Claudin-2 upregulation enhances intestinal permeability, immune activation, dysbiosis, and mortality in sepsis.

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients.

Claudin-2 protects against colitis-associated cancer by promoting colitis-associated mucosal healing.

Patients with inflammatory bowel disease (IBD) are susceptible to colitis-associated cancer (CAC). Chronic inflammation promotes the risk for CAC. In contrast, mucosal healing predicts improved prognosis in IBD and reduced risk of CAC. However, the molecular integration among colitis, mucosal healing, and CAC remains poorly understood. Claudin-2 (CLDN2) expression is upregulated in IBD; however, its role in CAC is not known. The current study was undertaken to examine the role for CLDN2 in CAC. The AOM/DSS-induced CAC model was used with WT and CLDN2-modified mice. High-throughput expression analyses, murine models of colitis/recovery, chronic colitis, ex vivo crypt culture, and pharmacological manipulations were employed in order to increase our mechanistic understanding. The Cldn2KO mice showed significant inhibition of CAC despite severe colitis compared with WT littermates. Cldn2 loss also resulted in impaired recovery from colitis and increased injury when mice were subjected to intestinal injury by other methods. Mechanistic studies demonstrated a possibly novel role of CLDN2 in promotion of mucosal healing downstream of EGFR signaling and by regulation of Survivin expression. An upregulated CLDN2 expression protected from CAC and associated positively with crypt regeneration and Survivin expression in patients with IBD. We demonstrate a potentially novel role of CLDN2 in promotion of mucosal healing in patients with IBD and thus regulation of vulnerability to colitis severity and CAC, which can be exploited for improved clinical management.

Knock Out of CGN and CGNL1 in MDCK Cells Affects Claudin-2 but Has a Minor Impact on Tight Junction Barrier Function.

Cingulin (CGN) and paracingulin (CGNL1) are cytoplasmic proteins of tight junctions (TJs), where they play a role in tethering ZO-1 to the actomyosin and microtubule cytoskeletons. The role of CGN and CGNL1 in the barrier function of epithelia is not completely understood. Here, we analyzed the effect of the knock out (KO) of either CGN or CGNL1 or both on the paracellular permeability of monolayers of kidney epithelial (MDCK) cells. KO cells displayed a modest but significant increase in the transepithelial resistance (TER) of monolayers both in the steady state and during junction assembly by the calcium switch, whereas the permeability of the monolayers to 3 kDa dextran was not affected. The permeability to sodium was slightly but significantly decreased in KO cells. This phenotype correlated with slightly increased mRNA levels of claudin-2, slightly decreased protein levels of claudin-2, and reduced junctional accumulation of claudin-2, which was rescued by CGN or CGNL1 but not by ZO-1 overexpression. These results confirm previous observations indicating that CGN and CGNL1 are dispensable for the barrier function of epithelia and suggest that the increase in the TER in clonal lines of MDCK cells KO for CGN, CGNL1, or both is due to reduced protein expression and junctional accumulation of the sodium pore-forming claudin, claudin-2.

Kidney stones, hypercalciuria, and recent insights into proximal tubule calcium reabsorption.

PURPOSE OF REVIEW: Most kidney stones are composed of calcium, and the greatest risk factor for kidney stone formation is hypercalciuria. Patients who form kidney stones often have reduced calcium reabsorption from the proximal tubule, and increasing this reabsorption is a goal of some dietary and pharmacological treatment strategies to prevent kidney stone recurrence. However, until recently, little was known about the molecular mechanism that mediates calcium reabsorption from the proximal tubule. This review summarizes newly uncovered key insights and discusses how they may inform the treatment of kidney stone formers. RECENT FINDINGS: Studies examining claudin-2 and claudin-12 single and double knockout mice, combined with cell culture models, support complementary independent roles for these tight junction proteins in contributing paracellular calcium permeability to the proximal tubule. Moreover, a family with a coding variation in claudin-2 causing hypercalciuria and kidney stones have been reported, and reanalysis of Genome Wide Association Study (GWAS) data demonstrates an association between noncoding variations in CLDN2 and kidney stone formation. SUMMARY: The current work begins to delineate the molecular mechanisms whereby calcium is reabsorbed from the proximal tubule and suggests a role for altered claudin-2 mediated calcium reabsorption in the pathogenesis of hypercalciuria and kidney stone formation.

