Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells

Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.     

Cyathula prostrata ethanol extract activates the extrinsic pathway of apoptosis in HeLa and U937 cell lines

Cyathula prostrata is a medicinal plant that is used in tropical Africa, Asia and Australia to treat many ailments including rheumatic fever, dysentery, wounds and eye trouble (Burkill, 1995). Sowemimo et al. (2009) reported cytotoxicity against a cervical cancer cell line, HeLa, at a concentration of 250 μg/mL. Due to the reported cytotoxicity, the mode of cell death caused by an ethanolic C. prostrata extract was investigated. Dose–response assays were carried out using HeLa and U937 cell lines and IC₅₀ values of 100.8 μg/mL and 64.43 μg/mL, respectively, were achieved. Cisplatin was used as a positive control for all experiments. Cytotoxicity of the plant extract was not evident when treating normal peripheral blood mononuclear cells. Progression of cells through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. Arrest of cells in the G0/G1 phase of the cell cycle was evident after 24 h of exposure to the plant extract in both cell lines, but not due to increases in p21 levels. Caspase 8 activation was evident and no depolarisation of the mitochondrial membrane and cytochrome c release was seen in both cell lines. Phosphatidylserine translocation was investigated and confirmed the onset of apoptosis. Thus, it is deduced that exclusive activation of the extrinsic pathway of apoptosis is caused by the treatment of HeLa and U937 cells with ethanolic C. prostrata.     

Mechanotransduction activates RhoA in the neighbors of apoptotic epithelial cells to engage apical extrusion

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Protein trafficking in the exocytic pathway of polarized epithelial cells

Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.     

Hydrogen peroxide activation of ERK5 confers resistance to Jurkat cells against apoptosis induced by the extrinsic pathway

Reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂) exhibit both pro-survival and pro-death signaling in leukemic cells. We examined the effect of exogenous H₂O₂ on Fas ligand (FasL) -induced apoptosis in Jurkat cells. H₂O₂ applied prior to (pre-conditioning) and during (post-conditioning) FasL stimulation attenuated early apoptosis through activation of EKR5. H₂O₂ increased the activated caspase-8 sequestered in the mitochondria thereby decreasing cell death through the extrinsic apoptotic pathway. In addition, inhibition of a protein tyrosine phosphatase likely explains the post-conditioning requirement for H₂O₂. Given that chemotherapeutic agents used for the treatment of acute lymphoblastic leukemia are thought to work partly through production of ROS, a simultaneous inhibition of the ERK5 pathway may abrogate the ROS-initiated pro-survival signaling for an enhanced cell kill.     

Helicobacter pylori infection activates NF-kappaB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-kappaB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-kappaB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-kappaB signaling pathway via NF-kappaB-inducing kinase and IkappaB kinase-beta activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IkappaBalpha superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-kappaB. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 microM), decreased caspase-3 activity and apoptosis in NF-kappaB-suppressed cells infected with H. pylori. These results suggest that NF-kappaB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.     

Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induces the generation of reactive oxygen species (ROS) by upregulating the expression of NADPH oxidase 2 and its regulatory proteins (p22(phox), p67(phox), p40(phox), p47(phox), and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to the activation of mTOR signaling, as pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of the type I insulin-like growth factor receptor (IGFR) β subunit, which was abrogated by NAC. Wortmannin, a phosphoinositide 3'-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1, as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of the mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/PI3K and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for the prevention of Cd-induced neurodegenerative diseases.     

Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.     

Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.     

Elevated calcium in preneoplastic cells activates NF-kappa B and confers resistance to apoptosis

Early preneoplastic cells (sup+) exhibit increased susceptibility to apoptosis, which is lost in late stage preneoplastic cells (sup-). Sup+ cells, which undergo apoptosis when cultured in low serum, show little or no DNA binding activity to nuclear factor (NF)-kappa B either in 10% or 0.2% serum. In contrast sup- cells, which are resistant to apoptosis in low serum, show a sustained constitutive activation of NF-kappa B. The constitutive activation of NF-kappa B observed in sup- cells is not due to loss of I kappa B alpha. We considered that the activation of NF-kappa B in sup- cells might be secondary to an increase in cytosolic Ca(2+), since sup- cells have a cytosolic Ca(2+) level that is double that in sup+ cells. In support of a role for Ca(2+), lowering cytosolic Ca(2+) in sup- cells by addition of the cell-permeable Ca(2+) chelator 1,2 bis(O-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) reduced cytosolic Ca(2+) by approximately 31% relative to untreated sup- cells, concomitant with a 65% reduction in NF-kappa B DNA binding activity and a reduction in I kappa B kinase (IKK) activity. In sup- cells in low serum, addition of BAPTA-AM also resulted in a significant ( approximately 50%) increase in caspase-3 activity. Raising extracellular Ca(2+) in sup+ cells resulted in a slight activation of I kappa B kinase and in enhanced NF-kappa B DNA binding activity. Using proteasome and calpain inhibitors, we determined that the basal activity of NF-kappa B in sup- cells is largely proteasome-independent, but sensitive to calpain inhibitors. Taken together these data suggest that the elevated Ca(2+) in sup- cells causes a modest activation of IKK, which likely contributes to the enhanced basal activation of NF-kappa B in sup- cells; however, the predominant effect of Ca(2+) appears to be mediated by Ca(2+)-enhanced degradation by calpain.     

