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Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na+, Cl−, and Ca2+, and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.

Background Barrier function is essential for the maintenance of normal intestinal function. Dysregulation of the intestinal barrier underlies a wide range of disorders. Aim Previously, we found that sodium butyrate (NaB) decreased the molecular permeability of intestinal barrier in vivo model, but the mechanism by which NaB facilitated the tightness of tight junctions (TJs) in small intestinal epithelium needed further studies. Methods In vitro culture of the cdx2-IEC monolayer was used to mimic barrier function. The TJs were assessed by transepithelial electrical resistance (TEER) and paracellular flux of fluorescein isothiocyanate-conjugated dextran 40,000 (FD-40), Western blot, Q-RT-PCR, and immunofluorescence. Promoter and chromatin immunoprecipitation (ChIP) assays were also done to analyze the Claudin-1 gene. Results NaB decreased FD-40 flux, increased TEER and TJ protein Claudin-1 expression, induced ZO-1 and Occludin redistribution in cellular membrane, and reversed the damage effect after calcium (Ca 2+ ) switch assay. Silencing Claudin-1 prevented protective function of NaB from enhancing intestinal barrier integrity. Further studies demonstrated that NaB increased Claudin-1 transcription by facilitating the interaction between transcription factor SP1 and a specific motif within the promoter region of Claudin-1. This SP1 binding motif was located upstream of the coding region (−138 to −76 bp) and indispensable for the transcription of Claudin-1 following NaB treatment. ChIP assay confirmed the association between SP1 and Claudin-1 promoter, and the elimination of the SP1 binding site by point mutation resulted in a significant loss of Claudin-1 transcription after NaB dealing. Conclusions NaB enhanced intestinal barrier function through increasing Claudin-1 transcription via facilitating the association between SP1 and Claudin-1 promoter.

Diabetic nephropathy is the most common cause of end-stage renal disease. Shu Wakino and colleagues now show that high-glucose conditions in the renal proximal tubules result in downregulation of Sirt1 expression there and in the glomeruli, resulting in epigentic upregulation of Claudin-1 in the glomeruli and thus proteinuria. They also show that genetic or chemical targeting of Sirt1 in the kidney is sufficient to improve kidney function in a mouse model of diabetic nephropathy. Sirtuin 1 (Sirt1), a NAD + -regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese ( db / db ) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in humans. It was recently noted that the characteristics of epidermal barrier functions critically influence the pathological features of AD. Evidence suggests that claudin-1 (CLDN1), a major component of tight junctions (TJs) in the epidermis, plays a key role in human AD, but the mechanism underlying this role is poorly understood. One of the main challenges in studying CLDN1’s effects is that Cldn1 knock-out mice cannot survive beyond 1 d after birth, due to lethal dehydration. Here, we established a series of mouse lines that express Cldn1 at various levels and used these mice to study Cldn1’s effects in vivo. Notably, we discovered a dose-dependent effect of Cldn1’s expression in orchestrating features of AD. In our experimental model, epithelial barrier functions and morphological changes in the skin varied exponentially with the decrease in Cldn1 expression level. At low Cldn1 expression levels, mice exhibited morphological features of AD and an innate immune response that included neutrophil and macrophage recruitment to the skin. These phenotypes were especially apparent in the infant stages and lessened as the mice became adults, depending on the expression level of Cldn1. Still, these adult mice with improved phenotypes showed an enhanced hapten-induced contact hypersensitivity response compared with WT mice. Furthermore, we revealed a relationship between macrophage recruitment and CLDN1 levels in human AD patients. Our findings collectively suggest that CLDN1 regulates the pathogenesis, severity, and natural course of human AD.

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with inside-out and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1β expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci . In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment.

Claudin-1 (CLDN1), a tight junctional protein, is highly expressed in lung cancer cells and may contribute to chemoresistance. A drug which decreases CLDN1 expression could be a chemosensitizer for enhancing the efficacy of anticancer drugs, but there is no such drug known. We found that PMTPV, a short peptide, which mimics the structure of second extracellular loop (ECL2) of CLDN1, can reduce the protein level of CLDN1 without affecting the mRNA level in A549 cells derived from human lung adenocarcinoma. The PMTPV-induced decrease in CLDN1 expression was inhibited by monodansylcadaverine, a clathrin-mediated endocytosis inhibitor, and chloroquine, a lysosome inhibitor. Quartz crystal microbalance assay showed that PMTPV can directly bind to the ECL2 of CLDN1. In transwell assay, PMTPV increased fluxes of Lucifer yellow (LY), a paracellular flux marker, and doxorubicin (DXR), an anthracycline anticancer drug, without affecting transepithelial electrical resistance. In three-dimensional spheroid culture, the size and cell viability were unchanged by short peptides, but the fluorescence intensity of hypoxia probe LOX-1 was decreased by PMTPV. PMTPV elevated the accumulation and cytotoxicity of DXR in the spheroids. Similar results were observed by knockdown of CLDN1. Furthermore, the sensitivities to cisplatin (CDDP), docetaxel, and gefitinib were enhanced by PMTPV. The level of CLDN1 expression in CDDP-resistant cells was higher than that in parental A549 cells, which was reduced by PMTPV. PMTPV restored the toxicity to DXR in the CDDP-resistant cells. Our data suggest that PMTPV may become a novel chemosensitizer for lung adenocarcinoma.

