Transmembrane Tight Junction Proteins Research Articles

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

Abstract B245: Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody.

Abstract In metastatic colorectal cancer (CRC) patients, chemotherapy in combination with targeted therapies such as Avastin, Erbitux, and Vectibix (monoclonal antibodies) has improved response rates with prolongation of progression free survival and overall survival. Despite these major therapeutic progresses, at least 30 to 50% of patients remain non-responders. Our project aimed to identify and validate new molecular targets of CRC. Comparison of gene expression profiles between normal mucosa and primary tumor of CRC patients followed by immunohistochemistry analysis identified claudin-1 (CLDN1) as potential therapeutic target for antibody-targeted therapy. CLDN1 is a major tight junction transmembrane protein, already found overexpressed in CRC in several studies and associated with tumor progression. We have generated monoclonal antibodies (mAb) directed against extracellular domains of CLDN1 and demonstrated their specificity against human CLDN1 in vitro and in vivo. Then, using mice xenografted with the CRC cell line SW620 we tested the therapeutic effect of the 6F6C3mAb. The mice were treated by i.p. injections with vehicle or 6F6C3mAb at two different doses. The results showed that 6F6C3mAb treated-groups had a significant (p= 0,018) reduced growth compared to the control group and this effect was dose-dependent. The in vitro effects of the binding of 6F6C3mAb to CLDN1 were also assessed by clonogenic assays. Treatment by 6F6C3mAb induced a significant (p&amp;lt; 0.05) reduction of the number of colony-forming cells for CLDN1high colorectal cancer cells (SW620, Caco2) but also for CLDN1high ovarian and pancreatic cancer cells (PANC1, BXPC3, SKOV3, IGROV1). Thus, 6F6C3mAb binding had a profound effect on cancer cell survival. In conclusion, CLDN1 seems to be a promising target for antibody-based therapy. The 6F6C3mAb is able to inhibit tumor growth and tumor survival by binding the extracellular domains of CLDN1. It can be used in the treatment of cancer diseases such as colorectal cancer as well in adjuvant as in metastatic setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B245. Citation Format: Adeline Ayrolles-Torro, Nadia Vezzio-Vie, Vincent Denis, Florence Boissiere-Michot, Véronique Garambois, Muriel Busson, Imade Ait Arsa, Caroline Mollevi, Martine Pugniere, Frédéric Bibeau, Pierre Martineau, Céline Gongora, Maguy Del Rio. Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B245.

Extracellular signal-regulated kinase1/2-dependent changes in tight junctions after ischemic preconditioning contributes to tolerance induction after ischemic stroke

Less disruption of the blood-brain barrier (BBB) after severe ischemic stroke is one of the beneficial outcomes of ischemic preconditioning (IP). However, the effect of IP on tight junctions (TJs), which regulate paracellular permeability of the BBB, is not well understood. In the present study, we examined IP-induced changes in TJs before and after middle cerebral artery occlusion (MCAO) in mice, and the association between changes in TJs and tolerance to a subsequent insult. After IP, we found decreased levels of transmembrane TJ proteins occludin and claudin-5, and widened gaps of TJs with perivascular swelling at the ultrastructural level in the brain. An inflammatory response was also observed. These changes were reversed by inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) via the specific ERK1/2 inhibitor U0126. After MCAO, reduced brain edema and inflammatory responses were associated with altered levels of angiogenic factors and cytokines in preconditioned brains. Pretreatment with U0126 reversed the neuroprotective effects of IP against MCAO. These findings suggest that ERK1/2 activation has a pivotal role in IP-induced changes in TJs and inflammatory response, which serve to protect against BBB breakdown and inflammation after ischemic stroke.

Claudin-2 pore function requires an intramolecular disulfide bond between two conserved extracellular cysteines

Claudins constitute a family of tight junction transmembrane proteins whose first extracellular loop (ECL1) determines the paracellular permeability and ion selectivity in epithelia. There are two cysteines in the ECL1 that are conserved among all claudins. We hypothesized that these extracellular cysteines are linked by an intramolecular disulfide bond that is necessary for correct pore folding and function. To test this, we mutated C54 and C64 in claudin-2, either individually or together to alanine or serine, and generated stable Madin-Darby canine kidney (MDCK) I Tet-off cell lines. Immunoblotting showed a higher molecular mass band in the mutants with a single cysteine mutation, consistent with a claudin-2 dimer, suggesting that the two conserved cysteines normally form an intramolecular disulfide bond in wild-type claudin-2. By immunofluorescent staining, the alanine mutants were mislocalized intracellularly, while the serine mutants were expressed at the tight junction. Thus dimerization of both C54A and C64A did not require tight junction expression, suggesting that C54 and C64 are located near an intermolecular interface involved in cis-interaction. The conductance and Na(+) permeability of the serine mutants were markedly lower than the wild type, but there was no difference between the single mutants and the double mutant. We conclude that the disulfide bond between the conserved extracellular cysteines in claudin-2 is necessary for pore formation, probably by stabilizing the ECL1 fold, but is not required for correct protein trafficking. We further speculate that this role is generalizable to other claudin family members.

