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FTY720 or fingolimod is a known functional antagonist of sphingosine-1-phosphate (S1P), and it is effective in treating multiple sclerosis and preventing inflammatory bowel disease (IBD). Evidence shows that its use in mice can increase the susceptibility to mucosal infections. Despite the significant contribution of S1P to barrier function, the effect of the administration of FTY720 on the mucosal barrier has never been investigated. In this study, we looked into how FTY720 therapy affected the function of the gut barrier susceptibility. Administration of FTY720 to C57BL/6 mice enhances the claudin-2 expression and reduces the expression of claudin-4 and occludin, as studied by qPCR, Western blot, and immunofluorescence. FTY720 inhibits the Akt-mTOR pathway to decrease occludin and claudin-4 expression and increase claudin-2 expression. FTY720 treatment induced increased colonic inflammation, with notably greater immune cell infiltration, colon histopathology, and increased production of TNF-α, IFN-γ, CXCL-1, and CXCL-2 than that in control mice. Taking into account the close association of "the leaky gut" and gut dysbiosis among the major diseases, we therefore can infer that the vigilance of gut pathology should be maintained, where FTY720 is used as a treatment option. [ABSTRACT FROM AUTHOR]

Claudin-4 is a key component of tight junctions, which play an important role in the formation of the epidermal barrier by forming a circumferential network in the granular layer that serves as a gatekeeper of the paracellular pathway. The aim of this study is to illustrate claudin-4 immunohistochemical staining patterns of different blistering disorders. We collected 35 cases, including two Hailey–Hailey disease, one Darier disease, three Grover disease, one acantholytic acanthoma, two warty dyskeratoma, 11 pemphigus vulgaris (PV) including six mucosal PV, and two pemphigus foliaceus. For comparison, we included five cases of normal skin, five eczema, and three bullous pemphigoid cases. Claudin-4 demonstrated weak-to-moderate expression in keratinocytes located in the stratum granulosum, keratinocytes surrounding hair follicles, and adnexal glands. Further, claudin-4 exhibited moderate-to-strong membranous staining in disrupted keratinocytes surrounding and within the acantholytic and bullous areas in 16/22 of the acantholytic cases (not seen in the six cases of mucosal PV) and all three bullous pemphigoids. This finding suggests that claudin-4 is upregulated in these conditions, which may be a compensatory response to the disrupted barrier function. This finding could shed light on the molecular mechanisms underlying disrupted barrier function in blistering disorders, independent of the specific underlying disease mechanism. [ABSTRACT FROM AUTHOR]

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelialmonolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCd (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability. [ABSTRACT FROM AUTHOR]

FTY720 or fingolimod is a known functional antagonist of sphingosine-1-phosphate (S1P), and it is effective in treating multiple sclerosis and preventing inflammatory bowel disease (IBD). Evidence shows that its use in mice can increase the susceptibility to mucosal infections. Despite the significant contribution of S1P to barrier function, the effect of the administration of FTY720 on the mucosal barrier has never been investigated. In this study, we looked into how FTY720 therapy affected the function of the gut barrier susceptibility. Administration of FTY720 to C57BL/6 mice enhances the claudin-2 expression and reduces the expression of claudin-4 and occludin, as studied by qPCR, Western blot, and immunofluorescence. FTY720 inhibits the Akt–mTOR pathway to decrease occludin and claudin-4 expression and increase claudin-2 expression. FTY720 treatment induced increased colonic inflammation, with notably greater immune cell infiltration, colon histopathology, and increased production of TNF-α, IFN-γ, CXCL-1, and CXCL-2 than that in control mice. Taking into account the close association of “the leaky gut” and gut dysbiosis among the major diseases, we therefore can infer that the vigilance of gut pathology should be maintained, where FTY720 is used as a treatment option.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1. [ABSTRACT FROM AUTHOR]

This review article examines some new and some problem areas in mesothelial pathology, four of which are discussed, as follows. (1) The concept of mesothelioma in situ: this lesion is defined as a single layer of bland mesothelial cells without evidence of invasion, but that have lost BAP1 and/or MTAP by immunohistochemistry. Benign reactions can exactly mimic mesothelioma in situ, but a hint to the correct diagnosis is a story of recurrent pleural effusions/ascites of unknown aetiology without radiological or direct visual evidence of tumour. (2) The nature of well‐differentiated papillary mesothelial tumour (WDPMT): WDPMT has a long history of arguments regarding its behaviour, and this uncertainty can now be seen to arise, in part, from the observation that some forms of mesothelioma in situ microscopically look exactly like WDPMT. Hence, it is recommended to always run at least a BAP1 stain on any lesion that looks like WDPMT. Both flat and WDPMT‐like mesothelioma in situ are strongly associated with eventual development of invasive mesothelioma, but this process is relatively slow. (3) New immunostains for separating mesothelioma from other tumours: here, it is proposed that in most cases, and particularly when the differential is epithelioid mesothelioma versus non‐small cell lung cancer, one can make this separation with extremely high sensitivity and specificity using just two stains: HEG1 and claudin‐4. (4) Markers for separating benign from malignant mesothelial proliferations: this topic is briefly reviewed, with an indication of which markers are generally accepted and the best utilisation and possible limitations of each marker. [ABSTRACT FROM AUTHOR]

