Claudin-1 Research Articles

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity

ABSTRACT The mammalian endometrium is covered by the lumenal epithelium (Le), which directly interacts with the blastocyst and plays an important role in the establishment of reciprocal crosstalk between the embryo and receptive uterus during implantation. However, the effect of the blastocyst on uterine differentiation during the window of receptivity is far from well understood. Through transcriptomic profiling of the uterine Le isolated by laser capture microdissection (LCM), it was demonstrated that global gene expression changes occurred in Le between pseudopregnant mice without embryos and pregnant mice with embryos. Some differentially expressed genes, including upregulated Areg (amphiregulin), Ihh (Indian hedgehog), Lifr (leukemia inhibitory factor receptor) and downregulated Msx1 (msh homeobox 1), Pgr (progesterone receptor), and Gata2 (GATA binding protein 2) in pregnant mice, have been reported to regulate the establishment of uterine receptivity. Besides, we found that blastocysts induced an increase in both the number and acidification of lysosome, consistent with enhanced lysosomal hydrolase activity in uterine Le. Further exploration uncovered that blastocyst-derived IGF2 was involved into the activation of epithelial STAT3 to induce lysosomal hydrolase expression, and inhibition of lysosomal function derails both uterine receptive maker gene expressions and embryo implantation. Finally, based on the proteomic data of both epithelia and the separated lysosome, it was revealed that CLDN1 (claudin 1) and MUC1 (mucin 1, transmembrane), two well-known downregulated molecules for successful implantation, are degraded by epithelial lysosome. In brief, our data demonstrated that blastocysts induced normal epithelium differentiation with lysosome activation to promote the uterine epithelial differentiation for embryo implantation. Abbreviations ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17β-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

Claudin-3 facilitates the progression and mediates the tumorigenic effects of TGF-β in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a significantly malignant and lethal brain tumor with an average survival time of less than 12months. Several researches had shown that Claudin-3 (CLDN3) is overexpressed in various cancers and might be important in their growth and spread. In this study, we used qRT-PCR, western blotting, immunohistochemistry, and immunofluorescence staining assays to investigate the expression levels of various proteins. To explore the proliferation abilities of GBM cells, we conducted the CCK-8 and EdU-DNA formation assays. Wound healing and transwell assays were used to investigate the capacities of invasion and migration of GBM cells. Additionally, we constructed an intracranial xenograft model of GBM to study the in vivo role of CLDN3. Our study devoted to investigate the function of CLDN3 in the pathogenesis and progression of GBM. Our study revealed that CLDN3 was upregulated in GBM and could stimulate tumor cell growth and epithelial-mesenchymal transition (EMT) in both laboratory and animal models. We also discovered that CLDN3 expression could be triggered by transforming growth factor-β (TGF-β) and reduced by specific inhibitors of the TGF-β signaling pathway, such as ITD-1. Further analysis revealed that increased CLDN3 levels enhanced TGF-β-induced growth and EMT in GBM cells, while reducing CLDN3 levels weakened these effects. Our study demonstrated the function of CLDN3 in facilitating GBM growth and metastasis and indicated its involvement in the tumorigenic effects of TGF-β. Developing specific inhibitors of CLDN3 might, therefore, represent a promising new approach for treating this devastating disease.

Impaired intestinal permeability in patients with multiple sclerosis.

