Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions. American Journal of Physiology

Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA.
AJP Gastrointestinal and Liver Physiology (Impact Factor: 3.8). 07/2001; 280(6):G1280-8.
Source: PubMed


Acetaldehyde-induced cytotoxicity is an important factor in pathogenesis of alcohol-related diseases; however, the mechanism of this toxicity is unknown. We recently showed that acetaldehyde increases epithelial paracellular permeability. We asked whether protein tyrosine phosphorylation via modulation of tyrosine kinases and/or PTPases is a mechanism involved in acetaldehyde-induced disruption of the tight junctions in the Caco-2 cell monolayer. Immunofluorescence localization of occludin and ZO-1 showed disruption of the tight junctions in acetaldehyde-treated cell monolayer. Administration of genistein prevented acetaldehyde-induced permeability. Acetaldehyde increased tyrosine phosphorylation of three clusters of proteins with molecular masses of 30-50, 60-90, and 110-150 kDa; three of these proteins were ZO-1, E-cadherin, and beta-catenin. Acetaldehyde reduced PTPase activity in plasma membrane and soluble fractions, whereas tyrosine kinase activity remained unaffected. Treatment with acetaldehyde resulted in a 97% loss of protein tyrosine phosphatase (PTP)1B activity and a partial reduction of PTP1C and PTP1D activities. These results strongly suggest that acetaldehyde inhibits PTPases to increase protein tyrosine phosphorylation, which may result in disruption of the tight junctions.