Background: Claudins are a clan of proteins that are the most important component of tight junctions. The claudin-4 expression has been linked to tumour cell invasion and progression in a variety of primary malignancies. Evaluation of lymphovascular density (LVD) correlates with tumour aggressiveness and may correlate with prognosis. D2-40 is a highly specific marker of lymphatic vessels. Aims: To evaluate the claudin-4 expression in relation to LVD by D2-40 expression and with clinicopathological parameters in prostatic adenocarcinoma. Settings and Design: Prospective study Materials and Methods: 39 cases of prostatic adenocarcinoma were taken, the D2-40 and claudin-4 immunohistochemical stains were performed and correlation was done with clinicopathological parameters. Statistical Analysis Used: Statistical analyses such as mean, median, standard deviation, Mann–Whitney U test, Fischer exact test, Spearman's rank-order correlation coefficient, Chi-square test and T-test were used. Results: The claudin-4 expression was seen higher in cases with higher Gleason score but it was statistically non-significant (P = 0.778). The claudin-4 expression did not correlate with any clinicopathological parameters. LVD in the peritumoral area was significantly higher as compared to the intratumoral area (P = 0.005). Intratumoral LVD and perineural invasion were found to be statistically significant (P = 0.048). Conclusion: The claudin-4 expression may correlate with adverse prognostic parameters. Higher lymphatic vessels can be responsible for the higher metastatic potential of prostatic adenocarcinomas. [ABSTRACT FROM AUTHOR]

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary‐cultured murine intestinal epithelial cells and discovered that AM upregulates claudin‐4 expression. In a mouse model of dextran sulfate sodium‐induced colitis, AM administration also enhanced claudin‐4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα‐mediated downregulation of claudin‐4 and loss of cell–cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin‐4 expression.

Introduction: N6-methyladenosine (m6A) is an emerging epigenetic modification, which plays a crucial role in the development of cancer. Nevertheless, the underlying mechanism of m6A-associated proteins and m6A modification in gallbladder cancer remains largely unknown. Materials and methods: The Gene Expression Omnibus database and tissue microarray were used to identify the key m6A-related gene in gallbladder cancer. The function and mechanism of IGF2BP3 were further investigated by knockdown and overexpression techniques in vitro and in vivo. Results: We found that IGF2BP3 was elevated and correlated with poor prognosis in gallbladder cancer, which can be used as an independent prognostic factor for gallbladder cancer. IGF2BP3 accelerated the proliferation, invasion and migration of gallbladder cancer cells in vitro and in vivo. Mechanistically, IGF2BP3 interacted with and augmented the stability of CLDN4 mRNA by m6A modification. Enhancement of CLDN4 reversed the inhibitory effect of IGF2BP3 deficiency on gallbladder cancer. Furthermore, we demonstrated that IGF2BP3 promotes the activation of NF-κB signaling pathway by up-regulation of CLDN4. Overexpression of IGF2BP3 in gallbladder cancer cells obviously promoted the polarization of immunosuppressive phenotype in macrophages. Besides, Gallbladder cancer cells-derived IGF2BP3 up-regulated the levels of STAT3 in M2 macrophages, and promoted M2 polarization. Conclusions: We manifested IGF2BP3 promotes the aggressive phenotype of gallbladder cancer by stabilizing CLDN4 mRNA in an m6A-dependent manner and induces macrophage immunosuppressive polarization, which might offer a new theoretical basis for against gallbladder cancer.

Claudin-4, a member of the claudin multigene family, participates in events associated with mesenchymal-like activity of cancerous cells. Claudin-4 expression is upregulated in cervical cancer tissue compared with that in adjoining non-neoplastic tissue. However, the mechanisms that regulate Claudin-4 expression in cervical cancer are poorly understood. Moreover, whether Claudin-4 contributes to the migration and invasion of cervical cancer cells remains unclear. By western blotting, reverse transcription-qPCR, bioinformatics analysis, dual-luciferase reporter assay, chromatin immunoprecipitation assay, wound healing assay and Transwell migration/invasion assay, the present study confirmed that Claudin-4 was a downstream target of Twist1, a helix-loop-helix transcriptional factor, the activity of which has a positive correlation with Claudin-4 expression. Mechanistically, Twist1 directly binds to Claudin-4 promoter, resulting in the transactivation of expression. The depletion of the Twist1-binding E-Box1 domain on Claudin-4 promoter via CRISPR-Cas9 knockout system downregulates Claudin-4 expression and suppresses the ability of cervical cancer cells to migrate and invade by elevating E-cadherin levels and lowering N-cadherin levels. Following activation by transforming growth factor-ß, Twist1 induces Claudin-4 expression, thus enhancing migration and invasion of cervical cancer cells. In summary, the present data suggested that Claudin-4 was a direct downstream target of Twist1 and served a critical role in promoting Twist1-mediated cervical cancer cell migration and invasion. [ABSTRACT FROM AUTHOR]