A number of recent studies have shown that the intestinal microbiome, part of the brain-gut axis, is implicated in the pathophysiology of multiple sclerosis. An essential part of this axis, is the intestinal barrier and gastrointestinal disorders with intestinal barrier dysregulation appear to be linked to CNS demyelination, and hence involved in the etiopathogenesis of multiple sclerosis (MS). The aim of this study was to evaluate the integrity of the intestinal barrier in patients with clinically definite multiple sclerosis (CDMS) and clinically isolated syndrome (CIS) using two serum biomarkers, claudin-3 (CLDN3), a component of tight epithelial junctions, and intestinal fatty acid binding protein (I-FABP), a cytosolic protein in enterocytes. Serum levels of CLDN3 in 37 MS patients and 22 controls, and serum levels of I-FABP in 46 MS patients and 51 controls were measured using commercial ELISA kits. Complete laboratory tests excluded the presence of gluten-related disorders in all subjects. Thirty MS patients received either disease-modifying drugs (DMD), immunosuppression (IS) or corticosteroid treatment. CLDN3 levels were only significantly higher in the MS patients treated with DMD or IS compared to the control group (P=0.006). There were no differences in I-FABP serum levels between the groups. Serum CLDN3 levels did not correlate with serum I-FABP levels in CDMS, in CIS patients or controls. In multiple sclerosis patients, the intestinal epithelium may be impaired with increased permeability, but without significant enterocyte damage characterized by intracellular protein leakage. Based on our data, CLDN3 serum levels appear to assess intestinal dysfunction in MS patients but mainly in treated ones.

Claudin expression in pulmonary adenoid cystic carcinoma and mucoepidermoid carcinoma.

Background: Although the expression of tight junction protein claudins (CLDNs) is well known in common histological subtypes of lung cancer, it has not been investigated in rare lung cancers. The aim of our study was to examine the expression of different CLDNs in pulmonary salivary gland tumors. Methods: 35 rare lung cancers including pathologically confirmed 12 adenoid cystic carcinomas (ACCs) and 23 mucoepidermoid carcinomas (MECs) were collected retrospectively. Immunohistochemical (IHC) staining was performed on formalin fixed paraffin embedded (FFPE) tumor tissues, and CLDN1, -2, -3, -4, -5, -7, and -18 protein expressions were analyzed. The levels of immunopositivity were determined with H-score. Certain pathological characteristics of ACC and MEC samples (tumor grade, presence of necrosis, presence of blood vessel infiltration, and degree of lymphoid infiltration) were also analyzed. Results: CLDN overexpression was observed in both tumor types, especially in CLDN2, -7, and -18 IHC. Markedly different patterns of CLDN expression were found for ACC and MEC tumors, especially for CLDN1, -2, -4, and -7, although none of these trends remained significant after correction for multiple testing. Positive correlations between expressions of CLDN2 and -5, CLDN3 and -4, and CLDN5 and -18 were also demonstrated. Tumors of never-smokers presented lower levels of CLDN18 than tumors of current smokers (p-value: 0.003). Conclusion: This is the first study to comprehensively describe the expression of different CLDNs in lung ACC and MEC. Overexpression of certain CLDNs may pave the way for targeted anti-claudin therapy in these rare histological subtypes of lung cancer.

Daucosterol confers protection against T-2 toxin induced blood-brain barrier toxicity through the PGC-1α-mediated defensive response in vitro and in vivo

T-2 toxin is a common environmental pollutant and contaminant in food and animal feed that represents a great challenge to human and animal’ health throughout the world. Using natural compounds to prevent the detrimental effects of T-2 toxin represents an attractive strategy. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a critical regulator in various cellular processes. Recently, PGC-1α activation has been reported to confer protection against neurological injuries. We aimed to identify a potent PGC-1α activator from plants as a chemopreventive compound and to demonstrate the efficacy of the compound in attenuating T-2 toxin-induced blood-brain barrier (BBB) toxicity. We identified daucosterol, which binds directly to the 71–74 (−1100 to −1000 bp) position of the second promoter of human PGC-1α by hydrogen bonding. An in vitro and in vivo T-2 toxin induced BBB injury model revealed that this compound can protect against this injury by increasing transepithelial/transendothelial electrical resistance, reducing sodium fluorescein (NaF) infiltration and increasing the expression of tight junction–related proteins (zonula occludens-1 (ZO-1), occludin (OCLN), claudin-5 (CLDN5)) expression. In conclusion, we identified daucosterol as representing a novel of PGC-1α activators and illustrated the mechanism of specific binding site. Furthermore, we have demonstrated the feasibility of using natural compounds targeting PGC-1α as a therapeutic approach to protect humans from environmental insults that may occur daily such as lipopolysaccharide.

