Hepatocyte growth factor/scatter factor decreases the expression of occludin and transendothelial resistance (TER) and increases paracellular permeability in human vascular endothelial cells.

Hepatocyte growth factor/scatter factor (HGF/SF), a multi function cytokine has been shown to regulate the expression of cell adhesion molecules in endothelial cells. In the current study, we examined the effects of HGF/SF on the function of tight junctions and the expression of occludin in these cells. Stimulation of human vascular endothelial cells with HGF/SF resulted in a concentration dependent increase in the paracellular permeability as measured using fluorescence labelled dextran but a decrease in the transendothelial cell resistance (TER) of the endothelial cells. Western blotting revealed that HGF/SF decreased the level of occludin in the cells, a primary tight junction forming protein. Immunofluorescence study indicated that shortly after treatment with HGF/SF there was a disturbance of the distribution of occludin and then a reduction in the staining of the molecule. It is concluded that HGF/SF decreases the expression of occludin, resulting in the functional change of tight junction.     

Biphasic effects of 17-beta-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells

Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction function are an early and key event during the vascular spread of cancer and inflammation. This study sought to determine the role of estrogen in the regulation of tight junctions and expression of molecules making tight junctions in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17-beta-estradiol induced a concentration- and time-dependent biphasic effect on tight junction. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17-beta-estradiol at lower concentrations (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 min and the second drop occurring 48 h after 17-beta-estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17-beta-estradiol, in line with changes of TER. This study shows that 17-beta-estradiol can induce concentration- and time-related biphasic effects on tight junction functions expression of occludin in endothelial cells and that this perturbation of tight junction functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.     

Antagonistic effect of NK4 on HGF/SF induced changes in the transendothelial resistance (TER) and paracellular permeability of human vascular endothelial cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a multi-function cytokine that has been shown to regulate the expression of cell adhesion molecules in human endothelial cells. It is also a key cytokine in the development and progression of cancer, particularly during metastasis. NK4 is a variant of HGF/SF that has already been shown to be antagonistic to HGF/SF. This study shows that HGF/SF decreased transendothelial resistance (TER) and increased paracellular permeability in human vascular endothelial cells can that such effects can be inhibited by addition of the NK4 variant. In addition, HGF/SF-stimulated invasion of endothelium by breast cancer cells was inhibited by the addition of NK4. Western blotting revealed that HGF/SF decreased the protein level, and increased tyrosine phosphorylation of ZO-1, but did not cause a change in level of occludin or claudin-1, both molecules involved in tight junction function. RT-PCR revealed that addition of HGF/SF caused no change in signal for claudin-5 or junctional adhesion molecule (JAM), but there was a decrease in the signal for claudin-1. NK4 was able to prevent the decrease in levels of ZO-1 protein by HGF/SF.     

Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.     

Multiple domains of occludin are involved in the regulation of paracellular permeability

Tight junctions form selective paracellular diffusion barriers that regulate the diffusion of solutes across epithelia and constitute intramembrane diffusion barriers that prevent the intermixing of apical and basolateral lipids in the extracytoplasmic leaflet of the plasma membrane. In MDCK cells, previous expression experiments demonstrated that occludin, a tight junction protein with four transmembrane domains, is critically involved in both of these tight junction functions and that its COOH-terminal cytoplasmic domain is of functional importance. By expressing mutant and chimeric occludin that exert a dominant negative effect on selective paracellular diffusion, we now demonstrate that the extracytoplasmic domains and at least one of the transmembrane domains are also critically involved in selective paracellular permeability. Multiple domains of occludin are thus important for the regulation of paracellular permeability. Expression of chimeras containing at least one transmembrane domain of occludin also resulted in an enhanced intracellular accumulation of claudin-4, another transmembrane protein of tight junctions, suggesting that the two proteins may cooperate in the regulation of paracellular permeability.     

Enhanced tight junction function in human breast cancer cells by antioxidant, selenium and polyunsaturated lipid

Paracellular permeability (PCP) is governed by tight junctions (TJs) in epithelial cells, acting as cell-cell adhesion structures, the aberration of which is known to be linked to the dissociation and metastasis of breast cancer cells. This study hypothesized that the function of TJs in human breast cancer cells can be augmented by gamma linolenic acid (GLA), selenium (Se), and iodine (I) in the presence of 17-beta-estradiol, as these molecules are known to increase TJ functions in endothelial cells, using assays of trans-epithelial resistance (TER), PCP, immunofluorescence, and in vitro invasion and motility models. GLA, I, and Se individually increased TER of MDA-MB-231 and MCF-7 human breast cancer cells. The combination of all three agents also had a significant increase in TER. Addition of GLA/Se/I reduced PCP of both breast cancer cell lines. GLA/Se/I reversed the effect of 17-beta-estradiol (reduced TER, increased PCP). Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h, there was increasing relocation to breast cancer cell-cell junctions of occludin and ZO-1 in MCF-7 cells. Moreover, treatment with GLA/Se/I, alone or in combination, significantly reduced in vitro invasion of MDA-MB-231 cells through an endothelial cell barrier (P < 0.0001) and reduced 17-beta-estradiol induced breast cancer cell motility (P < 0.0001). Our previous work has demonstrated that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells; this has now proved to be true of human breast cancer cells. This combination also completely reversed the effect of 17-beta-estradiol in these cells.     

Occludin modulates transepithelial migration of neutrophils

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.     

Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.     