Background: Epithelial cell adhesion molecule (EpCAM) is frequently used to distinguish carcinoma from background mesothelial cells during cytologic examination of body cavity fluids. Previously, the authors identified one malignant mesothelioma case with strong and diffuse membranous EpCAM staining, making it indistinguishable from carcinoma. Methods: In this study, the authors evaluated all available effusion specimens from patients with malignant mesothelioma, including the above‐mentioned index case, obtained at Stanford Health Care, from 2011 to 2021 (N = 17) as well as control cases (N = 5). Analyses included an immunohistochemistry (IHC) assay for EpCAM and claudin‐4, a multiplexed immunofluorescent (IF) assay for EpCAM, and an RNA in situ hybridization assay targeting EpCAM. Results: The authors detected EpCAM positivity of variable intensity and percentage in four malignant mesothelioma cases (23.5%; although only two showed positivity for the epithelial‐specific IHC marker MOC31 in ≥40% of cells) and claudin‐4 negativity in all cases, with two cases displaying focal and weak claudin‐4 staining in <1% of cells. Multiplexed IF staining on the cases with EpCAM IHC positivity showed strong, membranous EpCAM staining in one of four cases. RNA in situ hybridization also was used to assess the correlation between EpCAM positivity by IHC/IF and RNA expression levels. Strong EpCAM RNA expression was detected in the three malignant mesothelioma cases. Conclusions: The current findings revealed that a subset of epithelioid malignant mesothelioma cases mimic or exhibit the immunophenotypic features of carcinoma when evaluating for EpCAM only. Additional biomarker testing, such as claudin‐4, may help avoid this potential pitfall to yield accurate diagnoses. The authors examined epithelial biomarkers in effusion specimens of 17 malignant mesothelioma cases and five benign cases. By combining immunohistochemistry for epithelial cell adhesion molecule (EpCAM) and claudin‐4, a multiplexed immunofluorescent assay for EpCAM, and RNA in situ hybridization targeting EpCAM, this study revealed that a subset of epithelioid malignant mesothelioma cases mimic or exhibit features of carcinoma when evaluating for EpCAM only. [ABSTRACT FROM AUTHOR]

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Cryptococcal species often cause lung infections and are the main cause of fungal meningitis. Claudin-4 appears to be a major structural component that maintains a tight alveolar barrier and prevents fluid and electrolyte leakage into the alveolar space. We aimed to determine whether S7-tetrahydrocurcumin (THC) could clearance of C. deneoformans and regulate claudin-4 expression during C. deneoformans infection. We investigated the effect of THC on C. deneoformans infection and its possible mechanism in vivo. Transmission electron microscopy was used to observe the ultrastructure of the lung tissue and the invasion of Cryptococcus. To clarify the effect of THC, we examined claudin-4, c-Jun, and Smad2 expression. We also measured claudin-4 expression in pulmonary specimens from clinical patients. THC reduced cryptococcal cell invasion in the lungs, improved alveolar exudation, and reduced inflammation. Pretreatment with THC suppressed c-Jun and Smad2 expression, resulting in significantly increased claudin-4 levels. In contrast, the expression of claudin-4 in clinical specimens from patients with cryptococcal infection was higher than that in normal specimens. THC enhanced the clearance of C. deneoformans during infection in vivo. We investigated the expression of claudin-4 and the possible mechanism of THC against C. deneoformans infection. [ABSTRACT FROM AUTHOR]

Objective: We aimed to explore the relationship between peripheral blood circulating tumor cells (CTCs) and the expression of Claudin-4 in patients with breast cancer, and further explore the potential impact on clinical prognosis and risk assessment.Methods: We classified and enumerated circulating tumor cells in the blood of breast cancer patients by CTC-enriched in situ hybridization and the detection of Claudin-4 expression by immunohistochemistry. We carried out an analysis of the correlation between the two and the comparison of their impact on clinical parameters and prognosis.Results: There were 38 patients with a low expression of Claudin-4 and 27 patients with a high expression of Claudin-4. Compared with Claudin-4 low-expression patients, the number of CTCs was higher in patients with high Claudin-4 expression (11.7 vs. 7.4, p < 0.001). High Claudin-4 expression was associated with a lower count of epithelial CTCs (E-CTCs) (3.4 vs. 5.0, p = 0.033), higher counts of mesenchymal CTCs (M-CTC) (4.4 vs. 1.1, p < 0.001), and epithelial/mesenchymal CTCs (E/M-CTCs) (4.0 vs. 3.5, p = 0.021). The intensity of Claudin-4 was positively correlated with CTC (rs = 0.43, p = 0.001). Multivariate COX regression analysis showed that CTC counts (HR = 1.3, p < 0.001), Claudin-4 (HR = 4.6, p = 0.008), and Lymphatic metastasis (HR = 12.9, p = 0.001) were independent factors for poor prognosis. COX regression of CTC classification showed that epithelial/mesenchymal CTCs (E/M-CTC) (HR = 1.9, p = 0.001) and mesenchymal CTCs (M-CTC) (HR = 1.5, p = 0.001) were independent influencing factors of adverse reactions in breast cancer patients.Conclusion: The number of CTC in breast cancer is positively correlated with the expression of Claudin-4. High CTC counts and a high proportion of M-CTCs correlated with Claudin-4 expression. CTC counts and Claudin-4 expression were independent predictors of poor prognosis in breast cancer patients.

Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary‐cultured murine intestinal epithelial cells and discovered that AM upregulates claudin‐4 expression. In a mouse model of dextran sulfate sodium‐induced colitis, AM administration also enhanced claudin‐4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα‐mediated downregulation of claudin‐4 and loss of cell–cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin‐4 expression. [ABSTRACT FROM AUTHOR]