Loss of NKCC1 function increases epithelial tight junction permeability by upregulating claudin-2 expression.

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2, the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

SOX9 in Keratinocytes Regulates Claudin 2 Transcription during Skin Aging.

In order to prove that SOX9 in keratinocytes regulates claudin 2 transcription during skin aging, the skin of 8-week-old and 24-month-old mice is sequenced to obtain a differentially expressed gene SOX9. The gene is mainly expressed in keratinocytes, and it increases first and then decreases from newborn to aging. Six core sequences of SOX9 and claudin 2 are predicted from Jaspar. The double Luciferase Report shows that overexpression of SOX9 induces the full-length promoter of claudin 2 significantly and has no effect on the mutation and cleavage plasmid without SOX9 response. Claudin 2 is consistent with SOX9 in the skin of mice of different ages, and SOX9 is strongly positively correlated with claudin 2. Finally, overexpression of SOX9 and claudin 2 will delay PM2.5-induced keratinocyte senescence. The silencing of claudin 2 leads to the loss of SOX9 function. It is clearly evident that SOX9 can affect the transcription of claudin 2, which increases first and then decreases in the process of mice from newborn to aging. SOX9 inhibits proinflammatory mediators, increases antioxidant capacity, and restores keratin differentiation. It can effectively prevent melanin deposition and delay aging.

Increase in Anticancer Drug-Induced Toxicity by Fisetin in Lung Adenocarcinoma A549 Spheroid Cells Mediated by the Reduction of Claudin-2 Expression.

Claudin-2 (CLDN2), a component of tight junction, is involved in the reduction of anticancer drug-induced toxicity in spheroids of A549 cells derived from human lung adenocarcinoma. Fisetin, a dietary flavonoid, inhibits cancer cell growth, but its effect on chemosensitivity in spheroids is unknown. Here, we found that fisetin (20 µM) decreases the protein level of CLDN2 to 22.3%. Therefore, the expression mechanisms were investigated by real-time polymerase chain reaction and Western blotting. Spheroids were formed in round-bottom plates, and anticancer drug-induced toxicity was measured by ATP content. Fisetin decreased the phosphorylated-Akt level, and CLDN2 expression was decreased by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting the inhibition of PI3K/Akt signal is involved in the reduction of CLDN2 expression. Hypoxia level, one of the hallmarks of tumor microenvironment, was reduced by fisetin. Although fisetin did not change hypoxia inducible factor-1α level, it decreased the protein level of nuclear factor erythroid 2-related factor 2, a stress response factor, by 25.4% in the spheroids. The toxicity of doxorubicin (20 µM) was enhanced by fisetin from 62.8% to 40.9%, which was rescued by CLDN2 overexpression (51.7%). These results suggest that fisetin can enhance anticancer drug toxicity in A549 spheroids mediated by the reduction of CLDN2 expression.

The Increase of miR-195-5p Reduces Intestinal Permeability in Ulcerative Colitis, Modulating Tight Junctions' Expression.

Defects in the intestinal epithelial barrier functions characterize inflammatory conditions such as Inflammatory Bowel Disease (IBD). Overexpression of pro-inflammatory cytokines such as TNF-α, IL-1B, IL-6 and INF-γ trigger epithelial damage. These cytokines are due to upregulation of claudin-2 (CLDN2) that form a pore channel, resulting in redistribution of TJs and an alteration of barrier permeability. Recently, we demonstrated that miR-195-5p is able to regulate CLDN2 and indirectly also CLDN1 in intestinal epithelial cells. Now, we aimed to investigate the modulation of miR-195-5p on the expression of CLDN2 and other TJs under inflammatory conditions induced by TNF-α. We demonstrated that miR-195-5p also modulated the expression of CLDN2 levels after stimulation with TNF-α. In addition, we discovered the role of miR-195-5p in the integrity of the intestinal barrier and in promoting the restoration of the intestinal epithelial. Moreover, we established that replacement of miR-195-5p attenuated the colonic inflammatory response in DSS-induced, colitis and it reduced colonic permeability. In conclusion, our data revealed the role of miR-195-5p in intestinal inflammation in ulcerative colitis, suggesting a potential pharmacological target for new therapeutic approaches.

MiRNA-182-5p aggravates experimental ulcerative colitis via sponging Claudin-2.