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    • "EtOH-induced depletion of these proteins from the detergent-insoluble fractions of distal colon is more severe in ALDH2-deficient mice, indicating that acetaldehyde accumulation may have a direct impact on the functions of these tight junction proteins. Previous studies demonstrated that acetaldehyde disrupts adherens junctions in Caco-2 cell monolayers (Atkinson and Rao, 2001; Sheth et al., 2007) and human colonic mucosa by increasing tyrosine phosphorylation of E-cadherin and b-catenin. Loss of junctional distribution of E-cadherin and b-catenin and decline in their levels in detergent-insoluble fractions of distal colon in EtOH-fed wild-type mice indicated that EtOH feeding disrupts adherens junctions. "
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    ABSTRACT: Acetaldehyde, the toxic ethanol (EtOH) metabolite, disrupts intestinal epithelial barrier function. Aldehyde dehydrogenase (ALDH) detoxifies acetaldehyde into acetate. Subpopulations of Asians and Native Americans show polymorphism with loss-of-function mutations in ALDH2. We evaluated the effect of ALDH2 deficiency on EtOH-induced disruption of intestinal epithelial tight junctions and adherens junctions, gut barrier dysfunction, and liver injury. Wild-type and ALDH2-deficient mice were fed EtOH (1 to 6%) in Lieber-DeCarli diet for 4 weeks. Gut permeability in vivo was measured by plasma-to-luminal flux of FITC-inulin, tight junction and adherens junction integrity was analyzed by confocal microscopy, and liver injury was assessed by the analysis of plasma transaminase activity, histopathology, and liver triglyceride. EtOH feeding elevated colonic mucosal acetaldehyde, which was significantly greater in ALDH2-deficient mice. ALDH2(-/-) mice showed a drastic reduction in the EtOH diet intake. Therefore, this study was continued only in wild-type and ALDH2(+/-) mice. EtOH feeding elevated mucosal inulin permeability in distal colon, but not in proximal colon, ileum, or jejunum of wild-type mice. In ALDH2(+/-) mice, EtOH-induced inulin permeability in distal colon was not only higher than that in wild-type mice, but inulin permeability was also elevated in the proximal colon, ileum, and jejunum. Greater inulin permeability in distal colon of ALDH2(+/-) mice was associated with a more severe redistribution of tight junction and adherens junction proteins from the intercellular junctions. In ALDH2(+/-) mice, but not in wild-type mice, EtOH feeding caused a loss of junctional distribution of tight junction and adherens junction proteins in the ileum. Histopathology, plasma transaminases, and liver triglyceride analyses showed that EtOH-induced liver damage was significantly greater in ALDH2(+/-) mice compared to wild-type mice. These data demonstrate that ALDH2 deficiency enhances EtOH-induced disruption of intestinal epithelial tight junctions, barrier dysfunction, and liver damage. Copyright © 2015 by the Research Society on Alcoholism.
    Alcoholism Clinical and Experimental Research 07/2015; 39(8). DOI:10.1111/acer.12777 · 3.21 Impact Factor
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    • "Others and we have shown that AcH can disrupt intestinal epithelial TJs and thereby increases paracellular permeability (Atkinson and Rao, 2001; Elamin et al., 2012; Rao, 1998, 2008). Although the precise mechanism is ill defined, roles for reactive oxygen species (ROS; Banan et al., 2000, 2003), protein tyrosine kinase (Atkinson and Rao, 2001; Sheth et al., 2003), and protein phosphatase 2A have been reported (Dunagan et al., 2012). In addition to barrier dysfunction, accumulation of AcH and subsequent ROS generation in colonic mucosa of alcoholics is suggested to play a key role in the pathogenesis of colorectal hyperproliferation and carcinogenesis (Seitz and Homann, 2007; Seitz and Stickel, 2007) and to be an important determinant for blood AcH levels and subsequent liver injury (Salaspuro, 1996). "
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    ABSTRACT: Acetaldehyde (AcH) is mutagenic and can reach high concentrations in colonic lumen after ethanol consumption and is associated with intestinal barrier dysfunction and an increased risk of progressive cancers, including colorectal carcinoma. Snail, the transcription factor of epithelial-mesenchymal transition, is known to down-regulate expression of tight junction (TJ) and adherens junction (AJ) proteins, resulting in loss of epithelial integrity, cancer progression, and metastases. As AcH is mutagenic, the role of Snail in the AcH-induced disruption of intestinal epithelial TJs deserves further investigation. Our aim was to investigate the role of oxidative stress and Snail activation in AcH-induced barrier disruption in Caco-2 monolayers. The monolayers were exposed from the apical side to AcH ± L-cysteine. Reactive oxygen species (ROS) generation and Snail activation were assessed by ELISA and immunofluorescence. Paracellular permeability, localization, and expression of ZO-1, occludin, E-cadherin, and β-catenin were examined using transepithelial electrical resistance (TEER), fluorescein isothiocyanate-labeled dextran 4 kDa (FITC-D4), immunofluorescence, and ELISA, respectively. Involvement of Snail was further addressed by inhibiting Snail using small interfering RNA (siRNA). Exposure to 25 μM AcH increased ROS generation and ROS-dependently induced Snail phosphorylation. In addition, AcH increased paracellular permeability (decrease in TEER and increase in FITC-D4 permeation) in association with redistribution and decrease of TJ and AJ protein levels, which could be attenuated by L-cysteine. Knockdown of Snail by siRNA attenuated the AcH-induced redistribution and decrease in the TJ and AJ proteins, in association with improvement of the barrier function. Our data demonstrate that oxidative stress-mediated Snail phosphorylation is likely a novel mechanism contributing to the deleterious effects of AcH on the TJ and AJ, and intestinal barrier function.
    Alcoholism Clinical and Experimental Research 08/2013; 38(2). DOI:10.1111/acer.12234 · 3.21 Impact Factor
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    • "This process is considered to play a key role in the pathogenesis of ALD [2], [3]. There is increasing evidence that ethanol [4], [5], [6] and to a greater extent, its oxidative metabolite, acetaldehyde [7], [8] can disrupt the TJs and increase paracellular permeability in Caco-2 cell monolayers by mechanisms involving e.g. inducible nitric oxide synthase (iNOS)-mediated reactive oxygen species (ROS) generation [5], [6], and protein tyrosine phosphorylation [9]. "
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    ABSTRACT: Evidence is accumulating that ethanol and its oxidative metabolite, acetaldehyde, can disrupt intestinal epithelial integrity, an important factor contributing to ethanol-induced liver injury. However, ethanol can also be metabolized non-oxidatively generating phosphatidylethanol and fatty acid ethyl esters (FAEEs). This study aims to investigate the effects of FAEEs on barrier function, and to explore the role of oxidative stress as possible mechanism. Epithelial permeability was assessed by paracellular flux of fluorescein isothiocyanate-conjugated dextran using live cell imaging. Cell integrity was evaluated by lactate dehydrogenase release. Localization and protein levels of ZO-1 and occludin were analyzed by immunofluorescence and cell-based ELISA, respectively. Intracellular oxidative stress and cellular ATP levels were measured by dichlorofluorescein and luciferase driven bioluminescence, respectively. In vitro, ethyl oleate and ethyl palmitate dose dependently increased permeability associated with disruption and decreased ZO-1 and occludin protein levels, respectively, and increased intracellular oxidative stress without compromising cell viability. These effects could partially be attenuated by pretreatment with the antioxidant, resveratrol, pointing to the role of oxidative stress in the FAEEs-induced intestinal barrier dysfunction. These findings show that FAEEs can induce intestinal barrier dysfunction by disrupting the tight junctions, most likely via reactive oxygen species-dependent mechanism.
    PLoS ONE 03/2013; 8(3):e58561. DOI:10.1371/journal.pone.0058561 · 3.23 Impact Factor
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