Background: Claudin‐4 is a sensitive and specific marker for carcinoma in effusion cytology. The authors examined the diagnostic use of claudin‐4 versus MOC‐31 and Ber‐EP4 by comparing their sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in differentiating carcinoma from mesothelioma and benign/mesothelial hyperplasia in effusion specimens. Methods: This retrospective study comprised a cohort of 229 cytology specimens, including 211 effusion fluid and 18 fine‐needle aspiration specimens. Cytologic categories included 134 carcinoma, 28 mesothelioma, 46 indefinite (suspicious and atypical), and 21 benign. Cell block sections were stained for claudin‐4 and compared with those previously stained for MOC‐31 and Ber‐EP4. Indefinite cases were further reclassified based on clinical and pathologic findings into benign (26 cases), mesothelioma (11 cases), and carcinoma (nine cases). Results: None of the mesotheliomas (0/39) or benign effusions (0/47) were positive for claudin‐4, whereas 134 of the 143 carcinoma specimens were positive. Compared to MOC‐31 and Ber‐EP4, claudin‐4 had the highest specificity and PPV (100% for each), followed by Ber‐EP4. Claudin‐4 showed high sensitivity (93.7%), albeit lower than MOC‐31. MOC‐31 had the lowest specificity and PPV but the highest sensitivity and NPV. Ber‐EP4 had the lowest sensitivity (91.6%). Conclusions: Claudin‐4 can be used as a single marker for carcinoma with high sensitivity and superior specificity compared with MOC‐31 and Ber‐EP4. Mesothelial lineage can be ruled out when claudin‐4 is positive. In equivocal cytology samples with few scattered cells of interest, a panel of claudin‐4 and Ber‐EP4 results in the highest combined sensitivity and specificity. The findings in this study demonstrate the high sensitivity and superior specificity of claudin‐4 as a single marker for carcinoma in cytology specimens and its ability to rule out mesothelial lineage with high certainty. The authors also identify the highly sensitive and specific combination of claudin‐4 and Ber‐EP4 in diagnosing equivocal specimens previously categorized as atypical or suspicious. [ABSTRACT FROM AUTHOR]

Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target. [ABSTRACT FROM AUTHOR]

Due to the lack of timely and accurate screening modalities and treatments, most pancreatic cancer (PCa) patients undergo fatal PCa progression within a short period since diagnosis. The claudin(CLDN) family is expressed specifically as tight junction structure in a variety of tumors, including PCa, and affects tumor progression by changing the cell junctions. Thus far, many of the 27 members of the claudin family, including claudin-18.2 and claudin-4, have significantly aberrantly expression in pancreatic tumors. In addition, some studies have confirmed the role of some claudin proteins in the diagnosis and treatment of pancreatic tumors. By targeting different targets of claudin protein and combining chemotherapy, further enhance tumor cell necrosis and inhibit tumor invasion and metastasis. Claudins can either promote or inhibit the development of pancreatic cancer, which indicates that the diagnosis and treatment of different kinds of claudins require to consider different biological characteristics. This literature summarizes the functional characteristics and clinical applications of various claudin proteins in Pca cells, with a focus on claudin-18.2 and claudin-4. [ABSTRACT FROM AUTHOR]

Separation of mesothelioma from metastatic carcinoma requires immunohistochemical support, with small batteries of stains recommended as a starting‐point, but these numbers commonly expand to 10, 12 or more stains, a process that is not only expensive but frequently generates anomalous or confounding results, leading to even more stains. Here we review data on HEG1 clone SKM9‐2, a new (now commercially available) mesothelioma marker and claudin‐4, a broad‐spectrum carcinoma marker, to ask whether these two stains are sufficient, by themselves, to separate mesotheliomas from non‐small‐cell lung (NSCLC) as well as other carcinomas. Data for HEG1, derived from four laboratories, showed membrane staining in 393 of 434 (91%) epithelioid/biphasic mesotheliomas and one of 360 (0.3%) NSCLC (sensitivity 91%, specificity 99.7%). Reports from seven laboratories evaluating claudin‐4 in NSCLC showed positivity in 469 of 502 (93%) carcinomas and weak positivity in five of 463 (1.0%) epithelioid/biphasic mesotheliomas (sensitivity 93%, specificity 98.9%). Comparable results were found with carcinomas from other sites, except for serous and thyroid carcinomas, some of which react with HEG1 but are also positive for claudin‐4. For sarcomatoid mesotheliomas, HEG1 sensitivity is modest and staining sometimes difficult to interpret. We hypothesise that the combination of HEG1 and claudin‐4 immunostaining will potentially allow the separation of epithelioid/biphasic mesotheliomas from NSCLC carcinomas with high accuracy using only two immunostains in most cases. This combination will probably also work for carcinomas from most other sites, but more reports on HEG1 SKM9‐2 staining of carcinomas other than NSCLC are needed. This approach would greatly simplify the diagnosis of mesothelioma. [ABSTRACT FROM AUTHOR]

Simple Summary: Feline idiopathic cystitis is a common disease in domestic cats. The pathogenesis of the illness is not completely understood, but the role of various stress factors and the concomitant release of stress hormones, such as norepinephrine, is strongly suggested. Therefore, the examination of this hormone is essential to gain a deeper knowledge of the development of the disease. In the present study, a novel cell culture of uroepithelial cells from a feline urinary bladder was established to serve as a proper model for studying the effects of a norepinephrine triggered stress reaction. Acute, 1 h norepinephrine exposure affected uroepithelial cells by increasing metabolic activity, inducing a proinflammatory response, triggering oxidative stress, and decreasing the barrier integrity of the cultured cells. The results of this study underline that stress-associated norepinephrine release has a direct molecular effect on the uroepithelial cells; the reaction of these cells may play an important mediatory role in the pathogenesis of the disease. The established cell culture model can be a good tool for further in vitro investigations related to urinary disorders of cats. Feline idiopathic cystitis (FIC) is one of the most common urinary tract disorders in domestic cats. As stress is suggested to play a key role in the pathogenesis of FIC, the effects of norepinephrine (NE) as a stress mediator were investigated on a novel feline primary uroepithelial cell culture, serving as an in vitro model of the disease. The uroepithelial cells gained from the mucosa of the bladder of a euthanized cat were cultured for 6 days and were acutely exposed to NE (10, 100, and 1000 µM) for 1 h. NE increased the metabolic activity of the cultured cells and elevated the extracellular concentrations of the pro-inflammatory mediators interleukin-6 (IL-6) and stromal cell derived factor 1 (SDF-1), confirming that NE can trigger an inflammatory response in the uroepithelium. Cellular protein carbonyl levels were increased by NE exposure, while malondialdehyde and glucose regulated protein 78 concentrations remained unchanged, indicating that NE may provoke the oxidative damage of proteins without inducing lipid peroxidation or endoplasmic reticulum stress. Further, it can be strongly suggested that an acute NE challenge might diminish the barrier function of uroepithelial cells, as reflected by the decreased glycosaminoglycan concentration, claudin-4 protein expression, and reduced TER values of the NE-treated cell cultures. Based on these results, short-term NE exposure mimicking acute stress can provoke an inflammatory response and decrease the barrier integrity of cultured feline uroepithelial cells. Hence, it is highly expected that stress-associated NE release may play an important mediatory role in the pathogenesis of FIC. [ABSTRACT FROM AUTHOR]