Association of Claudin-1 overexpression with immune exclusion in nasopharyngeal carcinoma and its potential as a novel therapeutic target.

151 Background: Claudin-1 (CLDN1) is a tight junction protein overexpressed in several cancers, where it is involved in the tumor microenvironment remodeling and stiffening of the tumor-stroma interface. This potentially prevents penetration of tumor infiltrating immune cells, in turn hindering the efficacy of immune checkpoint inhibitors (ICI). Given recent evidence supporting ICI efficacy in nasopharyngeal carcinoma (NPC), we investigated whether CLDN1 is overexpressed in NPC and its association with the tumor immune microenvironment (TME). Methods: 131 NPC patients treated at a single institution were prospectively recruited. TME profiling was performed using deconvolutional methods (CIBERSORT, quanTIseq) on tumor transcriptomes to estimate proportions of immune cell subsets, and expression of immune-related genes and pathways. Transcriptome profiling was performed on tumors with >70% cellularity and normal controls using Illumina Truseq (CA). We stratified tumors by their median CLDN1 expression and compared the immune profile of CLDN1high versus CLDN1low tumors. Cox regression model was used to analyze disease-free survival (DFS) as the primary clinical endpoint. Results: We observed CLDN1 overexpression in NPC versus normal tissues ( P=0.0046) that was independent of known prognostic clinical parameters e.g., TNM-stage and EBV DNA levels. Consistent with our hypothesis, CLDN1 expression was negatively correlated with total immune cell fraction in the tumor ( P<0.001). We observed reduced abundance of most immune subsets in CLDN1high tumors, notably CD8 T cells ( P<0.001), activated CD4 memory T cells ( P<0.001), and M1 and M2 macrophages ( P=0.048 and P<0.001). There was an overall downregulation of both stimulatory and suppressive GO immune-related gene-sets and genes in CLDN1high versus CLDN1low tumors, thereby corroborating our preceding observation of reduced immune infiltration in the TME in CLDN1 overexpressing NPC. Our findings were validated in a published dataset of 113 NPC tumors (Zhang et al., 2017). Of clinical relevance, reduced immune infiltration in the TME was prognostic for DFS; HR of CLDN1high, CD8 T celllow (stratified by median) NPC was 2.1 (95% CI:0.9-5.0) when compared with CLDN1low, CD8 T cellhigh NPC. Conclusions: Here, we report the overexpression of CLDN1 in NPC that is ubiquitous across disease stages and associated with an immune-low TME. This influence on the TME is associated with an inferior outcome of NPC patients. Given the role of the tumor TME in ICI response, our findings provide a rationale for future trials investigating combination of CLDN1 targeting with ICI to reverse this immune exclusion effect, potentially leading to improved outcomes.

LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3.

The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unraveled a potentially novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2, and LoVo) with either knockdown or overexpression of LIN28B, we identified claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and posttranscriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a potentially novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B/CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by posttranscriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.

Downregulation of CLDN1 impairs trophoblast invasion and endovascular trophoblast differentiation in early-onset preeclampsia

IntroductionTo investigate the role of claudin-1 (CLDN1) in trophoblast invasion and endovascular trophoblast (enEVT) differentiation in early-onset preeclampsia (EOPE). MethodsThe expression and localization of CLDN1 in normal (n = 18) and EOPE (n = 20) placental tissues were detected by immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT‒PCR) and Western blotting. Next, invasion, migration and tube formation assays were performed to explore the involvement of CLDN1 in trophoblast invasion and enEVT differentiation in trophoblast cell lines (HTR8/SVneo). Then, invasion and enEVT markers were analyzed via Western blotting and qRT‒PCR, respectively. Finally, we established an EOPE mouse model to detect the Cldn1 protein level. ResultsCLDN1 expression was significantly decreased in EOPE placental tissues. Knockdown of CLDN1 suppressed HTR8/SVneo cell invasion, migration and the ability to penetrate the endothelial tube. Conversely, overexpression of CLDN1 promoted trophoblast invasion and the ability to invade the endothelial tube. Inhibition of CLDN1 decreased the protein expression of VIM and SNAIL along with downregulating IL1B and PECAM1 mRNA levels, while overexpression of CLDN1 gave the opposite results. In the EOPE mouse model, we found a decrease in Cldn1 expression in EOPE mouse placentas. DiscussionThese results suggest that the downregulation of CLDN1 in trophoblast cells is involved in the pathogenesis of early-onset preeclampsia by affecting trophoblast invasion and enEVT differentiation.

