Geetha Samak

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

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.

Protein phosphatase 2A plays a role in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers

Evidence indicates that PP2A (protein phosphatase 2A) interacts with epithelial tight junctions and negatively regulates the integrity of the tight junction. In the present study, the role of PP2A in the hydrogen peroxide-induced disruption of the tight junction was examined in Caco-2 cell monolayers. Hydrogen peroxide-induced decrease in electrical resistance and increase in inulin permeability was associated with the dephosphorylation of occludin on threonine residues. The hydrogen peroxide-induced decrease in electrical resistance, increase in inulin permeability and redistribution of occludin and ZO (zonula occludens)-1 from the intercellular junctions were significantly attenuated by selective inhibitors of PP2A (okadaic acid and fostriecin) and by knockdown of PP2A-Calpha (the catalytic subunit of PP2A). The PP2A-Calpha protein and PP2A activity were co-immunoprecipitated with occludin, and this co-immunoprecipitation was rapidly increased by hydrogen peroxide. Hydrogen peroxideinduced increase in co-immunoprecipitation of PP2A-Calpha with occludin was prevented by PP2, a Src kinase inhibitor. GST (glutathione transferase)-pull down assays using recombinant GST-Occludin-C (C-terminal tail of occludin) and the purified PP2A showed that PP2A binds to the C-terminal domain of occludin; Src-induced tyrosine phosphorylation of GST-Occludin-C enhanced this binding. The present study shows that hydrogen peroxide increases the association of PP2A with occludin by a Src kinase-dependent mechanism, and that PP2A activity is involved in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers.

Protection and Restitution of Gut Barrier by Probiotics: Nutritional and Clinical Implications

Probiotics are beneficial bacteria present in various dietary components and many of these colonize in the human and animal intestine. In the gut probiotics help the host by assisting in maintenance of normal mucosal homeostasis. Probiotics not only help maintain normal function of the gut mucosa, but also protect mucosa from injurious factors such as toxins, allergens and pathogens. The beneficial effect of probiotics is mediated by multiple mechanisms, including cytoprotection, cell proliferation, cell migration, resistance to apoptosis, synthesis of proteins and gene expression. One of the important cytoprotective effects of probiotics in the intestinal mucosa is to strengthen the epithelial tight junctions and preservation of mucosal barrier function. Probiotics not only enhance barrier function by inducing synthesis and assembly of tight junction proteins, but also preventing disruption of tight junctions by injurious factors. Bioactive factors released by probiotics trigger activation of various cell signaling pathways that lead to strengthening of tight junctions and the barrier function. This article reviews and summarizes the current understanding of various probiotics that are involved in the protection of gut barrier function, highlights the cellular and molecular mechanisms involved in the protective effect and addresses the clinical implications of probiotic supplementation.

c-Jun NH2-terminal kinase-2 mediates osmotic stress-induced tight junction disruption in the intestinal epithelium

Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of Jnk(-/-) and Jnk2(-/-) mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and JNK1(-/-) mice, but not in JNK2(-/-) mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium.

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

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.

ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.

CaV1.3 Channels and Intracellular Calcium Mediate Osmotic Stress-induced N-terminal c-Jun Kinase Activation and Disruption of Tight Junctions in Caco-2 Cell Monolayers

We investigated the role of a Ca2+ channel and intracellular calcium concentration ([Ca2+]i) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-mediated intracellular Ca2+ depletion. Depletion of extracellular Ca2+ at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca2+ depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca2+ depletion partially reduced osmotic stress-induced rise in [Ca2+]i, whereas thapsigargin and extracellular Ca2+ depletion together resulted in almost complete loss of rise in [Ca2+]i. L-type Ca2+ channel blockers (isradipine and diltiazem) or knockdown of the CaV1.3 channel abrogated [Ca2+]i rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca2+ depletion, thapsigargin, or CaV1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca2+ depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that CaV1.3 and rise in [Ca2+]i play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca2+]i mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca2+ also contributes to osmotic stress-induced tight junction disruption.

Role of Glutamine in Protection of Intestinal Epithelial Tight Junctions.

Glutamine, a conditionally essential amino acid, is consumed predominantly in the gastrointestinal tract as a source of energy, particularly under the conditions of trauma, sepsis and surgery. In this article, we discuss the unique role of glutamine in the preservation of epithelial barrier function in the gastrointestinal tract. Glutamine supplementation protects the gastrointestinal mucosal homeostasis during total parenteral nutrition, diarrhea, radiation injury, starvation, sepsis and trauma. A significant body of evidence indicates that glutamine preserves the gut barrier function and prevents permeability to toxins and pathogens from the gut lumen into mucosal tissue and circulation. Recent studies demonstrated that the mucosal barrier protective effect of glutamine relates to its effect on preservation of epithelial tight junction integrity. The current understanding of glutamine-mediated protection of intestinal epithelial tight junction integrity and the potential mechanisms involved in this protective effect of glutamine are discussed.

Epidermal growth factor protects the apical junctional complexes from hydrogen peroxide in bile duct epithelium

The tight junctions of bile duct epithelium form a barrier between the toxic bile and liver parenchyma. Disruption of tight junctions appears to have a crucial role in the pathogenesis of various liver diseases. In this study, we investigated the disruptive effect of hydrogen peroxide and the protective effect of epidermal growth factor (EGF) on the tight junctions and adherens junctions in the bile duct epithelium. Oxidative stress in NRC-1 and Mz-ChA-1 cell monolayers was induced by administration of hydrogen peroxide. Barrier function was evaluated by measuring electrical resistance and inulin permeability. Integrity of tight junctions, adherens junctions and the actin cytoskeleton was determined by imunofluorescence microscopy. Role of signaling molecules was determined by evaluating the effect of specific inhibitors. Hydrogen peroxide caused a rapid disruption of tight junctions and adherens junctions leading to barrier dysfunction without altering the cell viability. Hydrogen peroxide rapidly increased the levels of p-MLC (myosin light chain) and c-Src(pY418). ML-7 and PP2 (MLCK and Src kinase inhibitors) attenuated hydrogen peroxide-induced barrier dysfunction, tight junction disruption and reorganization of actin cytoskeleton. Pretreatment of cell monolayers with EGF ameliorated hydrogen peroxide-induced tight junction disruption and barrier dysfunction. The protective effect of EGF was abrogated by ET-18-OCH3 and the Ro-32-0432 (PLCγ and PKC inhibitors). Hydrogen peroxide increased tyrosine phosphorylation of ZO-1, claudin-3, E-cadherin and β-catenin, and pretreatment of cells with EGF attenuated tyrosine phosphorylation of these proteins. These results demonstrate that hydrogen peroxide disrupts tight junctions, adherens junctions and the actin cytoskeleton by an MLCK and Src kinase-dependent mechanism in the bile duct epithelium. EGF prevents hydrogen peroxide-induced tight junction disruption by a PLCγ and PKC-dependent mechanism.

Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism

The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.