(1) Background. In the differential diagnosis between sarcomatoid carcinoma (SC) and sarcomatoid mesothelioma (SM), we aimed to investigate the role of Claudin-4 and BAP1, a panel recently used to distinguish conventional carcinoma from epithelioid mesothelioma. (2) Methods. We collected 41 surgical pleural biopsies of SM, 46 surgical resections of SC from different sites and 49 pleural biopsies of normal/hyperplastic mesothelium. All the cases were tested for Claudin-4 and BAP1 using immunohistochemistry. The statistical calculations of the sensitivity, specificity and positive and negative predictive values were performed. (3) Results: Claudin-4 was negative in 41/41 SMs, while it was positive in 18/36 (50.1%) SCs (eight diffusely, 10 focally) within their sarcomatous component. BAP1 was lost in 23/41 SMs, while it was regularly expressed in 46/46 SCs. All the cases of the normal/hyperplastic mesothelium were negative for Claudin-4 and retained the regular expression of BAP1. The Claudin-4 expression was useful for detecting SC (sensitivity, 39.1%; specificity, 100%) and the BAP1 loss was useful for diagnosing SM (sensitivity, 56.1%; specificity, 100%). (4) Conclusions. The staining for Claudin-4 and BAP1 exhibited a low/moderate sensitivity in diagnosing SC and SM (39.1% and 56.1%, respectively), but a very high specificity (100%). Claudin-4 was expressed only in SC and BAP1 loss was noted only in SM. [ABSTRACT FROM AUTHOR]

Introduction: Claudin-4 has been described as a highly sensitive immunocytochemical marker for detection of metastatic carcinoma cells in effusion cytology specimens. This study aims to challenge the performance of claudin-4 in different types of malignancies and low cellularity specimens, by comparison with other markers in a large cohort of carcinomatous effusion specimens., Methodology: Cell block preparations from peritoneal and pleural fluid specimens were retrieved, with malignant (carcinoma) diagnoses confirmed by review of hospital diagnosis code and pathology reports. Claudin-4, BerEP4, CEA, and MOC31 immunocytochemistry were performed and scored by expression proportion and intensity. Tumor cellularity was assessed for subgroup analysis of low cellularity specimens., Results: Totally 147 specimens (70 pleural, 77 peritoneal) of 68 lung, 62 breast, 9 gynecological, and 7 gastrointestinal carcinomas were retrieved. The average proportion expression of claudin-4 was highest (89.6%, vs. CEA 40.5%, BerEp4 18.6%, MOC31 16.8%) and the percentage of strong expression was highest for claudin-4 (72.1%). Expression levels of claudin-4 were consistently higher than other markers in subgroups of all primary sites. The difference was more significant for low cellularity specimens. High (≥50%) proportion expression was seen for 96.61% of cases for claudin-4 (vs. BerEp4 8.77%, CEA 46.55%, MOC31 8.77%, p < 0.001). These factors contributed to a low concordance between claudin-4 and BerEp4, CEA and MOC31 (K = 0.010-0.043)., Conclusion: Claudin-4 is more sensitive than CEA, BerEp4 and MOC31, suitable for low cellularity specimens of most types of metastatic carcinoma and is a robust immunocytochemical marker for carcinoma that can be used solitarily., (© 2024 The Author(s). Diagnostic Cytopathology published by Wiley Periodicals LLC.)

[Display omitted] Previously, we reported that the combined use of spermine (SPM) and sodium taurocholate (STC) (SPM–STC) significantly improves the oral absorption of rebamipide (BCS class IV) and pulmonary absorption of interferon-α without any harmful histopathological changes in the gastrointestinal tract and lungs, respectively. In the present study, we examined the effect of SPM–STC on the transport of fluorescein isothiocyanate-labeled dextrans (FDs) across Caco-2 cell monolayers and attempted to clarify the mechanisms underlying the transport enhancement caused by SPM–STC. SPM–STC were found to significantly enhance the transport of FDs, while the treatment with SPM–STC was not harmful, and the decrease in transepithelial electrical resistance was transient and reversible. The voltage-clamp study clearly indicated that the opening of the paracellular route could be mainly responsible for the enhanced transport of FD-4. As for the mechanisms, it was found that SPM–STC caused a significant increase in membrane fluidity, which would lead to the enhanced transport of small-molecule drugs such as rebamipide. Since SPM–STC increased intracellular Ca2+ via Ca2+ uptake through Ca2+ channels and Ca2+ release from the endoplasmic reticulum stimulated by the IP 3 pathway, the subsequent possible activation of the MLCK signaling pathway would have led to the contraction of the actin–myosin ring. The rearrangement of tight junction-constituting proteins induced through the MAPK pathway has also been suggested as a possible mechanism for opening tight junctions. Claudin-4, a key protein constituting the tight junction, merged with F-actin along with the plasma membrane, was significantly decreased, which would be at least partial structural evidence for the tight-junction opening. [ABSTRACT FROM AUTHOR]