Sanguinarine-based isoquinoline alkaloids modulated the gut-brain axis and enhanced growth performance and gut integrity in natural heat stress broiler chickens

Persistent hot and humid tropical climate induces heat stress (HS), negatively impacting gut integrity with inflammation and tight junction degradation in broilers. As integrating operations of the digestive tract and hypothalamus in avian are regulated by feed intake, the effect of HS may be eased by feed supplementation. Herein, the effect of isoquinoline alkaloids (IQ) supplementation from Macleaya cordata on integrating gut-brain axis and gut integrity was assessed. 720 male broilers (Ross 308) were treated with IQ at null (control), 60 (IQ60), and 100 mg/kg (IQ100). They were reared in open production house between April and May in Southern Thailand with 33.0–38.0°C (average 35.0°C), 75–80% relative humidity (RH), and 160–200 heat stress index (HI). From day 1 to 42, IQ supplementation positively affected growth performance where body weight (BW), body weight gain (BWG), and feed intake (FI) were significantly higher in birds fed IQs than in the control group (P<0.05), and feed conversion ratio (FCR) was significantly lower in the IQ group (P<0.05). IQ improved gut integrity by reducing inflammation with significant down-regulation of nitric oxide synthase (iNOS), cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), and nuclear factor κB (NF-κB) in the ileum (P<0.05). It strengthened tight junction by upregulates occludin (OCLD), claudin-1 (CLDN1), tight junction proteins-1 (TJP-1) and mucin-2 (MUC-2) in the jejunum (P<0.05). As a result, serum fluorescein isothiocyanate-dextran (FITC-d) and corticosterone levels were significantly lower (P<0.05). In terms of the gut-brain axis, IQ supplementation reduces ghrelin and cholecystokinin (CCK) mRNA expression in the jejunum and hypothalamus (P<0.05), which downregulated the expression of pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotropin-releasing hormone (CRH), and melanocortin four receptor (MC4R). It was the first evidence that IQ influences on modulating the gut-brain axis to suppress anorexigenic regulation in broilers with HS exposure.

CLDN6 Suppresses Migration and Invasion of MCF-7 and SKBR-3 Breast Cancer Cells by Blocking the SMAD/Snail/MMP-2/9 Axis.

The study examined the mechanisms of action of signal protein claudin 6 (CLDN6) on migration and invasion of breast cancer cell lines MCF-7 and SKBR-3. To this end, the signal proteins SMAD were blocked with their inhibitor SB431542, the genes CLDN6 and SNAIL were knocked down with short hairpin RNAs, and MMP2 and MMP9 were inhibited with TIMP-1. Expressions of MMP2 and MMP9 mRNAs were evaluated by reverse transcription PCR, Expressions of MMP-2, MMP-9, E-cadherin, N-cadherin, and vimentin were examined by Western blotting. Migration and invasion were analyzed by scratch test and Matrigel invasion assay. SB431542 inhibited expression of MMP2 and MMP9 in both cell lines. Single use of SB431542 inhibited expression of MMP-2/MMP-9 and corresponding mRNAs, but subsequent silencing of CLDN6 gene reversed this effect. TIMP-1 reversed down-regulation of E-cadherin, upregulation of N-cadherin and vimentin, facilitation of migration and invasion evoked by CLDN6 knocking down. Silencing of SNAIL gene inhibited migration and invasion, upregulated the expression of E-cadherin, and down-regulated expression of MMP2, MMP 9, N-cadherin, and vimentin. Thus, CLDN6 suppresses the epithelial-mesenchymal transition, migration, and invasion via blocking SMAD/Snail/MMP-2/9 signaling pathway in MCF-7 and SKBR-3 cancer cell lines.

