KHDRBS3 promotes paclitaxel resistance and induces glycolysis through modulated MIR17HG/CLDN6 signaling in epithelial ovarian cancer.

Paclitaxel (PTX) resistance contributes to mortality in epithelial ovarian cancer (EOC). Aerobic glycolysis is elevated in the tumor environment and may influence resistance to PTX in EOC. KH domain-containing, RNA-binding signal transduction-associated protein 3 (KHDRBS3) is an RNA binding protein that is up-regulated in EOC, but its underlying mechanism in EOC is unclear. Here, we investigate the role of KHDRBS3 in glycolysis and increased resistance to PTX. Expression of KHDRBS3 and Claudin (CLDN6) were measured in EOC tissue and cells by quantitative real-time PCR, western blotting and immunohistochemistry. The biological functions of KHDRBS3, MIR17HG and CLDN6 were examined using MTT, colony formation, apoptosis and seahorse assays in vitro. For in vivo experiments, a xenograft model was used to investigate the effects of KHDRBS3 and MIR17HG in EOC. Here, we investigate the role of KHDRBS3 in glycolysis and increased resistance to PTX. The expression of KHDRBS3 was up-regulated in PTX-resistant cells. KHDRBS3 knockdown restrained the IC50 of PTX, cell proliferation, colony formation and glycolysis in SKOV3-R and A2780-R cells in vitro and enhanced PTX sensitivity in a xenograft mouse model in vivo. KHDRBS3 interacts with lncRNA MIR17HG, which is down-regulated in EOC tissue and cells. The effect of KHDRBS3 overexpression on PTX resistance and glycolysis was rescued by MIR17HG overexpression. Additionally, MIR17HG interacts with the 3'UTR of CLDN6 and negatively regulates CLDN6 expression. MIR17HG overexpression suppressed the IC50 of PTX and glycolysis by targeting CLDN6. Our results reveal a KHDRBS3-MIR17HG-CLDN6 regulatory axis that contributes to enhanced glycolysis in EOC and represents a potential target for therapy.

Reduced Claudin-12 Expression Predicts Poor Prognosis in Cervical Cancer.

BACKGROUND: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. METHODS: We generated a monoclonal Ab (mAb) against human/mouse CLDN12 and verified its specificity. By performing immunohistochemical staining and semiquantification, we evaluated the relationship between CLDN12 expression and clinicopathological parameters in tissues from 138 cases of cervical cancer. RESULTS: Western blot and immunohistochemical analyses revealed that the established mAb selectively recognized the CLDN12 protein. Twenty six of the 138 cases (18.8%) showed low CLDN12 expression, and the disease-specific survival (DSS) and recurrence-free survival rates were significantly decreased compared with those in the high CLDN12 expression group. We also demonstrated, via univariable and multivariable analyses, that the low CLDN12 expression represents a significant prognostic factor for the DSS of cervical cancer patients (HR 3.412, p = 0.002 and HR 2.615, p = 0.029, respectively). CONCLUSIONS: It can be concluded that a reduced CLDN12 expression predicts a poor outcome for cervical cancer. The novel anti-CLDN12 mAb could be a valuable tool to evaluate the biological relevance of the CLDN12 expression in diverse cancer types and other diseases.

High dose lithium chloride causes colitis through activating F4/80 positive macrophages and inhibiting expression of Pigr and Claudin-15 in the colon of mice.

OBJECTIVE: Lithium chloride (LiCl) was a mood stabilizer for bipolar affective disorders and it could activate Wnt/ß-catenin signaling pathway both in vivo and in vitro. Colon is one of a very susceptible tissues to Wnt signaling pathway, and so it would be very essential to explore the toxic effect of a high dose of LiCl on colon. METHODS: C57BL/6 mice were injected intraperitoneally with 200 mg/kg LiCl one dose a day for 5 days to activate Wnt signal pathway in intestines. H&E staining was used to assess the colonic tissues of mice treated with high dose of LiCl. The expression of inflammation-associated genes and tight junction-associated genes in colons was measured using qPCR, Western blot and immunostaining methods. The gut microbiome was tested through 16S rDNA gene analysis. RESULTS: The differentiation of enteroendocrine cells in colon was inhibited by treatment of 200 mg/kg LiCl. The F4/80 positive macrophages in colon were activated by high dose of LiCl, and migrated from the submucosa to the lamina propria. The expression of pro-inflammatory genes TNFα and IL-1ß was increased in the colon of high dose of LiCl treated mice. Clostridium_sp_k4410MGS_306 and Prevotellaceae_UCG_001 were specific and predominant for the high dose of LiCl treated mice. The expression of IgA coding genes, Pigr and Claudin-15 was significantly decreased in the colon tissues of the high dose of LiCl treated mice. CONCLUSION: 200 mg/kg LiCl might cause the inflammation in colon of mice through activating F4/80 positive macrophages and inhibiting the expression of IgA coding genes in plasma cells and the expression of Pigr and Claudin-15 in colonic epithelial cells, providing evidences for the toxic effects of high dose of LiCl on colon.