Background: Claudins are important transmembrane proteins in tight junction. The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin-4, a tight junction protein, is overexpressed in breast tumor cells compared to normal epithelial cells, which generally corresponds to loss in polarity and can provide valuable information about biology of the tumor. A prospective clinical study was conducted to assess the expression claudin-4 in patients with breast cancer and its correlation with hormone receptors - estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)-neu.Materials and Methods: The study included 102 biopsy-proven breast carcinoma patients. The biopsy samples were evaluated immunohistochemistry for expression of ER, PR, HER2-neu, and claudin-4. The expression of claudin-4 was correlated with ER, PR, and HER2-neu.Results: In the study, we found that out of 26 cases of high claudin-4, 25 cases (96.15%) were ER negative and P < 0.001, which was significant. Similar results were found with PR-negative cases. Whereas, out of 76 cases with low claudin-4, 54 cases (71.05%) were HER2-neu negative and P = 0.022, which was significant.Conclusions: Claqudin-4 expression has a negative correlation with ER and PR and has a positive correlation with HER2-neu. Hence, it can be effectively utilized as a prognostic and therapeutic marker in breast cancer in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: Brain metastasis occurs in primary cancers, such as breast cancer, and is correlated with mortality. There are limited options available for treatment, but Clostridium perfringens Enterotoxin (CPE) and its interaction with Claudin-4, a possible diagnostic biomarker for breast cancer, can provide a molecular pathway basis for the development of treatment options for metastatic brain cancer. Analysis of the literature reveals that Claudin-4 plays an important role as a receptor for CPE, allowing for the disruption of cell membrane permeability, an influx of calcium ions, and subsequent cell death. The negligible presence of Claudin-4 in normal brain cancer cells and the high abundance of Claudin-4 in breast cancer cells metastasized to the brain, allow for the targeted binding of CPE to tumor cells in the brain. We show that the C-terminal of CPE conjugated to nanoparticles that cross the blood–brain barrier could serve as a drug delivery tool to treat metastatic cells in the brain. Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE's high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast–brain metastatic tumors. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the effects of radiation on paracellular pathway of rat submandibular glands (SMGs) and the mechanism of increasing secretion following treatment with pilocarpine. Materials and Methods: In situ irradiation models of SMGs in Wistar rats were conducted, and the glands were exposed to X‐radiation at a single dose of 20 Gy. Pilocarpine was intraperitoneally injected 60 min prior to radiation and continuous 6 days postirradiation for a total of 7 days. Salivary secretion, histological changes, pro‐inflammatory cytokines, alterations in tight junctions (TJs), and functional membrane proteins aquaporin‐5 (AQP5) and claudin‐4 mediated by the muscarinic acetylcholine M3 subtype receptor were determined at 1 and 12 weeks after irradiation. Results: Salivary secretion of the irradiated glands was reduced at 1 and 12 weeks. As well, acinar cell numbers, TJ width, and the levels of M3 receptor and AQP5 were decreased. In contrast, tumor necrosis factor‐α, interleukin 6, interleukin 1α, and the expression of the TJ protein claudin‐4 were significantly increased in irradiated SMGs. Notably, all the alterations were attenuated by pilocarpine treatment. Conclusions: Pilocarpine could improve the secretory function of irradiated rat SMGs via reducing inflammation, ameliorating the structural injury of TJs, and attenuating the up‐regulation of claudin‐4 expression. [ABSTRACT FROM AUTHOR]

Shinhee Park,1 Pureun-Haneul Lee,2 Ae Rin Baek,1 Jong Sook Park,1 Junehyuk Lee,1 Sung-Woo Park,1 Do Jin Kim,1 An-Soo Jang1 1Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, Bucheon, Republic of Korea; 2Department of Interdisciplinary Program in Biomedical Science Major, Soonchunhyang Graduate School, Soonchunhyang University Bucheon Hospital, Bucheon, Republic of KoreaCorrespondence: An-Soo JangDivision of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, 170 Jomaru-ro, Bucheon, 14584, Republic of KoreaTel +82 32 621 5143Fax +82 32 621 6950Email jas877@schmc.ac.krPurpose: Chronic obstructive pulmonary disease (COPD) imposes a major healthcare burden. A tight junction protein, claudin-4 (CLDN4), may play a protective role in acute lung injury, but its role in COPD is unclear. To investigate the relationship between CLDN4 and COPD, we evaluated the association of CLDN4 with the clinical parameters of COPD, including exacerbations.Patients and Methods: We analyzed a cohort of 30 patients with COPD and 25 healthy controls and evaluated their clinical parameters, including lung function. The plasma CLDN4 level in stable and exacerbated COPD was measured.Results: The COPD patients were all males and predominantly smokers; their initial lung function was poorer than the healthy controls. The mean CLDN4 plasma level was 0.0219 ± 0.0205 ng/mg in the control group, 0.0086 ± 0.0158 ng/mg in the stable COPD group (COPD-ST) and 0.0917 ± 0.0871 ng/mg in the exacerbated COPD (COPD-EXA) group. The plasma CLDN4 level was significantly lower in the COPD-ST than the control group, but was significantly elevated in the COPD-EXA group. The plasma CLDN4 level was inversely correlated with forced vital capacity and forced expiratory volume in 1 second in the COPD-EXA group (r=0.506, P=0.001 and r=0.527, P< 0.001, respectively).Conclusion: The plasma CLDN4 level is closely correlated with COPD exacerbations and decreased lung function. This suggests that CLDN4 has potential as a severity marker for COPD.Keywords: claudin-4, COPD, lung function

