated (phosphorylated) in inflamed colon tissues from Mefv-/-, which also had increased expression of stem cell markers (OLFM4, BMI1, and MSI1) compared with colons from control mice. Administration of rIL18 to Mefv-/- mice reduced epithelial permeability, intestinal inflammation, the severity of colitis, and colon tumorigenesis. CONCLUSIONS:In studies with DSS-induced colitis, we found that pyrin (MEFV) is required for inflammasome activation and IL18 maturation, which promote intestinal barrier integrity and prevent colon inflammation and tumorigenesis. Strategies to increase activity of MEFV or IL18 might be developed for the treatment of IBD and prevention of colitis-associated tumorigenesis.
| S-EPMC5847456 | biostudies-literature