scholarly journals Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism

2012 ◽  
Vol 303 (12) ◽  
pp. G1356-G1364 ◽  
Author(s):  
Mitzi Dunagan ◽  
Kamaljit Chaudhry ◽  
Geetha Samak ◽  
R. K. Rao
Keyword(s):  
Tight Junctions ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Intercellular Junctions ◽  
Ethanol Administration ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Inhibitory Peptide ◽  
Cell Monolayers ◽  
Epithelial Tight Junctions

Acetaldehyde is accumulated at high concentrations in the colonic lumen following ethanol administration. Previous studies demonstrated that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability. In the present study, we investigated the role of PP2A in the acetaldehyde-induced disruption of intestinal epithelial tight junctions. Caco-2 cell monolayers were exposed to 200–600 μM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance and inulin permeability. Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability and redistribution of occludin and ZO-1 from the intercellular junctions. Treatment of cells with fostriecin (a PP2A-selective inhibitor) or knockdown of PP2A by siRNA blocked acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. The effects of fostriecin and acetaldehyde were confirmed in mouse intestine ex vivo. Acetaldehyde-induced tight junction disruption and barrier dysfunction were also attenuated by a PP2A-specific inhibitory peptide, TPDYFL. Coimmunoprecipitation studies showed that acetaldehyde increased the interaction of PP2A with occludin and induced dephosphorylation of occludin on threonine residues. Fostriecin and TPDYFL significantly reduced acetaldehyde-induced threonine dephosphorylation of occludin. Acetaldehyde failed to change the level of the methylated form of PP2A-C subunit. However, genistein (a tyrosine kinase inhibitor) blocked acetaldehyde-induced association of PP2A with occludin and threonine dephosphorylation of occludin. These results demonstrate that acetaldehyde-induced disruption of tight junctions is mediated by PP2A translocation to tight junctions and dephosphorylation of occludin on threonine residues.

scholarly journals Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

2011 ◽  
Vol 437 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Suneet Jain ◽  
Takuya Suzuki ◽  
Ankur Seth ◽  
Geetha Samak ◽  
Radhakrishna Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Intercellular Junctions ◽  
Cell Monolayers ◽  
Terminal Domain ◽  
Tight Junction Regulation ◽  
Epithelial Tight Junctions ◽  
Zona Occludens

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin–Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.

scholarly journals Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism

2008 ◽  
Vol 294 (4) ◽  
pp. G1060-G1069 ◽  
Author(s):  
A. Seth ◽  
Fang Yan ◽  
D. Brent Polk ◽  
R. K. Rao
Keyword(s):  
Hydrogen Peroxide ◽  
Tight Junction ◽  
Map Kinase ◽  
Tight Junctions ◽  
Barrier Function ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Cell Monolayers ◽  
Dependent Mechanism

Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and β-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCβI and PKCε. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

scholarly journals Berberine improves intestinal epithelial tight junctions by upregulating A20 expression in IBS-D mice

2019 ◽  
Vol 118 ◽  
pp. 109206 ◽  
Author(s):  
Qiuke Hou ◽  
Shuilian Zhu ◽  
Changrong Zhang ◽  
Yongquan Huang ◽  
Yajuan Guo ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Intestinal Epithelial ◽  
Epithelial Tight Junctions

scholarly journals Disruption of Tight Junctions and Induction of Proinflammatory Cytokine Responses in Colonic Epithelial Cells by Campylobacter jejuni

2006 ◽  
Vol 74 (12) ◽  
pp. 6581-6589 ◽  
Author(s):  
Ming L. Chen ◽  
Zhongming Ge ◽  
James G. Fox ◽  
David B. Schauer
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Campylobacter Jejuni ◽  
Transmembrane Protein ◽  
Intercellular Junctions ◽  
Subcellular Fractionation ◽  
Intestinal Epithelial ◽  
Intracellular Location ◽  
Mitogen Activated Protein ◽  
Triton X