Marisa Meloni, Paolo Buratti, Francesco Carriero, Laura Ceriotti VitroScreen, In Vitro Innovation Center, Milan, 20149, ItalyCorrespondence: Laura Ceriotti Via Mosè Bianchi, 103, Milan, 20149, ItalyTel +39 02 89077608Email laura.ceriotti@vitroscreen.comPurpose: A novel experimental model based on a 3D reconstructed human oesophageal epithelium model (HO2E) has been developed to investigate the structural and functional changes of the oesophageal epithelium following exposure to a solution of HCl 0.1 N (pH = 1.2) mirroring GERD microenvironment condition.Methods: The barrier structure modification after the exposure to the acid solution on HO2E tissues was investigated immediately after damage induction and after 1 hour post incubation and compared to HO2E tissues exposed to phosphate buffered saline solution. Immunofluorescence (IF) was applied to quantify the expression and localization of barrier function proteins: Claudin-1 (CLDN-1), Claudin-4 (CLDN-4), Zonulin-1 (ZO-1), E-Cadherin and Mucin-1 (MUC1). Barrier functionality was measured by TEER.Results: In the acidic microenvironment, TEER measurement has shown some limitations and results were not applicable, whereas the evaluation of protein localization and quantification provided clear and robust evidence of the damage which occurred to the epithelium barrier structure. CLDN-4 expression significantly decreased after exposure to acid. ZO-1 protein appeared upregulated immediately after exposure to HCl and was mainly localized in the cytoplasm and not on the cell membrane. This different localization was also observed for CLND-1. CLDN-1, MUC1 and, to a lower extent, ZO-1 expression increased during the post-incubation period.Conclusion: The relevant tissue biomarkers identified, CLDN-1 and MUC1, can be used to monitor TJ structure and epithelial barrier recovery after acid-induced damage which, in our experimental conditions, were non-destructive and suitable for recovery studies. The established model can be useful to investigate the mechanism of action of formulations acting on this specific pathophysiological condition and/or designed to potentiate the physiological defense mechanisms of oesophageal mucosa.Keywords: gastro-oesophageal reflux disease, GERD, 3D reconstructed human oesophageal epithelium, Claudin-1, Claudin-4, E-Cadherin, Zonulin-1, Mucin-1, TEER measurement

Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.

BackgroundBronchopulmonary dysplasia (BPD) is characterized by impaired alveolar and microvascular development. Claudin-18 is the only known lung-specific tight junction protein affecting the development and transdifferentiation of alveolar epithelium.ObjectiveWe aimed to explore the changes in the expression of claudin-18, podoplanin, SFTPC, and the canonical WNT pathway, in a rat model of hyperoxia-induced BPD, and to verify the regulatory relationship between claudin-18 and the canonical WNT pathway by cell experiments.MethodsA neonatal rat and cell model of BPD was established by exposing to hyperoxia (85%). Hematoxylin and eosin (HE) staining was used to confirm the establishment of the BPD model. The mRNA levels were assessed using quantitative real-time polymerase chain reaction(qRT-PCR). Protein expression levels were determined using western blotting, immunohistochemical staining, and immunofluorescence.ResultsAs confirmed by HE staining, the neonatal rat model of BPD was successfully established. Compared to that in the control group, claudin-18 and claudin-4 expression decreased in the hyperoxia group. Expression of β-catenin in the WNT signaling pathway decreased, whereas that of p-GSK-3β increased. Expression of the AEC II marker SFTPC initially decreased and then increased, whereas that of the AEC I marker podoplanin increased on day 14 (P

Claudin-4, a protein with the structure of classic claudins most often found in cell-cell junctions, is frequently overexpressed in epithelial cancers where its localization has not been studied. In this study we aimed to find out where this membrane protein is localized in an ovarian tumor model, OVCAR3 cells, that express high levels of the protein. Immunohistochemical studies showed claudin-4 staining in a perinuclear region, at most plasma membranes and in cytoplasmic puncta. Native claudin-4 did not overlap with phosphorylated claudin-4, which was partially located in focal adhesions. Using claudin-4 BioID technology we confirmed that large amounts of claudin-4 are localized to the Golgi compartment, including in dispersed Golgi in cells where claudin-4 is partially knocked down and in dividing cells. Claudin-4 appears to be present in the vicinity of several types of cell-cell junctions, but there is no evidence that it forms tight junctions in these tumor cells. Both claudin-4, the Golgi marker GM130, and the plasma membrane receptor Notch2 were found in dispersed Golgi in dividing cells. This definition of the cellular architecture of claudin-4 should provide a framework for better understanding of the function of claudin-4 in tumor cells and its molecular interactions.