Transforming growth factor beta 1 induces tight junction disruptions and loss of transepithelial resistance across porcine vas deferens epithelial cells

Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%-99% decrease in basal transepithelial electrical resistance (R(TE), a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R(TE) significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.     

Flagellin-induced tolerance of the Toll-like receptor 5 signaling pathway in polarized intestinal epithelial cells

Salmonella typhimurium is a gram-negative enteric pathogen that invades the mucosal epithelium and is associated with diarrheal illness in humans. Flagellin from S. typhimurium and other gram-negative bacteria has been shown to be the predominant proinflammatory mediator through activation of the basolateral Toll-like receptor 5 (TLR5). Recent evidence has shown that prior exposure can render immune cells tolerant to subsequent challenges by TLR ligands. Accordingly, we examined whether prior exposure to purified flagellin would render human intestinal epithelial cells insensitive to future contact. We found that flagellin-induced tolerance is common to polarized epithelial cells and prevents further activation of proinflammatory signaling cascades by both purified flagellin and Salmonella bacteria but does not affect TNF-alpha stimulation of the same pathways. Flagellin tolerance is a rapid process that does not require protein synthesis, and that occurs within 1 to 2 h of flagellin exposure. Prolonged flagellin exposure blocks activation of the NF-kappaB, MAPK, and phosphoinositol 3-kinase signaling pathways and results in the internalization of a fraction of the basolateral TLR5 without affecting the polarity or total expression of TLR5. After removal of flagellin, cells require more than 24 h to fully recover their ability to mount a normal proinflammatory response. We have found that activation of phosphoinositol 3-kinase and Akt by flagellin has a small damping effect in the early stages of flagellin signaling but is not responsible for tolerance. Our study indicates that inhibition of TLR5-associated IL-1 receptor-associated kinase-4 activity occurs during the development of flagellin tolerance and is likely to be the cause of tolerance.     

Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.     

Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the “secretory” NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.     

Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells

The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ–) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ– clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ– and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ– monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ– monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 Å in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected. tight junction; occludin; Rho-GTP     

Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production.     

Aeroallergen Der p 2 induces apoptosis of bronchial epithelial BEAS-2B cells via activation of both intrinsic and extrinsic pathway

Background

Excessive apoptosis of airway epithelium is reported to induce airway remodeling and inhibited airway epithelium repair is highly associated with development of asthma and chronic obstructive pulmonary disease. Der p 2 is a major allergen derived from Dermatophagoides pteronyssinus and commonly causes airway hypersensitiveness and asthma; however, the connection between Der p 2 and epithelial apoptosis remains unclear. This study was aimed to explore whether Der p 2 induces apoptosis of airway epithelial cells and the underlying mechanisms.

Results

Our results showed that recombinant Der p 2 (rDP2) inhibited cell growth and induced apoptosis of human bronchial epithelial cell BEAS-2B. Further investigation revealed that rDP2 increased intracellular reactive oxygen species, level of cytosolic cytochrome c and cleavage of caspase-9 and caspase-3. rDP2 also induced activation of p38 mitogen-activated protein kinase (P38) and c-Jun N-terminal kinase (JNK), and triggered proapoptotic signals including decrease of Bcl-2, increase of Bax and Bak, and upregulation of Fas and Fas ligand. In parallel, rDP2 inhibited glycogen synthase kinase 3beta and consequently enhanced degradation of cellular (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP). Involvement of toll-like receptor (TLR)2 in rDP2-induced apoptosis was also demonstrated using specific small inhibitory RNA.

Conclusions

Our findings indicate that rDP2 suppresses cell growth and trigger apoptosis of BEAS-2B cells, which may attribute to induction of both intrinsic and extrinsic pathway via TLR2 and P38/JNK signaling and c-FLIP degradation. It suggests that Der p 2 may aggravate respiratory disorders through enhancement of apoptosis and the consequent airway injury.