The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks

Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.

Poly (I:C) induces human endothelial barrier dysfunction by disrupting tight junction expression of claudin-5 (P4197)

Abstract Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 ligand Poly (I:C), a synthetic analog of viral double-stranded RNA commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular cells. Poly (I:C) produced a dose- and time-dependent increase in endothelial monolayer permeability accompanied by a corresponding decrease in the expression of claudin-5, a transmembrane tight junction protein. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of internalized claudin-5 with lysosomal-associated membrane protein 1 (LAMP-1), a marker of lysosomes, suggesting enhanced lysosomal degradation of claudin-5. Poly (I:C) also induced a dose- and time-dependent cleavage of TLR3 and TRIF as well as activation of NF-kB and IRF3, but not IRF7 (MyD88-dependent pathway), consistent with the direct stimulation of the TLR3 signaling. Pronounced inflammatory response with the release of IL-6, IL-8, MCP-1 and RANTES and a hallmark TLR3-mediated type 1 IFN production (IFNbeta) was also observed. Together, these findings provide new insight on how virally-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

Detection of claudin‐4 in salivary gland neoplasms (a study utilizing RT‐PCR and immunohistochemistry)

Claudins are transmembrane proteins of tight junctions emerging as targets for diagnosis, prediction of prognosis, disease recurrence, and metastasis. Our goal was to evaluate expression of claudin-4 in the most common benign and malignant salivary gland neoplasms. Claudin-4 gene levels and protein expression were determined by real-time polymerase chain reaction (PCR), and immunohistochemistry in a total of 30 specimens containing normal salivary tissue, pleomorphic adenoma, Warthin's tumor, mucoepidermoid carcinoma, and adenoid cystic carcinoma. We identified down-regulation of claudin-4 gene levels and protein expression from normal control to benign salivary gland neoplasms and reached their lowest values in the malignant salivary gland neoplasms. Low claudin-4 expression could be considered as a sign of increasing cellular disorientation and invasion of salivary gland tumors.

A role for tricellulin in the regulation of gill epithelium permeability

The apical-most region of cell-to-cell contact in a vertebrate epithelium is the tight junction (TJ) complex. It is composed of bicellular TJs (bTJs) that bridge two adjacent epithelial cells and tricellular TJs (tTJs) that are points of contact between three adjoining epithelial cells. Tricellulin (TRIC) is a transmembrane TJ protein of vertebrates that is found in the tTJ complex. Full-length cDNA encoding rainbow trout TRIC was cloned and sequenced. In silico analysis of rainbow trout TRIC revealed a tetraspannin protein with several putative posttranslational modification sites. TRIC mRNA was broadly expressed in rainbow trout tissues and exhibited moderately greater abundance in the gill. In a primary cultured gill epithelium, TRIC localized to tTJs and TRIC protein abundance increased in association with corticosteroid-induced reductions in paracellular permeability. Sodium caprate was used to compromise cultured gill epithelium integrity by disrupting the tTJ complex. Sodium caprate treatment caused a reversible reduction in transepithelial resistance, caused an increase in paracellular permeability (as measured by [³H]PEG-4000 flux), and displaced TRIC from tTJs while leaving bTJs intact. Data from this study support the view that tTJs and the TJ protein TRIC 1) play a role in maintaining gill epithelium integrity and 2) contribute to the regulation of gill epithelium permeability.

Occludin Deficiency Exacerbates Ethanol‐Induced Colonic Barrier Dysfunction and Liver Damage