Claudin (CLDN) is a tight junction protein found in human epithelial cells and its altered expression is known to be associated with the progression of gastric cancer. We aimed to investigate the differential expression of CLDN-4 in early gastric cancer (EGC) according to its clinicopathological characteristics. We enrolled 53 patients with EGC who underwent surgical gastric resection from January 2007 to December 2018. The staining intensity of the tumor cells was scored as 0–3, and the percentage of staining was scored as 0–5; high expression was defined if the intensity plus percentage score was 7 or 8, and low expression was defined if the score was 0–6. Among the 53 patients, 16 (30.2%) showed low CLDN-4 expression, while 37 (69.8%) had high CLDN-4 expression. High CLDN-4 expression was significantly associated with intestinal-type EGC (low: 12.5% vs. high: 56.8%, p = 0.003), open-type atrophic change (low: 60.0% vs. high: 90.9%, p = 0.011), and the presence of synchronous tumors (0 vs. 32.4%, p = 0.010), and all 12 EGCs with synchronous tumors showed high CLDN-4 expression. However, expression of CLDN-3, a typical intestinal phenotype CLDN, was neither correlated with CLDN-4 expression nor associated with synchronous tumors. Taken together, high CLDN-4 expression may be considered as an auxiliary tool for screening synchronous tumors in patients with EGC. [ABSTRACT FROM AUTHOR]

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2′-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin β1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target. [ABSTRACT FROM AUTHOR]

Background & aimsThere is evidence from clinical studies that compromised intestinal epithelial permeability contributes to the development of nonalcoholic steatohepatitis (NASH), but the exact mechanisms are not clear. Mice with disruption of the gene (F11r) encoding junctional adhesion molecule A (JAM-A) have defects in intestinal epithelial permeability. We used these mice to study how disruption of the intestinal epithelial barrier contributes to NASH.MethodsMale C57BL/6 (control) or F11r(-/-) mice were fed a normal diet or a diet high in saturated fat, fructose, and cholesterol (HFCD) for 8 weeks. Liver and intestinal tissues were collected and analyzed by histology, quantitative reverse-transcription polymerase chain reaction, and flow cytometry. Intestinal epithelial permeability was assessed in mice by measuring permeability to fluorescently labeled dextran. The intestinal microbiota were analyzed using 16S ribosomal RNA sequencing. We also analyzed biopsy specimens from proximal colons of 30 patients with nonalcoholic fatty liver disease (NAFLD) and 19 subjects without NAFLD (controls) undergoing surveillance colonoscopy.ResultsF11r(-/-) mice fed a HFCD, but not a normal diet, developed histologic and pathologic features of severe NASH including steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis, whereas control mice fed a HFCD developed only modest steatosis. Interestingly, there were no differences in body weight, ratio of liver weight:body weight, or glucose homeostasis between control and F11r(-/-) mice fed a HFCD. In these mice, liver injury was associated with significant increases in mucosal inflammation, tight junction disruption, and intestinal epithelial permeability to bacterial endotoxins, compared with control mice or F11r(-/-) mice fed a normal diet. The HFCD led to a significant increase in inflammatory microbial taxa in F11r(-/-) mice, compared with control mice. Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r(-/-) mice fed a HFCD. Protein and transcript levels of JAM-A were significantly lower in the intestinal mucosa of patients with NAFLD than without NAFLD; decreased expression of JAM-A correlated with increased mucosal inflammation.ConclusionsMice with defects in intestinal epithelial permeability develop more severe steatohepatitis after a HFCD than control mice, and colon tissues from patients with NAFLD have lower levels of JAM-A and higher levels of inflammation than subjects without NAFLD. These findings indicate that intestinal epithelial barrier function and microbial dysbiosis contribute to the development of NASH. Restoration of intestinal barrier integrity and manipulation of gut microbiota might be developed as therapeutic strategies for patients with NASH.

• PM2.5 treatment enhances Shp2 level in mouse lungs and pulmonary epithelial cells. • PM2.5 impairs airway epithelial barrier function by reducing claudin-4 expression. • Inhibition of Shp2 activity reverses PM2.5-induced claudin-4 downregulation. • PM2.5 triggers Shp2-ERK1/2 pathway to impair airway epithelial barrier function. The impact of fine particulate matter (PM2.5) on public health has received increasing attention. Through various biochemical mechanisms, PM2.5 alters the normal structure and function of the airway epithelium, causing epithelial barrier dysfunction. Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) has been implicated in various respiratory diseases; however, its role in PM2.5-induced epithelial barrier dysfunction remains unclear. Herein, we assessed the regulatory effects of Shp2 on PM2.5-mediated epithelial barrier function and tight junction (TJ) protein expression in both mice and human pulmonary epithelial (16HBE) cells. We observed that Shp2 levels were upregulated and claudin-4 levels were downregulated after PM2.5 stimulation in vivo and in vitro. Mice were exposed to PM2.5 to induce acute lung injury, and disrupted epithelial barrier function, with decreased transepithelial electrical resistance (TER) and increased paracellular flux that was observed in 16HBE cells. In contrast, the selective inhibition or knockdown of Shp2 retained airway epithelial barrier function and reversed claudin-4 downregulation that triggered by PM2.5, and these effects may occur through the ERK1/2 MAPK signaling pathway. These data highlight an important role of Shp2 in PM2.5-induced airway epithelial barrier dysfunction and suggest a possible new course of therapy for PM2.5-induced respiratory diseases. [ABSTRACT FROM AUTHOR]

BackgroundThe EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased β-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2.ResultsWe found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and β-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-β-catenin upstream of PI3K and AKT.ConclusionsWe conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of β-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

BackgroundThe EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased β-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2.ResultsWe found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and β-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-β-catenin upstream of PI3K and AKT.ConclusionsWe conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of β-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.