Tight junction proteins play crucial roles in maintaining the integrity of intestinal mucosal barrier. MiRNA-182-5p is capable of targeting claudin-2 which is one of the vital tight junction proteins and the effect and mechanism of miRNA-182-5p was explored here in the DSS-induced colitis model. The pathological conditions were evaluated via hematoxylin and eosin staining. The gene expression level was assessed via PCR. Quantitative immunohistochemistry analysis was performed for the measurement of claudin-2. microRNA.org online tool was used for target gene prediction. Luciferase reporter assay and RNA pull-down assay were performed to detect the target of miRNA-182-5p. The inflammatory and oxidative stress level were measured using corresponding kits. MiRNA-182-5p was highly expressed in colitis model and miRNA-182-5p inhibitor exerted protective effects on colitis induced by DSS in mice. The protective effects includded improvement of pathological changes, increases in anti-inflammation and anti-oxidative genes, and up-regulation of TGF-ß1. Claudin-2 mRNA was predicted as the target of miRNA-182-5p, which was validated via luciferase reporter assay and RNA pull-down assay. Claudin-2 overexpression was found in miRNA-182-5p inhibitor group. Consistent with the role of miRNA-182-5p, claudin-2 overexpression also exerted protective effects on DSS-induced colitis in mice. Inhibition of miRNA-182-5p exerted protective effects on colitis via targeting and upregulating claudin-2. The findings in study provide a new therapeutic strategy for colitis treatment and lay the foundation for future study.

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1-deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.

Down-Regulation of Claudin-2 Expression by Cyanidin-3-Glucoside Enhances Sensitivity to Anticancer Drugs in the Spheroid of Human Lung Adenocarcinoma A549 Cells.

Claudin-2 (CLDN2), an integral membrane protein located at tight junctions, is abnormally expressed in human lung adenocarcinoma tissues, and is linked to drug resistance in human lung adenocarcinoma A549 cells. CLDN2 may be a target for the prevention of lung adenocarcinoma, but there are few compounds which can reduce CLDN2 expression. We found that cyanidin-3-glucoside (C3G), the anthocyanin with two hydroxyl groups on the B-ring, and cyanidin significantly reduce the protein level of CLDN2 in A549 cells. In contrast, pelargonidin-3-glucoside (P3G), the anthocyanin with one hydroxyl group on the B-ring, had no effect. These results suggest that cyanidin and the hydroxyl group at the 3-position on the B-ring play an important role in the reduction of CLDN2 expression. The phosphorylation of Akt, an activator of CLDN2 expression at the transcriptional level, was inhibited by C3G, but not by P3G. The endocytosis and lysosomal degradation are suggested to be involved in the C3G-induced decrease in CLDN2 protein expression. C3G increased the phosphorylation of p38 and the p38 inhibitor SB203580 rescued the C3G-induced decrease in CLDN2 expression. In addition, SB203580 rescued the protein stability of CLDN2. C3G may reduce CLDN2 expression at the transcriptional and post-translational steps mediated by inhibiting Akt and activating p38, respectively. C3G enhanced the accumulation and cytotoxicity of doxorubicin (DXR) in the spheroid models. The percentages of apoptotic and necrotic cells induced by DXR were increased by C3G. Our data suggest that C3G-rich foods can prevent the chemoresistance of lung adenocarcinoma A549 cells through the reduction of CLDN2 expression.

Claudin-2 pore causes leak that breaches the dam in intestinal inflammation.

The tight junction protein claudin-2 is upregulated in inflammatory bowel disease, and yet its deficit worsens infectious and chemical colitis. In this issue of the JCI, Raju and Shashikanth et al. examined the contribution of claudin-2 to immune-mediated colitis. The authors used transgenic mouse models to show that claudin-2 deficiency attenuated colitis progression as well as a leak barrier defect, albeit at the risk of intestinal obstruction. Further, inhibition of claudin-2 by targeting casein kinase 2 (CK2) also ameliorated colitis. The findings reveal unsuspected links between the pore and leak pathways of intestinal permeability and immune responses leading to colitis. They additionally suggest potential targets for therapeutic intervention in inflammatory bowel disease.

Kaempferide Enhances Chemosensitivity of Human Lung Adenocarcinoma A549 Cells Mediated by the Decrease in Phosphorylation of Akt and Claudin-2 Expression.

