Role In Tight Junction Formation Research Articles

Tim-3 promotes tube formation and decreases tight junction formation in vascular endothelial cells.

As a negative immune checkpoint molecule, T-cell immunoglobulin domain and mucin domain containing molecule-3 (Tim-3) has been found to serve a crucial role in immune escape and tumour progression. Previous studies have reported that Tim-3 is important to endothelial cells and it has also been demonstrated to be involved in numerous types of human diseases, including melanoma, lymphoma, rickettsial infection and atherosclerosis; however, its exact mechanism of action remains largely unknown. In the present study, Tim-3 was overexpressed in vascular endothelial human lung microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs), and in vitro assays were used to determine that Tim-3 promoted cell proliferation, migration, invasion and tube formation through activating cyclin D1 (CCND1), Ras homolog gene family member A and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). Additionally, Tim-3 decreased tight junction (TJ) formation and the transepithelial resistance (TER) of endothelial cells by decreasing the expression levels of TJ protein 2, Occludin and claudin 1 (CLND1). In conclusion, these findings suggested that Tim-3 may exert a positive role in angiogenesis and a negative role in TJ formation in vascular endothelial cells, which may provide novel strategies for the treatment of Tim-3-associated diseases.

Moderately Neutralizing Epitopes in Nonfunctional Regions Dominate the Antibody Response to Plasmodium falciparum EBA-140.

Plasmodium falciparum erythrocyte-binding antigen 140 (EBA-140) plays a role in tight junction formation during parasite invasion of red blood cells and is a potential vaccine candidate for malaria. Individuals in areas where malaria is endemic possess EBA-140-specific antibodies, and individuals with high antibody titers to this protein have a lower rate of reinfection by parasites. The red blood cell binding segment of EBA-140 is comprised of two Duffy-binding-like domains, called F1 and F2, that together create region II. The sialic acid-binding pocket of F1 is essential for binding, whereas the sialic acid-binding pocket in F2 appears dispensable. Here, we show that immunization of mice with the complete region II results in poorly neutralizing antibodies. In contrast, immunization of mice with the functionally relevant F1 domain of region II results in antibodies that confer a 2-fold increase in parasite neutralization compared to that of the F2 domain. Epitope mapping of diverse F1 and F2 monoclonal antibodies revealed that the functionally relevant F1 sialic acid-binding pocket is a privileged site inaccessible to antibodies, that the F2 sialic acid-binding pocket contains a nonneutralizing epitope, and that two additional epitopes reside in F1 on the opposite face from the sialic acid-binding pocket. These studies indicate that focusing the immune response to the functionally important F1 sialic acid binding pocket improves the protective immune response of EBA-140. These results have implications for improving future vaccine designs and emphasize the importance of structural vaccinology for malaria.

A look at tricellulin and its role in tight junction formation and maintenance

Tight junctions are elaborate networks of transmembrane and cytosolic proteins that regulate epithelial permeability. Tricellulin was the first tight junction protein found at tricellular tight junctions, the specialized structures occurring where three cells meet together. Here, we summarize the current knowledge about tricellulin (marvelD2), a MARVEL domain protein. We address tricellulin location at tricellular junctions, and establish the comparison with the other members of the MARVEL family, occludin (marvelD1) and marvelD3. The structure of tricellulin and its membrane folding, as well as the proposed molecular interactions of tricellulin with other tight junction proteins, together with the interplay between those proteins are also discussed. In addition, we address the role of tricellulin in barrier properties, discriminating the involvement of the protein in paracellular permeability at bicellular and at tricellular tight junctions. Moreover, the key importance of the protein for hearing is highlighted based on the fact that mutations in TRIC, the human tricellulin gene, lead to deafness. Furthermore, this review points to some of the aspects that still deserve clarification for a better understanding of the biology of tight junctions in general and of tricellulin in particular.