Epithelial tight junctions (TJs) confer barrier function in the intestinal mucosa. Occludin is one of the transmembrane proteins of TJs. Occludin knockout (OKO) mice showed no apparent anomaly in the intestinal epithelial TJs. Hence, the function of occludin is undetermined. Recent studies indicated the potential role of occludin in apoptosis and cell migration. Chronic ethanol feeding (CEF) increases colonic permeability and causes liver damage. We evaluated the susceptibility of OKO mice to CEF‐induced gut barrier dysfunction and liver damage. Wild type (WT) and OKO mice were fed a liquid diet containing ethanol (1% 2d, 2% 2d, 4% 1wk, 5% 1wk and 6% 1wk). Control mice were pair fed (PF) with isocaloric maltodextrin. CEF led to a significant loss of body weight in OKO mice, compared to WT mice. Colonic mucosal permeability in vivo, measured as plasma to lumen flux of FITC‐inulin, was ~3 fold greater in EF‐OKO mice. CEF induced redistribution of ZO‐1 from the intercellular junctions; redistribution was more severe in EF‐OKO mice. CEF increased liver weight and plasma ALT activity in OKO mice, but not in WT mice. H&amp;E, Oil red‐O and Sirius red staining showed that CEF induced greater levels of fat deposition, focal lesions and collagen accumulation in liver of EF‐OKO mice than in WT mice. In summary, results indicate that occludin deficiency increases susceptibility to CEF‐induced colonic barrier disruption and liver damage.

Homodimers of the Claudin‐2 Polymerize to Tight Junctions of Canine Kidney Epithelial Cells

Claudin‐2 (CLDN‐2) is a member of a family of 27 tight junction transmembrane proteins. Claudins regulate paracellular ion transport in mammalian epithelial cells by forming either a barrier or a size‐ and charge‐selective pore. CLDN‐2 forms a high‐conductance cation pore, and controls sodium ion permeability. Little is known about the structural basis for ion selectivity of claudins. Their molecular organization into multiprotein complexes at tight junctions is also poorly understood. Recent biochemical studies have suggested that CLDN‐2 can form stable dimers in Madin‐Darby canine kidney (MDCK) II cells, while our mutagenesis studies indicate that CLDN‐2 pores exist as high order multimers. In the current work, we overexpressed tandem homodimers of CLDN‐2 in high‐resistance MDCK I TetOff cells. By Western blotting, cells exhibited inducible expression of CLDN‐2 dimers (~33 kDa) in the absence of doxycycline (−dox), which was almost completely suppressed by doxycycline. By immunofluorescence, the dimeric species of CLDN‐2 were colocalized with ZO‐1 at the apical junctional complex, with some additional intracellular staining at perinuclear area. The transepithelial resistance of cells overexpressing dimers of CLDN‐2 (−dox) was 1.5–2 times lower than in the presence of doxycycline. Our results indicate that tandem dimers of CLDN‐2 polymerize to tight junctions and function normally as paracellular pores suggesting that the native stoichiometry of CLDN‐2 is a multiple of two subunits. Our study may help to understand how claudins are organized into oligomers at tight junctions. This study is supported by NIH grant U01‐GM094627

Peptide-enhanced oral delivery of therapeutic peptides and proteins

Systemic therapy upon oral delivery of biologics, such as peptide and protein drugs is limited due to their large molecular size, their low enzymatic stability and their inability to cross the intestinal epithelium. Ways to overcome the epithelial barrier include the use of peptide-based excipients such as the cell penetrating peptides (CPPs) and the tight junction modulating peptides (TJMPs), which are able to translocate across the cellular membranes in a non-disruptive way or reversibly modulate the integrity of intercellular tight junctions (TJs), respectively. However, because of the harsh environment throughout the gastrointestinal (GI) tract, chemical stability is an inherent challenge when employing amino acid-based excipients for oral delivery, and multiple approaches have been investigated to improve this. The exact mechanisms of transepithelial translocation are discussed, and it is believed that CPP-mediated translocation involves transcytosis and/or direct translocation through the epithelial cells; whereas TJMP-mediated translocation is dependent on interaction with transmembrane or peripheral TJ proteins. This review focuses on the CPPs and the TJMPs currently employed as excipients for oral delivery of peptide and protein drugs highlighting recent studies and the most promising compounds from these classes of peptide excipients.

Claudin Heterogeneity and Control of Lung Tight Junctions

Lung epithelial cells interconnected by tight junctions provide a barrier to the free diffusion of solutes into airspaces. Transmembrane tight junction proteins known as claudins are essential for epithelial barrier function. Claudins are regulated through interactions with each other that are coordinated with other transmembrane tight junction proteins and cytosolic scaffold proteins. Of the 14 claudins expressed by the alveolar epithelium, claudin-3, claudin-4, and claudin-18 are the most prominent; each confers unique properties to alveolar barrier function. In particular, a protective role for claudin-4 in preventing lung injury has emerged. By contrast, lung diseases that affect claudin expression and impair barrier function, including alcoholic lung syndrome and sepsis, prime the lung for pulmonary edema. Thus, approaches to restore and/or augment lung claudin expression provide potential targets for promoting healthy barrier function.