Dietary supplemental coated essential oils and organic acids mixture improves growth performance and gut health along with reduces Salmonella load of broiler chickens infected with Salmonella Enteritidis

BackgroundReducing Salmonella infection in broiler chickens by using effective and safe alternatives to antibiotics is vital to provide safer poultry meat and minimize the emergence of drug-resistant Salmonella and the spread of salmonellosis to humans. This study was to first evaluate the protective efficacy of feeding coated essential oils and organic acids mixture (EOA) on broiler chickens infected with Salmonella Enteritidis (S. Enteritidis, SE), and then its action mechanism was further explored.MethodsA total of 480 1-day-old Arbor Acres male chickens were randomly assigned into five treatments with six replicates, including non-challenged control fed with basal diet (A), SE-challenged control (B), and SE-infected birds fed a basal diet with 300 mg/kg of EOA (BL), 500 mg/kg of EOA (BM) and 800 mg/kg of EOA (BH), respectively. All birds on challenged groups were infected with Salmonella Enteritidis on d 13. ResultsFeeding EOA showed a reversed ability on negative effects caused by SE infection, as evidenced by decreasing the feed conversion rate (FCR) and the ratio of villus height to crypt depth (VH/CD) (P < 0.05), obviously decreasing intestinal and internal organs Salmonella load along with increasing cecal butyric acid-producing bacteria abundance (P < 0.05). Moreover, supplemental different levels of EOA notably up-regulated claudin-1 (CLDN-1), occludin (OCLN), zonula occludens-1 (ZO-1), mucin-2 (MUC-2), fatty acid binding protein-2 (FABP-2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), myeloid differential protein-88 (MyD88) and interleukin-6 (IL-6) mRNA levels in the ileum of the infected chickens after challenge, whereas down-regulated toll-like receptor-4 (TLR-4) mRNA levels (P < 0.05). Linear discriminant analysis combined effect size measurements analysis (LEfSe) showed that the relative abundance of g_Butyricicoccus, g_Anaerotruncus and g_unclassified_f_Bacillaceae significantly was enriched in infected birds given EOA. Also, phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis showed that alpha-linolenic acid metabolism, fatty acid metabolism and biosynthesis of unsaturated fatty acids were significantly enriched in the EOA group.ConclusionOur data suggest that the essential oils and organic acids mixture can be used as an effective strategy to ameliorate and alleviate Salmonella Enteritidis infection in broilers.

Alpha-tocopherylquinone differentially modulates claudins to enhance intestinal epithelial tight junction barrier via AhR and Nrf2 pathways

Defects in intestinal epithelial tight junctions (TJs) allow paracellular permeation of noxious luminal antigens and are important pathogenic factors in inflammatory bowel disease (IBD). We show that alpha-tocopherylquinone (TQ), a quinone-structured oxidation product of vitamin E, consistently enhances the intestinal TJ barrier by increasing barrier-forming claudin-3 (CLDN3) and reducing channel-forming CLDN2 in Caco-2 cell monolayers (invitro), mouse models (invivo), and surgically resected human colons (exvivo). TQ reduces colonic permeability and ameliorates colitis symptoms in multiple colitis models. TQ, bifunctionally, activates both aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. Genetic deletion studies reveal that TQ-induced AhR activation transcriptionally increases CLDN3 via xenobiotic response element (XRE) in the CLDN3 promoter. Conversely, TQ suppresses CLDN2 expression via Nrf2-mediated STAT3 inhibition. TQ offers a naturally occurring, non-toxic intervention for enhancement of the intestinal TJ barrier and adjunct therapeutics to treat intestinal inflammation.

The Src-Family Kinases SRC and BLK Contribute to the CLDN6-Adhesion Signaling.

Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk-/- and F9:Cldn6:Src-/- cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling.