Junctional Adhesion Molecule-C Is a Soluble Mediator of Angiogenesis

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed by endothelial cells (ECs) that plays a role in tight junction formation, leukocyte adhesion, and transendothelial migration. In the current study, we investigated whether JAM-C is found in soluble form and whether soluble JAM-C (sJAM-C) mediates angiogenesis. We found that JAM-C is present in soluble form in normal serum and elevated in rheumatoid arthritis (RA) serum. The concentration of sJAM-C is also elevated locally in RA synovial fluid compared with RA serum or osteoarthritis synovial fluid. sJAM-C was also present in the culture supernatant of human microvascular ECs (HMVECs) and immortalized human dermal microvascular ECs, and its concentration was increased following cytokine stimulation. In addition, sJAM-C cleavage from the cell surface was mediated in part by a disintegrin and metalloproteinases 10 and 17. In functional assays, sJAM-C was both chemotactic and chemokinetic for HMVECs and induced HMVEC tube formation on Matrigel in vitro. Neutralizing anti-JAM-C Abs inhibited RA synovial fluid-induced HMVEC chemotaxis and sJAM-C-induced HMVEC tube formation on Matrigel. sJAM-C also induced angiogenesis in vivo in the Matrigel plug and sponge granuloma models. Moreover, sJAM-C-mediated HMVEC chemotaxis was dependent on Src, p38, and PI3K. Our results show that JAM-C exists in soluble form and suggest that modulation of sJAM-C may provide a novel route for controlling pathological angiogenesis.

The Relationship of JAM2 Polymorphisms and Graft-Versus-Host-Disease (GVHD) after Allogeneic Stem Cell Transplantation

Introduction: The junctional adhesion molecule 2 (JAM2) gene plays a key role in tight junction formation in epithelial and endothelial cell. We studied JAM2 polymorphism in GVHD and non-GVHD patients after allogeneic stem cell transplantation.Methods: We tested 3 sites of JAM2 gene - rs2276220 (G/A), rs963103 (T/C), rs974680 (G/A) - and studied the possible link between the JAM2 polymorphism and the risk of GVHD. We studied 251 patients who had treated with allogeneic stem cell transplantation. The three-SNP block study was performed with rs2276220-rs963103-rs974680 haplotypes. The JAM2 gene was screened for polymorphisms by SNaPShot method. We used SAS version 8.1 (SAS Institute Inc., Cary, NC, USA) for statistic analysis. The odds ratio and p-value was adjusted with multiple logistic regression analysis about age, gender, stem cell source, disease status at the time of transplantation, and conditioning regimen.Results: The minor allele C of rs963103 (T/C) was dominant in patients with acute GVHD (Odds ratio (OR) 2.918, 95% confidence interval (CI) 1.178~7.233; p=0.0207). This phenomenon was distinct in gut GVHD (OR 1.684, 1.019~2.784; p=0.0419). The minor allele A of rs2276220 (G/A) was dominant in patients with infection after allogeneic stem cell transplantation (OR 6.058, 1.266~28.979; p=0.0241). The overall survival (OS) was associated with rs2276220 (hazard ratio (HR) 0.887, 95% CI 0.633~1.242; p=0.0346) and rs974680 (HR 0.969, 0.763~1.23; p=0.0193). The progression-free survival (PFS) was associated with rs2276220 (HR 0.761, 0.544~1.064; p=0.0095) and rs974680 (HR 0.551, 0.274~1.108; p=0.0382). In three-SNP block study which was performed with rs2276220- rs963103-rs974680 haplotypes, ATA (HR 0.844, 0.645~1.105; p=0.0016) and GTA (HR 5.568, 1.686~18.384; p=0.0048) was associated with OS. ATA (HR 0.796, 0.559~1.133; p=0.0039) and GTG (HR 1.339, 0.834~2.149; p=0.0353) was associated with PFS.Conclusions: The JAM2 gene polymorphism rs963103 was associated with acute GVHD especially with gut GVHD after allogeneic stem cell transplantation; with higher risk in carriers of the C allele. The JAM2 gene polymorphism rs2276220 was associated with infection, OS, and PFS after allogeneic stem cell transplantation; with higher risk in carriers of the A allele.

