Claudin-4 Modulates Autophagy via SLC1A5/LAT1 as a Mechanism to Regulate Micronuclei.

Genome instability is a hallmark of cancer crucial for tumor heterogeneity and is often a result of defects in cell division and DNA damage repair. Tumors tolerate genomic instability, but the accumulation of genetic aberrations is regulated to avoid catastrophic chromosomal alterations and cell death. In ovarian cancer tumors, claudin-4 is frequently upregulated and closely associated with genome instability and worse patient outcomes. However, its biological association with regulating genomic instability is poorly understood. Here, we used CRISPR interference and a claudin mimic peptide to modulate the claudin-4 expression and its function in vitro and in vivo. We found that claudin-4 promotes a tolerance mechanism for genomic instability through micronuclei generation in tumor cells. Disruption of claudin-4 increased autophagy and was associated with the engulfment of cytoplasm-localized DNA. Mechanistically, we observed that claudin-4 establishes a biological axis with the amino acid transporters SLC1A5 and LAT1, which regulate autophagy upstream of mTOR. Furthermore, the claudin-4/SLC1A5/LAT1 axis was linked to the transport of amino acids across the plasma membrane as one of the potential cellular processes that significantly decreased survival in ovarian cancer patients. Together, our results show that the upregulation of claudin-4 contributes to increasing the threshold of tolerance for genomic instability in ovarian tumor cells by limiting its accumulation through autophagy. SIGNIFICANCE: Autophagy regulation via claudin-4/SLC1A5/LAT1 has the potential to be a targetable mechanism to interfere with genomic instability in ovarian tumor cells.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Single-cell transcriptomics reveal metastatic CLDN4+ cancer cells underlying the recurrence of malignant pleural effusion in patients with advanced non-small-cell lung cancer.

BACKGROUND: Recurrent malignant pleural effusion (MPE) resulting from non-small-cell lung cancer (NSCLC) is easily refractory to conventional therapeutics and lacks predictive markers. The cellular or genetic signatures of recurrent MPE still remain largely uncertain. METHODS: 16 NSCLC patients with pleural effusions were recruited, followed by corresponding treatments based on primary tumours. Non-recurrent or recurrent MPE was determined after 3-6 weeks of treatments. The status of MPE was verified by computer tomography (CT) and cytopathology, and the baseline pleural fluids were collected for single-cell RNA sequencing (scRNA-seq). Samples were then integrated and profiled. Cellular communications and trajectories were inferred by bioinformatic algorithms. Comparative analysis was conducted and the results were further validated by quantitative polymerase chain reaction (qPCR) in a larger MPE cohort from the authors' centre (n = 64). RESULTS: The scRNA-seq revealed that 33 590 cells were annotated as 7 major cell types and further characterized into 14 cell clusters precisely. The cell cluster C1, classified as Epithelial Cell Adhesion Molecule (EpCAM)+ metastatic cancer cell and correlated with activation of tight junction and adherence junction, was significantly enriched in the recurrent MPE group, in which Claudin-4 (CLDN4) was identified. The subset cell cluster C3 of C1, which was enriched in recurrent MPE and demonstrated a phenotype of ameboidal-type cell migration, also showed a markedly higher expression of CLDN4. Meanwhile, the expression of CLDN4 was positively correlated with E74 Like ETS Transcription Factor 3 (ELF3), EpCAM and Tumour Associated Calcium Signal Transducer 2 (TACSTD2), independent of driver-gene status. CLDN4 was also found to be associated with the expression of Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Vascular Endothelial Growth Factor A (VEGFA), and the cell cluster C1 was the major mediator in cellular communication of VEGFA signalling. In the extensive MPE cohort, a notably increased expression of CLDN4 in cells from pleural effusion among patients diagnosed with recurrent MPE was observed, compared with the non-recurrent group, which was also associated with a trend towards worse overall survival (OS). CONCLUSIONS: CLDN4 could be considered as a predictive marker of recurrent MPE among patients with advanced NSCLC. Further validation for its clinical value in cohorts with larger sample size and in-depth mechanism studies on its biological function are warranted. TRIAL REGISTRATION: Not applicable.

The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function.

AIMS: Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. METHODS: ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. RESULTS: ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. CONCLUSION: ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.

Effect of sodium butyrate treatment at the basolateral membranes on the tight junction barrier function via a monocarboxylate transporter in goat mammary epithelial cells.

In lactating mammary glands, tight junctions (TJs) prevent blood from mixing with milk and maintain epithelial cell polarity, which is important for milk production. This study aimed to investigate the effect of sodium acetate and sodium butyrate (SB) stimulation direction on the TJ barrier function, which is measured with regard to transepithelial electrical resistance and fluorescein flux, in goat mammary epithelial cells. The expression and localization of the TJ proteins claudin-3 and claudin-4 were examined using Western blotting and immunofluorescence. SB treatment in the lower chamber of cell culture inserts adversely affected the TJ barrier function, whereas sodium acetate barely had any effect, regardless of stimulation direction. In addition, SB treatment in the lower chamber significantly upregulated claudin-3 and claudin-4, whereas TJ proteins showed intermittent localization. Moreover, SB induced endoplasmic reticulum (ER) stress. ARC155858, a monocarboxylate transporter-1 inhibitor, alleviated the adverse impact of SB on TJs and the associated ER stress. Interestingly, sodium ß-hydroxybutyrate, a butyrate metabolite, did not affect the TJ barrier function. Our findings indicate that sodium acetate and SB influence the TJ barrier function differently, and excessive cellular uptake of SB can disrupt TJs and induce ER stress.

Expression of Claudin-4 and D2-40 and their significance in prostatic adenocarcinoma.

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.

Acute Modulations in Stratum Corneum Permeability Barrier Function Affect Claudin Expression and Epidermal Tight Junction Function via Changes of Epidermal Calcium Gradient

Tight junction (TJ) is recognized as a second barrier of the skin. Altered expression of TJ proteins in various skin diseases characterized by the abnormal permeability barrier such as psoriasis suggests that TJ could be affected by stratum corneum (SC) barrier status. However, the physiological relationship between SC and TJ barrier remains to be investigated. Therefore, we examined the effect of SC barrier disruption on the expression of TJ proteins, claudin (Cldn)-1 and Cldn-4, and TJ barrier function in hairless mouse skin. We also investigated whether the alterations in epidermal Ca2+ affected TJ proteins expression in vivo. Repeated tape-stripping induced a sequential change of the expression and function of TJ. As early as 15-30 minutes after tape-stripping, downregulation of Cldn-1 and Cldn-4 immunoreactivity and protein level without change in mRNA level was found. This was accompanied by the abnormal leakage of lanthanum. However, by 1 hour Cldn-1 and Cldn-4 immunolocalization recovered along with normalized lanthanum permeation pattern. Moreover, the mRNA and protein levels of Cldn-1 and Cldn-4 were increased by 1 to 6 hours after tape-stripping. Inhibition of calcium loss by immersion of barrier-disrupted skin into a high Ca2+ solution prevented the dislocation of Cldn-1 and Cldn-4. Occlusion of barrier-disrupted skin delayed the restoration of Cldn-1 and Cldn-4. Our results suggest that the alteration of epidermal Ca2+ gradient caused by SC barrier perturbation affects the TJ structure and function and the faster recovery of TJ as compared to the SC barrier may imply the protective homeostatic mechanism of skin barrier.