Fructo-oligosaccharide-mediated alteration in claudin expression in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes and the glucan receptor gene CLEC7A

ObjectivesIndigestible carbohydrates may strengthen tight junctions (TJs) independently of intestinal bacteria. This study investigated whether indigestible carbohydrates (i.e., fructo-oligosaccharides [FOS]) promote TJs directly to intestinal absorptive Caco-2 cells and examined the association between the expression of genes constructing TJs and other genes using mRNA microarray analysis. MethodsCaco-2 cells at 1.0 × 105/mL were seeded in a type I collagen plate and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS); the cells reached confluence at 7 d after seeding. Ten days after the cells reached confluency, they were cultured for 24 h in 10% FCS-containing DMEM medium supplemented with 0%, 5%, or 10% FOS. We performed mRNA microarray to identify the genes whose expression was altered by FOS. Subsequently, quantitative reverse transcription polymerase chain reaction was performed for these altered genes, including CLEC7A encoding the glucan receptor, and for the claudin (CLDN) family genes. The expression of CLDN2, CLDN4, and CLEC7A proteins was assessed using western blot analysis. ResultsFOS decreased the mRNA and protein expression of CLDN2, which weakens TJs, and increased those of CLDN4, which strengthens TJs, in Caco-2 cells. FOS treatment (10%) reduced the mRNA expression of antioxidative genes and induced the expression of immune response–related genes, including CLEC7A, CCL2, and ITGA2. Furthermore, the expression of CLEC7A protein was enhanced by FOS. ConclusionsInduction of TJ-strengthening CLDN4 and reduction of TJ-weakening CLDN2 by FOS treatment in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes, including CLEC7A.

Effects of high incubation temperature on tight junction proteins in the yolk sac and small intestine of embryonic broilers

During the transition from incubation to hatch, the chicks shift from obtaining nutrients from the yolk sac to the intestine. The yolk sac tissue (YST) and small intestine serve as biological barriers between the yolk or gut contents and the blood circulation. These barriers must maintain structural integrity for optimal nutrient uptake as well as protection from pathogens. The objective of this study was to investigate the effect of high incubation temperature on mRNA abundance of the tight junction (TJ) proteins zona occludens 1 (ZO1), occludin (OCLN), claudin 1 (CLDN1), and junctional adhesion molecules A and 2 (JAMA, JAM2) and the heat shock proteins (HSP70 and HSP90) in the YST and small intestine of embryonic broilers. Broiler eggs were incubated at 37.5°C. On embryonic day 12 (E12), half of the eggs were switched to 39.5°C. YST samples were collected from E7 to day of hatch (DOH), while small intestinal samples were collected from E17 to DOH. The temporal expression of TJ protein mRNA from E7 to DOH at 37.5°C and the effect of incubation temperature from E13 to DOH were analyzed by one-way and two-way ANOVA, respectively and Tukey's test. Significance was set at P < 0.05. The temporal expression pattern of ZO1, OCLN, and CLDN1 mRNA showed a pattern of decreased expression from E7 to E13 followed by an increase to DOH. High incubation temperature caused an upregulation of ZO1 and JAM2 mRNA in the YST and small intestine. Using in situ hybridization, OCLN and JAMA mRNA were detected in the epithelial cells of the YST. In addition, JAMA mRNA was detected in epithelial cells of the small intestine, whereas JAM2 mRNA was detected in the vascular system of the villi and lamina propria. In conclusion, the YST expressed mRNA for TJ proteins and high incubation temperature increased ZO1 and JAM2 mRNA. This suggests that the TJ in the vasculature of the YST and intestine is affected by high incubation temperature.