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 Ca 2+ medium, the application of pan-protein kinase C inhibitor GF-109203X blocked the formation of tight junctions induced by protein kinase C agonist diacyglycerol. Gö6976, inhibitor of conventional protein kinase C, promoted the formation of tight junctions and occludin phosphorylation in cells cultivated in low Ca 2+ medium and attenuated the disruption of tight junction complex induced by the switch to low Ca 2+ medium. In addition, Gö6976 accelerated the occludin phosphorylation and the formation of tight junction barrier during assembly of tight junctions induced by Ca 2+ 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 Ca 2+ 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.

Claudin expression in gastric adenocarcinomas: a tissue microarray study with prognostic correlation

The claudins comprise a multigene family of integral membrane proteins, which play a major role in tight junction formation. Aberrations in the expression of certain claudins have been described in a number of malignancies. Our aims were to determine the expression pattern of claudins 1, 3, and 4 as well as ZO-1 in a large series of US patients with gastric cancer and to correlate expression with clinicopathologic and prognostic variables. Tissue microarrays were created from paraffinized samples from 146 patients with distal gastric adenocarcinomas (61 intestinal and 85 diffuse or mixed subtypes). In addition, cores of normal mucosa and intestinal metaplasia were taken from most cases. The microarrays were stained for claudins 1, 3, and 4 and ZO-1, and the intensity of staining was determined using a 3-point scale. Moderate claudin 1 and ZO-1 membranous staining were present, whereas only focal weak claudin 3 and 4 membranous staining was present in normal gastric epithelium. Moderate to strong staining of claudins 1, 3, 4, and ZO-1 was detected in 74%, 48%, 62%, and 74% of the intestinal but in only 46%, 24%, 45%, and 36% of the diffuse subtype of adenocarcinomas (P < .05). Cox multivariate analysis revealed that tumor stage, diffuse subtype, and moderate to strong claudin 4 staining were associated with decreased survival (P < .02). In conclusion, claudins 1, 3, and 4 and ZO-1 are strongly expressed in most gastric intestinal-type adenocarcinomas but less frequently in diffuse gastric cancers. The up-regulation of claudin expression during gastric carcinogenesis suggests their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.

Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells

The formation and maintenance of tight junctions is essential for the development of epithelial cell polarity. Recently, a number of conserved polarity-regulating proteins have been shown to localize to epithelial tight junctions, and to play a role in the regulation of tight junction formation. The Crumbs3/PALS1/PATJ protein complex localizes at epithelial tight junctions and interacts with the polarity-regulating protein complex of Par6/Par3/aPKC. Overexpression of Crumbs3 in MDCKII cells leads to a delay in tight junction formation in these cells, suggesting a role in the regulation of tight junction development. Here we report new evidence that Crumbs3 indeed plays an essential role in tight junction formation. Mammary MCF10A cells express little endogenous Crumbs3 and fail to form tight junctions when grown under standard tissue culture conditions. The staining pattern of ZO-1, a tight junction marker, is fragmented, and other tight junction markers show either fragmented junctional expression or diffuse cytoplasmic staining. Expression of exogenous Crumbs3 induces the formation of tight junction structures marked by smooth, continuous ZO-1 staining at apical cell-cell junctions. A number of other tight junction markers, including claudin-1 and occludin, are also recruited to these junctions. Analysis by transmission electron microscopy and measurements of the transepithelial electrical resistance confirm that these structures are functional tight junctions. Mutations in either the Crumbs3 PDZ binding motif or the putative FERM binding motif lead to defects in the ability of Crumbs3 to promote tight junction development. Our results suggest that Crumbs3 plays an important role in epithelial tight junction formation, and also provide the first known functional role for the mammalian Crumbs FERM binding domain.