Claudins (CLDNs) play crucial roles in the formation of tight junctions. We have reported that abnormal expression of CLDN2 confers chemoresistance in the spheroids of human lung adenocarcinoma A549 cells. A food composition, which can reduce CLDN2 expression, may function to prevent the malignant progression. Here, we found that ethanol extract of Brazilian green propolis (EBGP) and kaempferide, a major component of EBGP, decrease CLDN2 expression. In the two-dimensional culture model, EBGP decreased the tight junctional localization of CLDN2 without affecting that of zonula occludens-1, an adaptor protein, and enhanced paracellular permeability to doxorubicin, a cytotoxic anticancer drug. EBGP reduced hypoxic stress, and enhanced the accumulation and sensitivity of doxorubicin in the spheroid of A549 cells. Kaempferide dose-dependently decreased CLDN2 expression, although dihydrokaempferide and pinocembrin did not. The phosphorylation of Akt, a regulatory factor of CLDN2 expression, was inhibited by kaempferide but not by dihydrokaempferide. The 2,3-double bond in the C ring may be important to inhibit Akt. Kaempferide decreased the mRNA level and promoter activity of CLDN2, indicating that it inhibits the transcription of CLDN2. In accordance with EBGP, kaempferide decreased the tight junctional localization of CLDN2 and increased a paracellular permeability to doxorubicin, suggesting that it diminished the paracellular barrier to small molecules. In addition, kaempferide reduced hypoxic stress, and enhanced the accumulation and sensitivity of doxorubicin in the spheroids. In contrast, dihydrokaempferide did not improve the sensitivity to doxorubicin. Further study is needed using an animal model, but we suggest that natural foods abundantly containing kaempferide are candidates for the prevention of the chemoresistance of lung adenocarcinoma.

Claudin-15 forms a water channel through the tight junction with distinct function compared to claudin-2.

AIM: Claudin-15 is mainly expressed in the small intestine and indirectly involved in glucose absorption. Similar to claudin-2 and -10b, claudin-15 is known to form a paracellular channel for small cations. Claudin-2, but not claudin-10b, also forms water channels. Here we experimentally tested whether claudin-15 also mediates water transport and if yes, whether water transport is Na+ -coupled, as seen for claudin-2. METHODS: MDCK C7 cells were stably transfected with claudin-15. Ion and water permeability were investigated in confluent monolayers of control and claudin-15-expressing cells. Water flux was induced by an osmotic or ionic gradient. RESULTS: Expression of claudin-15 in MDCK cells strongly increased cation permeability. The permeability ratios for monovalent cations indicated a passage of partially hydrated ions through the claudin-15 pore. Accordingly, its pore diameter was determined to be larger than that of claudin-2 and claudin-10b. Mannitol-induced water flux was elevated in claudin-15-expressing cells compared to control cells. In contrast to the Na+ -coupled water flux of claudin-2 channels, claudin-15-mediated water flux was inhibited by Na+ flux. Consequently, water flux was increased in Na+ -free solution. Likewise, Na+ flux was decreased after induction of water flux through claudin-15. CONCLUSION: Claudin-15, similar to claudin-2, forms a paracellular cation and water channel. In functional contrast to claudin-2, water and Na+ fluxes through claudin-15 inhibit each other. Claudin-15 allows Na+ to retain part of its hydration shell within the pore. This then reduces the simultaneous passage of additional water through the pore.

Protective Effect of Kaempferol on LPS-Induced Inflammation and Barrier Dysfunction in a Coculture Model of Intestinal Epithelial Cells and Intestinal Microvascular Endothelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of intestinal mucosa and submucosa, characterized by the disruption of the intestinal epithelial barrier, increased production of inflammatory mediators, and excessive tissue injury. Intestinal epithelial cells, as well as microvascular endothelial cells, play important roles in IBD. To study the potential effects of kaempferol in IBD progress, we established a novel epithelial-endothelial cells coculture model to investigate the intestinal inflammation and barrier function. Data demonstrated an obvious increased transepithelial electrical resistance (TEER) (1222 ± 60.40 Ω cm2 vs 1371 ± 38.77 Ω cm2), decreased flux of FITC (180.8 ± 20.06 µg/mL vs 136.7 ± 14.78 µg/mL), and up-regulated occludin and claudin-2 expression in Caco-2 that was specifically cocultured with endothelial cells. Meanwhile, 80 µM kaempferol alleviated the drop of TEER, the increase of FITC flux, and the overexpression of interleukin-8 (IL-8) induced by 1 µg/mL lipopolysaccharide (LPS). Additionally, kaempferol also ameliorated the LPS-induced decrease of protein expression of zonula occludens-1 (ZO-1), occludin, and claudin-2, together with the inhibited protein expressions of the phosphorylation level of NF-κB and I-κB induced by LPS. Our results suggest that kaempferol alleviates the IL-8 secretion and barrier dysfunction of the Caco-2 monolayer in the LPS-induced epithelial-endothelial coculture model via inhibiting the NF-κB signaling pathway activation.

