Caveolae-mediated Internalization of Occludin and Claudin-5 during CCL2-induced Tight Junction Remodeling in Brain Endothelial Cells

Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.The blood-brain barrier is situated at the cerebral endothelial cells and their linking tight junctions. Increased brain endothelial barrier permeability is associated with remodeling of inter-endothelial tight junction (TJ)² complex and gap formation between brain endothelial cells (paracellular pathway) and/or intensive pinocytotic vesicular transport between the apical and basal side of brain endothelial cells (transcellular pathway) (1, 2). The transcellular pathway can be either passive or active and is characterized by low conductance and high selectivity. In contrast, the paracellular pathway is exclusively passive, being driven by electrochemical and osmotic gradients, and has a higher conductance and lower selectivity (3).Brain endothelial barrier paracellular permeability is maintained by an equilibrium between contractile forces generated at the endothelial cytoskeleton and adhesive forces produced at endothelial cell-cell junctions and cell-matrix contacts (1–3). A dynamic interaction among these structural elements controls opening and closing of the paracellular pathway and serves as a fundamental mechanism regulating blood-brain exchange. How this process occurs is under intense investigation. Two possible mechanisms may potentially increase paracellular permeability: phosphorylation of TJ proteins and/or endocytosis of transmembrane TJ proteins.Changes in TJ protein phosphorylation seem to be required to initiate increased brain endothelial permeability and a redistribution of most TJ proteins away from the cell border (4–8). Endocytosis may also be involved in remodeling TJ complexes between endothelial cells. Several types of endocytosis may be involved in TJ protein uptake, including clathrin- and caveolae-mediated endocytosis and macropinocytosis (for reviews, see Refs. 8 and 9–12). After first forming cell membrane-derived endocytotic vesicles, these vesicles fuse with early endosomes whose contents are further sorted for transport to lysosomes for degradation or recycling back to the plasma membrane for reuse (11).Although there is a lack of definitive knowledge regarding endocytotic internalization of brain endothelial cell TJ proteins, several studies on epithelial cells have indicated that occludin may be internalized via caveolae-mediated endocytosis whereas ZO-1, claudin-1, and junctional adhesion molecules-A may undergo macropinocytosis in response to stimuli such as TNF-α and INF-γ (13, 14). In contrast, there is evidence that Ca²⁺ may induce internalization of claudin-1 and occludin via clathrin-coated vesicles (8, 14–16). All of these studies pinpoint endocytosis as an underlying process in TJ complex remodeling and redistribution, and thus regulation of paracellular permeability in epithelial cells.The present study examines whether internalization of transmembrane TJ proteins could be one process by which adhesion between brain endothelial cells is changed during increased paracellular permeability. Our results show that a pro-inflammatory mediator, the chemokine CCL2, induces disassembly of the TJ complex by triggering caveolae-dependent internalization of transmembrane TJ proteins (occludin and claudin-5). Once internalized, occludin and claudin-5 are further processed to recycling endosomes awaiting return to the plasma membrane.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

Endocytosis of the apical junctional complex: mechanisms and possible roles in regulation of epithelial barriers

Tight junctions (TJ) and adherens junctions (AJ) regulate cell-cell adhesion and barrier function of simple polarized epithelia. These junctions are positioned in the apical end of the lateral plasma membrane and form the so-called apical junctional complex (AJC). Although initially seen as purely structural features, the AJC is now known to play important roles in cell differentiation and proliferation. The AJC is a highly dynamic entity, undergoing rapid remodeling during normal epithelial morphogenesis and under pathologic conditions. There is growing evidence that remodeling of the AJC is mediated by internalization of junctional proteins. This review summarizes what is known about endocytic pathways, intracellular destinations and signaling cascades involved in internalization of AJC proteins. Potential biological roles for AJC endocytosis in maintaining functional apical junctions, reversible opening of epithelial barrier and disruption of intercellular adhesion are also discussed.     

JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in approximately 50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (huDeltaC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huDeltaC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.     

A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein sigma1

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.     

Microtubules regulate disassembly of epithelial apical junctions

Background  

Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.     

Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia

Vascular endothelial growth factor (VEGF) and Angiopoietin 1 (Ang1) are both potent proangiogenic factors, but, whereas VEGF causes vascular permeability, Ang1 stabilizes blood vessels and protects them from VEGF-induced plasma leakage. The antivascular permeability mechanisms deployed by Ang1 are still undefined. Here, we demonstrate that Ang1 halts the ability of VEGF to induce the phosphorylation-dependent redistribution of the adhesion molecule VE-cadherin, thereby rescuing the endothelial barrier function. Ang1 inhibits the activation of Src by VEGF, the most upstream component of the pathway linking VEGF receptors to VE-cadherin internalization. Indeed, Ang1 promotes the activation of mDia through RhoA, resulting in the association of mDia with Src. This ultimately deprives VEGF receptors of an essential molecule required for promoting the disruption of endothelial cell-cell contacts and paracellular permeability.     

NaCl flux between apical and basolateral side recruits claudin-1 to tight junction strands and regulates paracellular transport

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.     