Relocalization of Junctional Adhesion Molecule A during Inflammatory Stimulation of Brain Endothelial Cells

Junctional adhesion molecule A (JAM-A) is a unique tight junction (TJ) transmembrane protein that under basal conditions maintains endothelial cell-cell interactions but under inflammatory conditions acts as a leukocyte adhesion molecule. This study investigates the fate of JAM-A during inflammatory TJ complex remodeling and paracellular route formation in brain endothelial cells. The chemokine (C-C motif) ligand 2 (CCL2) induced JAM-A redistribution from the interendothelial cell area to the apical surface, where JAM-A played a role as a leukocyte adhesion molecule participating in transendothelial cell migration of neutrophils and monocytes. JAM-A redistribution was associated with internalization via macropinocytosis during paracellular route opening. A tracer study with dextran-Texas Red indicated that internalization occurred within a short time period (~10 min) by dextran-positive vesicles and then became sorted to dextran-positive/Rab34-positive/Rab5-positive vesicles and then Rab4-positive endosomes. By ~20 min, most internalized JAM-A moved to the brain endothelial cell apical membrane. Treatment with a macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, or Rab5/Rab4 depletion with small interfering RNA oligonucleotides prevented JAM-A relocalization, suggesting that macropinocytosis and recycling to the membrane surface occur during JAM-A redistribution. Analysis of the signaling pathways indicated involvement of RhoA and Rho kinase in JAM-A relocalization. These data provide new insights into the molecular and cellular mechanisms involved in blood-brain barrier remodeling during inflammation.

ZO-1 Regulates Erk, Smad1/5/8, Smad2, and RhoA Activities to Modulate Self-Renewal and Differentiation of Mouse Embryonic Stem Cells

ZO-1/Tjp1 is a cytosolic adaptor that links tight junction (TJ) transmembrane proteins to the actin cytoskeleton and has also been implicated in regulating cell proliferation and differentiation by interacting with transcriptional regulators and signaling proteins. To explore possible roles for ZO-1 in mouse embryonic stem cells (mESCs), we inactivated the ZO-1 locus by homologous recombination. The lack of ZO-1 was found to affect mESC self-renewal and differentiation in the presence of leukemia-inhibiting factor (LIF) and Bmp4 or following removal of the growth factors. Our data suggest that ZO-1 suppresses Stat3 and Smad1/5/8 activities and sustains extracellular-signal-regulated kinase (Erk) activity to promote mESC differentiation. Interestingly, Smad2, critical for human but not mESC self-renewal, was hyperactivated in ZO-1(-/-) mESCs and RhoA protein levels were concomitantly enhanced, suggesting attenuation of the noncanonical transforming growth factor β (Tgfβ)/Activin/Nodal pathway that mediates ubiquitination and degradation of RhoA via the TJ proteins Occludin, Par6, and Smurf1 and activation of the canonical Smad2-dependent pathway. Furthermore, Bmp4-induced differentiation of mESCs in the absence of LIF was suppressed in ZO-1(-/-) mESCs, but differentiation down the neural or cardiac lineages was not disturbed. These findings reveal novel roles for ZO-1 in mESC self-renewal, pluripotency, and differentiation by influencing several signaling networks that regulate these processes. Possible implications for the differing relevance of Smad2 in mESC and human ESC self-renewal and how ZO-1 may connect to the different pathways are discussed.

Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-α, IL-4 and IFN-γ to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as GT values; (d) epidermal growth factor receptor (EGFR)-dependent mitogen-activated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-α (47±3%), IL-4 (43±1%) and INF-γ (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. GT values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm2) × 1000 in control cultures and in those exposed to TNF-α, IFN-γ and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.

The tight junction protein claudin-1 influences cranial neural crest cell emigration

The neural crest is a population of migratory cells that follows specific pathways during development, eventually differentiating to form parts of the face, heart, and peripheral nervous system, the latter of which includes contributions from placodal cells derived from the ectoderm. Stationary, premigratory neural crest cells acquire the capacity to migrate by undergoing an epithelial-to-mesenchymal transition that facilitates their emigration from the dorsal neural tube. This emigration involves, in part, the dismantling of cell-cell junctions, including apically localized tight junctions in the neuroepithelium. In this study, we have characterized the role of the transmembrane tight junction protein claudin-1 during neural crest and placode ontogeny. Our data indicate that claudin-1 is highly expressed in the developing neuroepithelium but is down-regulated in migratory neural crest cells, although expression persists in the ectoderm from which the placode cells arise. Depletion or overexpression of claudin-1 augments or reduces neural crest cell emigration, respectively, but does not impact the development of several cranial placodes. Taken together, our results reveal a novel function for a tight junction protein in the formation of migratory cranial neural crest cells in the developing vertebrate embryo.