Linear and circular CDKN2B-AS1 expression is associated with Inflammatory Bowel Disease and participates in intestinal barrier formation.

AIMS: The role of long non-coding RNA's (lncRNA) in the biology of ulcerative colitis (UC) is not well understood. We have previously detected changes in lncRNA's associated with UC. This study aims to characterize one specific lncRNA, CDKN2B-AS1 whose expression was downregulated in UC patients. MAIN METHODS: UC biopsies were used to determine the levels of linear and circular CDKN2B-AS1 relative to healthy controls. In situ hybridization was used to determine the localization of CKDN2B-AS1 in the colon. The intestinal epithelial cell line, Caco-2, was used to study the effects of shRNA mediated loss of CDKN2B-AS1. Transepithelial electrical resistance was used to measure barrier function. An RT-PCR array, immunoblots and immunohistochemistry were used to determine tight junction proteins that CDKN2B-AS1 regulates. KEY FINDINGS: CDKN2B-AS1 is transcribed into not only linear transcripts but also as circular RNA through back-splicing and both forms are decreased in IBD. CDKN2B-AS1 is expressed mainly in colonic epithelial cells. Cells with down-regulated CDKN2B-AS1 exhibited increased proliferation and no alterations in apoptosis. Targeting both the linear and circular transcripts of CDKN2B-AS1 with short hairpin RNAs enhanced barrier function. We subsequently determined that Claudin-2, a "leaky Claudin" known to decrease barrier function, was decreased in CDKN2B-AS1 knockdown cells. SIGNIFICANCE: This study identifies a novel lncRNA with both linear and circular transcripts affecting UC biology.

Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ-specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn-1, -2, or -3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including "kissing-point"-like structures representing homophilic trans-interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultrastructural analysis revealed a self-assembly of TJ strands and a cldn-specific strand morphology.-Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier-Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Afadin cooperates with Claudin-2 to promote breast cancer metastasis.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that the PDZ-binding motif of Claudin-2 is necessary for anchorage-independent growth of cancer cells and is required for liver metastasis. Several PDZ domain-containing proteins were identified that interact with the PDZ-binding motif of Claudin-2 in liver metastatic breast cancer cells, including Afadin, Arhgap21, Pdlim2, Pdlim7, Rims2, Scrib, and ZO-1. We specifically examined the role of Afadin as a potential Claudin-2-interacting partner that promotes breast cancer liver metastasis. Afadin associates with Claudin-2, an interaction that requires the PDZ-binding motif of Claudin-2. Loss of Afadin also impairs the ability of breast cancer cells to form colonies in soft agar and metastasize to the lungs or liver. Immunohistochemical analysis of Claudin-2 and/or Afadin expression in 206 metastatic breast cancer tumors revealed that high levels of both Claudin-2 and Afadin in primary tumors were associated with poor disease-specific survival, relapse-free survival, lung-specific relapse, and liver-specific relapse. Our findings indicate that signaling downstream from a Claudin-2/Afadin complex enables the efficient formation of breast cancer metastases. Moreover, combining Claudin-2 and Afadin as prognostic markers better predicts the potential of breast cancer to metastasize to soft tissues.

Homoectoine Protects Against Colitis by Preventing a Claudin Switch in Epithelial Tight Junctions.

BACKGROUND: Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins. AIM: To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice. METHODS/RESULTS: We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis. CONCLUSION: While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.

An intestinal paracellular pathway biased toward positively-charged macromolecules.

Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). While TNF-α/IFN-γ treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.