Occludin regulates actin cytoskeleton in endothelial cells

Occludin is a major membrane component of tight junctions of endothelial cells, though the role of this molecule is not fully understood. RLE cells, derived from rat lung endothelial cells, express a negligible level of occludin with clear expression of E-cadherin and ZO-1 at cell junctions. Introduction of occludin by transfection induced clear junctional expression of occludin with few or no changes of expression of E-cadherin and ZO-1. The paracellular barrier function, as determined by transelectrical resistance and flux of non-ionic small molecules, was not detectably upregulated. When cells expressing occludin were cocultured with RLE cells null for occludin, clear junctional expression of occludin was observed irrespective of the expression of occludin on the apposing cells. Cortical actin was developed at the site of these occludin positive cell junctions. Treatment of cells with an actin depolymerizing agent, mycalolide B, abolished junctional expression of occludin together with E-cadherin and circumferential actin. ZO-1 showed relative resistance to this actin depolymerizing treatment and was maintained at the cell junctions, though fragmentation of immunoreactivity was detectable. Collectively, junctional expression of occludin was not associated with paracellular barrier function in this cell line. There was, however, a close correlation of occludin with the actin cytoskeleton, indicating a role of occludin as an important molecule in the regulation of the actin cytoskeleton in endothelial cells.     

Claudins--key pieces in the tight junction puzzle

Tight junctions restrict the flow of ions and aqueous molecules between cells by forming a selective barrier to the paracellular pathway. Permeability of the tight junction barrier is determined by a class of transmembrane proteins known as claudins. The relationship between claudins and paracellular permeability is complex and determined not only by the profile of claudin expression but also by the arrangement of claudins and other proteins into tight junction strands. This review summarizes progress in understanding how claudins are assembled into tight junctions and how they interact with other tight junction proteins.     

Assembly of tight junction is regulated by the antagonism of conventional and novel protein kinase C isoforms

Apparently conflicting observations indicated that protein kinase C both may block and support the assembly of tight junctions. We therefore tested the hypothesis that different isoenzymes antagonistically affect tight junction proteins and function. Thus, by using specific inhibitors we investigated the involvement of conventional and novel protein kinase C of kidney tubule cells in tight junction assembly. In low Ca2+ medium, the application of pan-protein kinase C inhibitor GF-109203X blocked the formation of tight junctions induced by protein kinase C agonist diacyglycerol. G?6976, inhibitor of conventional protein kinase C, promoted the formation of tight junctions and occludin phosphorylation in cells cultivated in low Ca2+ medium and attenuated the disruption of tight junction complex induced by the switch to low Ca2+ medium. In addition, G?6976 accelerated the occludin phosphorylation and the formation of tight junction barrier during assembly of tight junctions induced by Ca2+ re-addition. This phosphorylation was accompanied by accelerated occludin incorporation into newly forming tight junctions and by reducing the paracellular permeability. In contrast, inhibitor of novel protein kinase C rottlerin blocked the occludin phosphorylation and the formation of tight junction barrier, both caused by re-addition of normal Ca2+ medium. It is concluded that the conventional protein kinase C alpha participates in tight junction disassembly while the novel protein kinase C epsilon plays a role in tight junction formation of kidney epithelial cells. The discovered antagonism contributes to a better understanding of the regulation of the structure and function of tight junctions and hence to that of the epithelial barrier.     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

src family kinases regulate renal epithelial paracellular permeability barrier through an occludin‐independent mechanism

Paracellular permeability is mediated by the epithelial cell tight junction. Studies in intestinal and other epithelia have suggested that the activity of src family kinases (SFKs) increases epithelial paracellular permeability through its action on the tight junction protein, occludin, but the involvement of SFKs and occludin in regulation of renal epithelial paracellular permeability is unclear. In this study, the role of SFKs in regulation of renal epithelial paracellular permeability and the involvement of occludin protein in this regulatory event was examined in two renal epithelial cell lines, LLC‐PK₁ (proximal tubule‐like) and MDCK (distal tubule‐like). The effect of broad spectrum SFK inhibitors on paracellular permeability of calcein and fluorescein‐dextran3000 were examined. SFK inhibitor treatment increased paracellular movement of both compounds in both renal epithelial cell lines. The SFK inhibitor effect was concentration‐dependent and, at low concentrations, was not associated with cell damage/death. Response to SFK inhibitors was acquired progressively after cell populations attained confluence suggesting maturation of the regulatory mechanism. Increased paracellular permeability was not associated with dramatic changes in total cell content of occludin protein, its partitioning between detergent‐soluble and ‐insoluble fractions, or its subcellular localization. Further, the SFK‐induced increase in paracellular permeability was unaffected by either occludin protein overexpression or occludin protein knockdown. These results demonstrate that SFK activity decreases paracellular permeability of renal epithelial cells, as opposed to its effect in intestinal epithelial cells, and that this regulation is not mediated by occludin protein. J. Cell. Physiol. 228: 1210–1220, 2013. © 2012 Wiley Periodicals, Inc.     

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

Tight junctions control paracellular permeability and cellpolarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro modelof renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rhosignaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells thancontrol junctions, whereas cells expressing activated Rho bettermaintained junctions during ATP depletion than control cells. ATPdepletion and Rho signaling altered phosphorylation signalingmechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoaminoacid content following ATP depletion, which was restored on recovery.Expression of Rho mutant proteins in MDCK cells also altered levels ofoccludin serine/threonine phosphorylation, indicating that occludin isa target for Rho signaling. We conclude that Rho GTPase signalinginduces posttranslational effects on tight junction components. Ourdata also demonstrate that activating Rho signaling protects tightjunctions from damage during ATP depletion.

     

A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 μM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.     

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.     

Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.