#5837 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE PERITONEAL MESOTHELIAL AND ENDOTHELIAL CELL BARRIER

Abstract Background and Aims Solute transport across cellular levels is mediated by paracellular and transcellular pores, channels and carriers. Knowledge on their cell specific expression, regulation and function in peritoneal dialysis (PD) is limited. Method Polarized primary (HPMC) and immortalized human peritoneal mesothelial (MeT-5A), microvascular (HCMEC) and umbilical vein endothelial (HUVEC) cells underwent RNA sequencing and gene enrichment analysis. Key findings were confirmed by western blotting (WB), confocal and single molecule localization microscopy (SMLM). Arteriolar transcriptome and proteome datasets of non-CKD, CKD5 and PD children (n = 6/group) underwent targeted transport pathways analysis. Key transporter proteins were quantified in parietal peritoneum (n = 20–30/group) and related to peritoneal transport rates available in 23 children. Barrier function was studied in vitro (transepithelial electrical resistance, TER, and molecular weight dependent flux), ex vivo and in vivo. Results Junction, transmembrane and transcytotic transporter expression was highly cell type specific on RNA and protein level. Sealing Claudin (CLDN)1 was only expressed in mesothelial cells, sealing CLDN5 in endothelial cells. TER which reflects functional junction status, was 50% lower in HCMEC compared to HUVEC and the two mesothelial cell lines; 4- and 10-kDa dextran permeability was higher in HCMEC. At nanoscale, SMLM yielded highest distance of junction molecules in HCMEC and different spatial organisation, reflecting the low TER. In sheep peritoneum, removal of the mesothelium abolished tissue TER. In mice, short-term LPS exposure to modify mesothelial permeability resulted in faster transperitoneal 4- and 70-kDa dextran transport, suggesting a specific barrier function of the mesothelium. In human parietal peritoneum, total endothelial surface area per section was age dependently 1.5- to 2-fold higher than the respective mesothelial surface area, and further increased with double-chamber PD fluid, due to major hypervascularisation. Tight junction proteins CLDN-1 to -5, and -15, ZO-1, occludin and tricellulin, and transcellular transporter ENaC, PIT1, and SGLT1 were detected in mesothelial and arteriolar endothelial cells. In CKD mesothelial CLDN-1 and arteriolar CLDN-2 and -3 were more abundant than in non-CKD controls, and PD patients had highest mesothelial and arteriolar CLDN-1 and mesothelial CLDN-2, lower mesothelial and arteriolar claudin-3 and lower arteriolar ENaC. D/P creatinine and D/D0 glucose correlated with arteriolar CLDN-2 and with mesothelial CLDN-4 and -15, which are pore forming junctions, and for creatinine with mesothelial PIT-1, a sodium/phosphate co-transporter. Conclusion We provide the first in-depth analysis of peritoneal determinants of solute transport. The molecular expression pattern of the mesothelial and endothelial cell barrier and transporter proteins differs substantially. Disruption of the mesothelial layer increases peritoneal solute absorption rate. In the human peritoneum, peritoneal transporter status is modified by CKD and PD, and pore forming junction proteins are associated with dialytic solute transport function. These represent a promising target for therapeutic intervention.

ATRT-17. CAR T CELL THERAPY TARGETING CLAUDIN-6 IS EFFECTIVE IN CELLULAR AND XENOGRAFT MODELS OF ATYPICAL TERATOID/RHABDOID TUMOR