Apical cargo traverses endosomal compartments on the passage to the cell surface

Epithelial polarity is based on intracellular sorting machinery that maintains the asymmetric distribution of lipids and proteins to the cell surface. Dependent on their lipid raft affinity, newly synthesized apical polypeptides are segregated into distinct vesicle populations subsequent to the passage through the Golgi apparatus. Using a combined fluorescence microscopic and biochemical approach, we found that lipid raft-associated sucrase-isomaltase (SI) as well as non-raft-associated lactase-phlorizin hydrolase (LPH) traverse endosomal compartments before entering the apical membrane. Fluorescent fusion proteins of both hydrolases were co-stained with Rab4-, Rab8- and Rab11-positive endosomes in polarized Madin-Darby canine kidney and non-polarized COS-1 cells. Immunoisolation of post-Golgi vesicles subsequent to different times of TGN release revealed that LPH and SI navigate in chronological order through Rab4-, Rab8- and Rab11-positive endosomes. Thereafter, the two hydrolases are segregated into distinct vesicle populations. In addition, apical membrane traffic could be significantly inhibited by RNA interference-mediated depletion of these guanosine triphosphatases. These results suggest that in epithelial cells, lipid raft-dependent and -independent apical cargo follow a transendosomal route.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

Junctional adhesion molecule-A regulates cell migration and resistance to shear stress

Junctional adhesion molecule-A (JAM-A) is an adhesive protein expressed in endothelial cells, epithelial cells, platelets, and some leukocytes. JAM-A localizes to the tight junctions between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesion and to the control of paracellular permeability. JAM-A also regulates cell motility, even though the quantitative biophysical features have not been characterized. In this study, we evaluated the role of JAM-A in the regulation of cell motility using JAM-A-expressing and JAM-A-deficient murine endothelial cells. We report that, in the absence of shear stress, JAM-A absence increases cell motility by increasing directional persistence but not cell speed. In addition, in the presence of shear stress, JAM-A absence increases protrusion extension in the direction of flow and increased downstream cellular displacement (while, conversely, decreasing upstream displacement). All these effects of JAM-A absence are mitigated by the microtubule-stabilizing compound taxol. A motility- and microtubule-related function, integrin-mediated adhesiveness, was only slightly reduced in JAM-A-deficient cells compared with JAM-A-expressing cells. However, overexpression of JAM-A in the JAM-A-deficient cells increased integrin adhesiveness to the same levels as those observed in taxol-treated JAM-A-deficient cells. Taken together, these data indicate that JAM-A regulates cell motility by cooperating with microtubule-stabilizing pathways.     

Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and beta-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and alpha-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.     

aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation

The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.     

Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v-src mutant. Shifting monolayers established at 40 degrees C (non-permissive temperature) to 34 degrees C (permissive temperature) rapidly reactivated v-Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v-src was recruited on apical vesicles, induced cortactin-associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3-kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 degrees C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v-src, Rab11, and rabankyrin-5 but not early endosome antigen 1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.     

Agonist-dependent internalization and trafficking of the human prostacyclin receptor: a direct role for Rab5a GTPase

The human prostacyclin receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5a(S34N) did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5a(Q79L) significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating prostacyclin signaling.     

Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.     

Endothelia of term human placentae display diminished expression of tight junction proteins during preeclampsia

This study explores the molecular composition of the tight junction (TJ) in human term placenta from normal women and from patients with preeclampsia, a hypertensive disorder of pregnancy. Maternal endothelial dysfunction is a critical characteristic of preeclampsia; hence, we have analyzed its impact on placental vessels. The study concentrates on the TJ because this structure regulates the sealing of the paracellular route. We have found that, in placental endothelial vessels, TJ components include the peripheral protein ZO–1 and the integral proteins occludin and claudins 1, 3, and 5. During preeclampsia, the amounts of occludin and ZO–1 exhibit no significant variation, whereas those of claudins 1, 3, and 5 diminish, suggesting the presence of leakier TJs in the endothelia of the preeclamptic placenta, possibly in response to the decreased perfusion of this organ during preeclampsia. We have unexpectedly found that, in normal placentae, the multinucleated syncytiotrophoblast layer displays claudin 4 at the basal surface of the plasma membrane, and claudin 16 along the apical and basolateral surfaces. The presence of membrane-lined channels that cross the syncytiotrophoblast constituting a paracellular pathway has been determined by transmission electron microscopy and by the co-immunolocalization of claudin 16 with the plasma membrane proteins Na⁺K⁺-ATPase and GP135. Since claudin 16 functions as a paracellular channel for Mg²⁺, its diffuse pattern in preeclamptic placentae suggests the altered paracellular transport of Mg²⁺ between the maternal blood and the placental tissue.This work was supported by grants 45691-Q from the Mexican Council for Science and Technology (CONACYT) and 2005/1/I/012 from the Research Promotion Fund of the Mexican Institute of Social Security (IMSS/FOFOI).     

Larazotide acetate promotes tight junction assembly in epithelial cells

Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.