A Peptidomimetic Tight Junction Modulator To Improve Regional Analgesia

The paracellular flux of solutes through tissue barriers is limited by transmembrane tight junction proteins. Within the family of tight junction proteins, claudin-1 seems to be a key protein for tightness formation and integrity. In the peripheral nervous system, the nerve fibers are surrounded with a barrier formed by the perineurium which expresses claudin-1. To enhance the access of hydrophilic pharmaceutical agents via the paracellular route, a claudin-1 specific modulator was developed. For this purpose, we designed and investigated the claudin-1 derived peptide C1C2. It transiently increased the paracellular permeability for ions and high and low molecular weight compounds through a cellular barrier model. Structural studies revealed a β-sheet potential for the functionality of the peptide. Perineurial injection of C1C2 in rats facilitated the effect of hydrophilic antinociceptive agents and raised mechanical nociceptive thresholds. The mechanism is related to the internalization of C1C2 and to a vesicle-like distribution within the cells. The peptide mainly colocalized with intracellular claudin-1. C1C2 decreased membrane-localized claudin-1 of cells in culture and in vivo in the perineurium of rats after perineurial injection. In conclusion, a novel tool was developed to improve the delivery of pharmaceutical agents through the perineurial barrier by transient modulation of claudin-1.

Opening of Epithelial Tight Junctions and Enhancement of Paracellular Permeation by Chitosan: Microscopic, Ultrastructural, and Computed-Tomographic Observations

This study investigates the effects of chitosan (CS) on the opening of epithelial tight junctions (TJs) and paracellular transport at microscopic, ultrastructural, and computed-tomographic levels in Caco-2 cell monolayers and animal models. Using immunofluorescence staining, CS treatment was observed to be associated with the translocation of JAM-1 (a trans-membrane TJ protein), resulting in the disruption of TJs; the removal of CS was accompanied by the recovery of JAM-1. Ultrastructural observations by TEM reveal that CS treatment slightly opened the apical intercellular space, allowing lanthanum (an electron-dense tracer) to stain the intercellular surface immediately beneath the TJs, suggesting the opening of TJs. Following the removal of CS, the TJs were completely recovered. Similar microscopic and ultrastructural findings were obtained in animal studies. CS nanoparticles were prepared as an insulin carrier. The in vivo fluorescence-microscopic results demonstrate that insulin could be absorbed into the systemic circulation, while most CS was retained in the microvilli scaffolds. These observations were verified in a biodistribution study following the oral administration of isotope-labeled nanoparticles by single-photon emission computed tomography. Above results reveal that CS is a safe permeation enhancer and is an effective carrier for oral protein delivery.

Unique Cell Type-Specific Junctional Complexes in Vascular Endothelium of Human and Rat Liver Sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

Effect of Inhibiting Interleukin‐1β with Neutralizing Antibody on Tight Junction Protein Expression after Brain Ischemia in Ovine Fetus

We tested the hypothesis that inhibiting interleukin-1β (IL-1β) with neutralizing monoclonal antibody (mAb) alters tight junction (TJ) protein expression after brain ischemia-reperfusion (I/R) in the ovine fetus. Ovine fetuses at 85% gestation were studied after 30 min of ischemia and 24 h of reperfusion. Ovine IL-1β mAb has been generated, and showed high sensitivity, specificity, and neutralizing effects to IL-1β protein in vitro. Intravenous placebo (PL) or IL-1β mAb (5 mg/kg) infusions were given to the fetuses 15 min and 4 h after brain ischemia. Occludin (Occl), claudin-1 (CL-1), and CL-5 protein expression were determined by Western immunoblot in non-ischemic (sham, n=5), I/R+PL (n=6), and I/R+IL-1β mAb-treated (n=7) groups in the cerebral cortex, caudate nucleus, cerebellum, and medulla. Results are expressed as ratio to an internal control protein. In the medulla, Occl expression decreased and CL-5 expression increased after I/R+IL-1β mAb treatment compared to the sham group (Fig., M ± SD, P<0.05), but CL-1 expression did not change. Similar patterns of TJ expression were not observed in the cerebral cortex, cerebellum or caudate nucleus (data not shown). Treatment with IL-1β mAb affects I/R-related transmembrane TJ protein expression in some, but not all brain regions studied. Alterations in BBB function after I/R and IL-1β mAb treatment may not be directly regulated by changes in transmembrane TJ proteins in the fetus. NIH-HD-057100