ABSTRACT Campylobacter jejuni is a leading cause of human enterocolitis and is associated with postinfectious complications, including irritable bowel syndrome and Guillain-Barré syndrome. However, the pathogenesis of C. jejuni infection remains poorly understood. Paracellular pathways in intestinal epithelial cells are gated by intercellular junctions (tight junctions and adherens junctions), providing a functional barrier between luminal microbes and host immune cells in the lamina propria. Here we describe alterations in tight junctions in intestinal epithelial monolayers following C. jejuni infection. Apical infection of polarized T84 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of the tight junctional transmembrane protein occludin from an intercellular to an intracellular location. Subcellular fractionation using equilibrium sucrose density gradients demonstrated decreased hyperphosphorylated occludin in lipid rafts, Triton X-100-soluble fractions, and the Triton X-100-insoluble pellet following apical infection. Apical infection with C. jejuni also caused rapid activation of NF-κB and AP-1, phosphorylation of extracellular signal-regulated kinase, Jun N-terminal protein kinase, and p38 mitogen-activated protein kinases, and basolateral secretion of the CXC chemokine interleukin-8 (IL-8). Basolateral infection with C. jejuni caused a more rapid decrease in TER, comparable redistribution of tight-junction proteins, and secretion of more IL-8 than that seen with apical infection. These results suggest that compromised barrier function and increased chemokine expression contribute to the pathogenesis of C. jejuni-induced enterocolitis.

scholarly journals Lactobacillus casei and Epidermal Growth Factor Prevent Osmotic Stress-Induced Tight Junction Disruption in Caco-2 Cell Monolayers

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3578
Author(s):  
Geetha Samak ◽  
Rupa Rao ◽  
Radhakrishna Rao
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Osmotic Stress ◽  
Tight Junction ◽  
Lactobacillus Casei ◽  
Kinase Inhibitor ◽  
Adherens Junction ◽  
Barrier Dysfunction ◽  
Cell Monolayers ◽  
Epidermal Growth

Osmotic stress plays a crucial role in the pathogenesis of many gastrointestinal diseases. Lactobacillus casei and epidermal growth factor (EGF) effects on the osmotic stress-induced epithelial junctional disruption and barrier dysfunction were investigated. Caco-2 cell monolayers were exposed to osmotic stress in the presence or absence of L. casei or EGF, and the barrier function was evaluated by measuring inulin permeability. Tight junction (TJ) and adherens junction integrity were assessed by immunofluorescence confocal microscopy. The role of signaling molecules in the L. casei and EGF effects was determined by using selective inhibitors. Data show that pretreatment of cell monolayers with L. casei or EGF attenuates osmotic stress-induced TJ and adherens junction disruption and barrier dysfunction. EGF also blocked osmotic stress-induced actin cytoskeleton remodeling. U0126 (MEK1/2 inhibitor), the MAP kinase inhibitor, blocked EGF-mediated epithelial protection from osmotic stress. In contrast, the L. casei-mediated epithelial protection from osmotic stress was unaffected by U0126, AG1478 (EGFR tyrosine kinase inhibitor), SP600125 (JNK1/2 inhibitor), or SB202190 (P38 MAP kinase inhibitor). On the other hand, Ro-32-0432 (PKC inhibitor) blocked the L. casei-mediated prevention of osmotic stress-induced TJ disruption and barrier dysfunction. The combination of EGF and L. casei is more potent in protecting the barrier function from osmotic stress. These findings suggest that L. casei and EGF ameliorate osmotic stress-induced disruption of apical junctional complexes and barrier dysfunction in the intestinal epithelium by distinct signaling mechanisms.

Development of intercellular junctions in the pulmonary epithelium of the foetal lamb

1978 ◽  
Vol 32 (1) ◽  
pp. 307-324
Author(s):  
E.E. Schneeberger ◽  
D.V. Walters ◽  
R.E. Olver
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Tight Junctions ◽  
Lung Development ◽  
Light And Electron Microscopy ◽  
Intercellular Junctions ◽  
Intercellular Spaces ◽  
Thin Sections ◽  
Epithelial Tight Junctions ◽  
Foetal Lamb