Abstract Novel therapies are needed to for the treatment of atypical teratoid/rhabdoid tumor (ATRT), an aggressive brain tumor that predominantly affects young children and has an average 5-year survival under 50%. Claudin-6 (CLDN6) is a tight junction protein present during development and is expressed in up to 70% of ATRT specimens but not in normal tissue, making it a promising immunotherapeutic target. CLDN6-targeted chimeric antigen receptor (CAR) T cells in combination with a CAR T cell-amplifying mRNA vaccine have demonstrated antitumor activity against other CLDN6-expressing cancers in pre-clinical and a phase I adult trial (NCT04503278; Haanen J et al AACR, 2022). To assess the effectiveness of CLDN6-targeted CAR T cells against ATRT, we utilized a second-generation mRNA CAR with a 4-1BB costimulatory domain and single-chain variable fragment against CLDN6 (Reinhard et al, 2020). CLDN6 expression in patient-derived ATRT specimens was profiled by RNAseq (mean FPKM 11.4) and immunohistochemistry (positive staining in 53% of specimens). Tumor-derived cell lines were assessed for CLDN6 expression by flow cytometry. Co-culture of CLDN6-directed mRNA CAR T cells resulted in tumor-specific cytotoxicity compared to CD19-directed control CAR T cells in CLDN6-positive ATRT cell lines 7316-2187 (92% versus 15% at 10:1, p &amp;lt; 0.0001; 86% versus 0% at 5:1, p&amp;lt;0.0001) and 7316-2141 (75% versus 7% at 10:1, p&amp;lt;0.0001; 53% versus 0% at 5:1, p&amp;lt; 0.0001). Patient-derived, orthotopic xenograft ATRT models were created through intracranial injection of tumor cells into the cerebellum of NSG mice. Following engraftment with 7316-2141, repeated intratumoral administration of mRNA CLDN6 CAR T cells resulted in significant tumor regression (-1.9x108 vs. +2.4x109 p/sec/cm2/sr, p&amp;lt;0.001) and improved survival compared to mRNA CD19 CAR T cells (median OS not reached vs. 13 days, p=0.002). This work highlights the potential for targeting CLDN6 via CAR T cell therapy in patients with ATRT as a novel therapeutic strategy.

Genome-wide identification, evolution and expression analysis of tight junction gene family and the immune roles of claudin5 gene in turbot (Scophthalmus maximus L.)

Tight junction proteins (TJs) are important component proteins that maintaining the structure and function of TJs, connecting to each other to form a TJ complex between cells, maintaining the biological homeostasis of the internal environment. In this study, a total of 103 TJ genes were identified in turbot according to our whole-transcriptome database. Transmembrane TJs were divided into seven subfamilies, including claudin (CLDN), occludin (OCLD), tricellulin (MARVELD2), MARVEL domain containing 3 (MARVELD3), junctional adhesion molecules (JAM), immunoglobulin superfamily member 5 (IGSF5/JAM4), blood vessel epicardial substance (BVEs). Moreover, the majority of homologous pairs of TJ genes showed highly conserved alongside length, exon/intron number and motifs. As for phylogenetic analysis for 103 TJ genes, eight of them have undergone a positive selection and JAMB-like has undergone the most neutral evolution. The expression patterns of several TJ genes showed the lowest expression levels in blood, while the highest expression levels were detected in intestine, gill and skin, which all belong to mucosal tissues. Meanwhile, most examined TJ genes showed down-regulated expression patterns during bacterial infection, while several TJ genes exhibited up-regulated expression patterns at a later stage (24 h). At the same time, several potential candidate genes (such as CLDN-15, CLDN-3, CLDN-12, CLDN-5 and OCLD) were significantly down-regulated, which may indicate their important functions that involved in the regulation of bacterial infection. Currently, there is little research on CLDN5 in the intestine, but it is highly expressed in the intestine and has significant changes in intestinal expression after bacterial infection. Thus, we knocked down CLDN5 by the method of lentiviral infection. The result showed CLDN5 was related to cell migration (wound healing) and apoptosis, and the method of dualluciferasereporterassay showed that the functions of CLDN5 could be regulated by miR-24. The study of TJs may lead to a better understanding of the function of TJs in teleost.

Expression of Cell-Adhesion Molecules in E. coli: A High Throughput Screening to Identify Paracellular Modulators.

Cell-adhesion molecules (CAMs) are responsible for cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Claudins (CLDNs), occludin (OCLN), and junctional adhesion molecules (JAMs) are CAMs' components of the tight junction (TJ), the single protein structure tasked with safeguarding the paracellular space. The TJ is responsible for controlling paracellular permeability according to size and charge. Currently, there are no therapeutic solutions to modulate the TJ. Here, we describe the expression of CLDN proteins in the outer membrane of E. coli and report its consequences. When the expression is induced, the unicellular behavior of E. coli is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). Our method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows high-throughput screening (HTS) of small-molecules for interactions with CAMs. Here, we focused on using iCLASP to identify paracellular modulators for CLDN2. Furthermore, we validated those compounds in the mammalian cell line A549 as a proof-of-concept for the iCLASP method.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.