Cigarette smoke is the leading cause of lung diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). While harmful effects of cigarette smoke on lung epithelial function are well established, the consequences of inhalation of aerosols from e-cigarettes are less clear and disputed. In this study, human lung adenocarcinoma epithelial cells (Calu-3) were treated with extracts of cigarette smoke (CSE) or e-cigarette vapor (EVE) and comparatively analyzed for barrier function and integrity as well as for mRNA and protein expression of components forming the apical junctional complex (AJC). In addition, potential proinflammatory and genotoxic effects were investigated. Our results clearly demonstrate that CSE induced considerable disruption of the epithelial barrier leading to hyperpermeability and reduced protein expression of AJC proteins, especially of claudin-1. In addition, exposure of cells to CSE induced pronounced proinflammatory effects and increased DNA double strand breaks. By contrast, EVE, even if applied undiluted, neither affected functional nor structural parameters. In summary, on the basis of our results obtained with the Calu-3 in vitro model, we propose that e-cigarette vapor may exert less harmful effects on lung epithelial cells compared to cigarette smoke.

BackgroundClaudin-1 is a membrane-tight junction protein and important for the sealing of the paracellular cleft in epithelial and endothelial cells. Differential expression of Claudin-1 is linked to disease outcome in various cancers.Material and methodsTo evaluate the potential relevance of Claudin-1 expression in prostate cancer, a tissue microarray containing samples of 17,747 tumors with annotated clinico-pathological and molecular data was immunohistochemically analyzed for Claudin-1 expression.ResultsIn normal prostate, glandular cells were always Claudin-1-negative while there was a strong staining of gland-surrounding basal cells. In contrast to normal prostatic glands, a positive Claudin-1 immunostaining, was found, however, in 38.7% of 12,441 interpretable cancers and was considered weak in 12.7%, moderate in 13.2%, and strong in 12.8% of cases. Positive Claudin-1 immunostaining was associated with favorable tumor features like low pT (p = 0.0032), low Gleason grade (p< 0.0001), and a reduced risk of PSA recurrence (p = 0.0005). A positive Claudin-1 staining was markedly more frequent in ERG-positive (63%) than in ERG-negative cancers (23%; p < 0.0001). Subset analyses revealed that all associations of Claudin-1 expression and favorable phenotype and prognosis were driven by ERG-positive cancers. Multivariate analyses revealed, however, that even in ERG-positive cancers, the prognostic impact of high Claudin-1 expression was not independent of established clinico-pathological parameters. Comparison with 12 previously analyzed chromosomal deletions identified conspicuous associations with PTEN and 12p13 deletions potentially indicating functional interactions.ConclusionThese data identify a peculiar role for Claudin-1 in prostate cancer. The protein is overexpressed in a fraction of prostate cancers and increased Claudin-1 expression levels predict a favorable prognosis in ERG-positive cancer.

The pilosebaceous unit is the site of a common chronic inflammatory skin disorder called acne. Tight junctions (TJ) are present in sebocytes and are primarily composed of claudin-1. When claudin-1 is knocked down, the TJ barrier leaks, and holocrine secretion is compromised. This leads to the accumulation of partially degenerated sebocytes in the pilosebaceous ducts and the creation of microcomedos, which can cause acne lesions. The study’s aim was to measure the claudin-1 gene and protein expression in acne patients’ blood samples and establish a correlation with the severity of the condition and their levels. 48 acne patients were involved and 48 matched control for age and sex. First samples were obtained from both groups and RNA was extracted from the whole blood sample followed by qPCR to measure the relative expression of the claudin-1 gene. Enzyme-linked immunosorbent assay (ELISA) was used to measure claudin-1 protein level. There were statistically significant lower relative expression of claudin-1 gene (p = 0.001) and its protein level (p = 0.001) in acne patients compared to controls (0.34 ± 0.32 vs 1.0 ± 0.0, 0.64 ± 0.62 vs 3.85 ± 2.14, respectively). There was a statistically significant inverse correlation between claudin-1 protein expression and acne severity (R = −0.374, p = 0.009). Computational and bioinformatics tools were used for enrichment analysis to better understand claudin-1 role in the context of the disease and provide possible therapeutic approaches. Claudin-1 dysregulation in acne patients, suggest that it have an impact on the severity and course of acne through affecting the epidermal barrier and facilitating inflammation.

This study was conducted to investigate impacts of dietary protein levels on gut bacterial community and gut barrier. The intestinal microbiota of finishing pigs, fed with 16%, 13% and 10% crude protein (CP) in diets, respectively, were investigated using Illumina MiSeq sequencing. The ileal bacterial richness tended to decrease when the dietary protein concentration reduced from 16% to 10%. The proportion of Clostridium_sensu_stricto_1 in ileum significantly decreased, whereas Escherichia-Shigella increased with reduction of protein concentration. In colon, the proportion of Clostridium_sensu_stricto_1 and Turicibacter increased, while the proportion of RC9_gut_group significantly decreased with the dietary protein reduction. Notably, the proportion of Peptostreptococcaceae was higher in both ileum and colon of 13% CP group. As for metabolites, the intestinal concentrations of SCFAs and biogenic amines decreased with the dietary protein reduction. The 10% CP dietary treatment damaged ileal mucosal morphology, and decreased the expression of biomarks of intestinal cells (Lgr5 and Bmi1), whereas the expression of tight junction proteins (occludin and claudin) in 13% CP group were higher than the other two groups. In conclusion, moderate dietary protein restriction (13% CP) could alter the bacterial community and metabolites, promote colonization of beneficial bacteria in both ileum and colon, and improve gut barrier function.