The integrity of epithelial tight junctions in foetal mammalian lungs is essential to maintain the unique ionic composition of lung liquid, and to prevent leakage of serum proteins into peripheral air spaces. In the present study the development of intercellular junctions of the lining epithelium of foetal lamb lungs during gestation was examined by light and electron microscopy. Both thin sections and freeze-fracture replicas were examined by electron microscopy. By 39 days of gestation, epithelial tight junctions consist of a minimum of 3.1 +/− 1.6 (s.D.) and a maximum of 5.8 +/− 2.0 discontinuous rows of particles and short segments of strands on P face ridges and in complementary E face grooves, while from 58 to 76 days they are composed of a network of 4.3 +/− 1.6 to 7.7 +/− 1.9 focally interrupted P face strands. Complementary replicas show that many of the discontinuities on the P face are due to separation of junctional particles on to the E face during fracturing, and not to an absence of junctional particles. From 76 days to term, epithelial tight junctions (exclusive of upper airway epithelium which was not examined) resemble those of adult lungs, and consist of a continuous network of 4.5 +/− 2.0 to 7.5 +/− 2.5 P face strands and complementary particle-free grooves. Permeability measurements, published elsewhere, indicate that the epithelium is functionally ‘tight’ from 69 days onwards. Tight junctions in peripheral air-space epithelium, therefore, are structurally continuous and functionally ‘tight’ early in foetal lung development, and form seals at one end of long, narrow intercellular spaces; these features may be important for coupled ion and water transport. When the bounding epithelial cells become flattened, these narrow intercellular spaces remain intact as a result of complex interdigitations of adjacent cell membranes. Desmosomes were present throughout gestation near the abluminal side of the tight junctions and occasionally near the base of the intercellular space. These junctions may serve to connect cells to each other at a time when tight junctions may be mechanically weak. In addition, gap junctions are associated with tight junctions from the glandular through the canalicular stages of lung development. They disappear by 120 days when the epithelial cells are differentiated.

scholarly journals Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers

2010 ◽  
Vol 433 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Sudhir Aggarwal ◽  
Takuya Suzuki ◽  
William L. Taylor ◽  
Aditi Bhargava ◽  
Radhakrishna K. Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Cell Monolayers ◽  
Differentiated Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ERK (extracellular-signal-regulated kinase) activation leads to disruption of tight junctions in some epithelial monolayers, whereas it prevents disruption of tight junctions in other epithelia. The factors responsible for such contrasting influences of ERK on tight junction integrity are unknown. The present study investigated the effect of the state of cell differentiation on ERK-mediated regulation of tight junctions in Caco-2 cell monolayers. EGF (epidermal growth factor) potentiated H2O2-induced tight junction disruption in under-differentiated cell monolayers, which was attenuated by the MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitor U0126. In contrast, EGF prevented H2O2-induced disruption of tight junctions in differentiated cell monolayers, which was also attenuated by U0126. Knockdown of ERK1/2 enhanced tight junction integrity and accelerated assembly of tight junctions in under-differentiated cell monolayers, whereas it had the opposite effect in differentiated cell monolayers. Regulated expression of wild-type and constitutively active MEK1 disrupted tight junctions, and the expression of dominant-negative MEK1 enhanced tight junction integrity in under-differentiated cells, whereas contrasting responses were recorded in differentiated cells. EGF prevented both H2O2-induced association of PP2A (protein phosphatase 2A), and loss of association of PKCζ (protein kinase Cζ), with occludin by an ERK-dependent mechanism in differentiated cell monolayers, but not in under-differentiated cell monolayers. Active ERK was distributed in the intracellular compartment in under-differentiated cell monolayers, whereas it was localized mainly in the perijunctional region in differentiated cell monolayers. Thus ERK may exhibit its contrasting influences on tight junction integrity in under-differentiated and differentiated epithelial cells by virtue of differences in its subcellular distribution and ability to regulate the association of PKCζ and PP2A with tight junction proteins.

Intraepithelial γδ+ Lymphocytes Maintain the Integrity of Intestinal Epithelial Tight Junctions in Response to Infection

2006 ◽  
Vol 131 (3) ◽  
pp. 818-829 ◽  
Author(s):  
Jane E. Dalton ◽  
Sheena M. Cruickshank ◽  
Charlotte E. Egan ◽  
Rainy Mears ◽  
Darren J. Newton ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Intestinal Epithelial ◽  
Epithelial Tight Junctions ◽  
Γδ Lymphocytes
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