Reduced occludin and claudin-7 expression is associated with urban locations and exposure to second-hand smoke in allergic rhinitis patients.

The breakdown of nasal epithelial barrier occurs in allergic rhinitis (AR) patients. Impairment of cell junction molecules including tight junctions (TJs) and desmosomes plays causative roles in the pathogenesis of AR. In this study, we investigated the transcript expression levels of TJs including occludin (OCLN), claudin-3 and -7 (CLDN3 and CLDN7), desmoglein 3 (DSG3) and thymic stromal lymphopoietin (TSLP) in AR patients (n = 30) and non-allergic controls (n = 30). Nasal epithelial cells of non-allergic controls and AR patients were collected to examine their mRNA expression levels, and to correlate with clinico-demographical and environmental parameters. We demonstrated that the expression of OCLN (p = 0.009), CLDN3 (p = 0.032) or CLDN7 (p = 0.004) transcript was significantly lower in AR patients compared with non-allergic controls. No significant difference was observed in the expression of DSG3 (p = 0.750) or TSLP (p = 0.991) transcript in AR patients compared with non-allergic controls. A significant association between urban locations and lower OCLN expression (p = 0.010), or exposure to second-hand smoke with lower CLDN7 expression (p = 0.042) was found in AR patients. Interestingly, none of the TJs expression was significantly associated with having pets, frequency of changing bedsheet and housekeeping. These results suggest that defective nasal epithelial barrier in AR patients is attributable to reduced expression of OCLN and CLDN7 associated with urban locations and exposure to second-hand smoke, supporting recent findings that air pollution represents one of the causes of AR.

E7 oncoprotein from human papillomavirus 16 alters claudins expression and the sealing of epithelial tight junctions.

Tight junctions (TJs) are cell­cell adhesion structures frequently altered by oncogenic transformation. In the present study the role of human papillomavirus (HPV) 16 E7 oncoprotein on the sealing of TJs was investigated and also the expression level of claudins in mouse cervix and in epithelial Madin­Darby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins ­1 and ­10 in the cervix of 7­month­old transgenic K14E7 mice treated with 17ß­estradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin­1 expression in the cervix of 2­month­old K14E7 mice, and claudin­10 accumulated at the border of cells in the upper layer of the cervix in FvB mice treated with E2, and in K14E7 mice treated with or without E2. These changes were accompanied by an augmented paracellular permeability of the cervix in 2­ and 7­month­old FvB mice treated with E2, which became more pronounced in K14E7 mice treated with or without E2. In MDCK cells the stable expression of E7 increased the space between adjacent cells and altered the architecture of the monolayers, induced the development of an acute peak of transepithelial electrical resistance accompanied by a reduced expression of claudins ­1, ­2 and ­10, and an increase in claudin­4. Moreover, E7 enhances the ability of MDCK cells to migrate through a 3D matrix and induces cell stiffening and stress fiber formation. These observations revealed that cell transformation induced by HPV16 E7 oncoprotein was accompanied by changes in the pattern of expression of claudins and the degree of sealing of epithelial TJs.

The role of cldnh during the early retinal development in zebrafish.

Claudin-3, an integral component of tight junction, has recently been shown to be expressed in retinal ganglion cells, retinal pigment cells, and retinal vascular endothelial cells. However, the role of claudin-3 in the development of the neural retina and its vessels remains undefined. This study aimed to investigate the role of zebrafish claudin-h (cldnh), the closest ortholog of mouse and human claudin-3, in the development of the neural retina and its vessels. Cldnh levels in green fluorescent protein transgenic zebrafish were genetically manipulated by cldnh morpholino oligonucleotide (MO) and cldnh mRNA to investigate gene function. The expression of cldnh was analyzed using polymerase chain reaction and immunofluorescence staining. The altered morphological, cellular and molecular events in the cldnh MO-morphant eyes were detected using hematoxylin-eosin staining, fluorescent dye injection, confocal in vivo imaging, BrdU labeling, TUNEL assay, RNA sequencing, and Western blot. We demonstrated that the cldnh protein was expressed in the neural retina and the hyaloid vessel which is the predecessor of the retinal vessel in zebrafish. Cldnh knockdown delayed lamination of the neural retina and reduced its thickness, which might be associated with the downregulation of the retinal development-related genes of atoh7, pcdh17, crx, neurod1, insm1a, sox9b and cdh11, and the upregulation of the cell cycle and apoptosis-associated genes of tp53, cdkn1a and casp8. Cldnh knockdown also reduced the density and interrupted the lumenization of the hyaloid vessels, which might be owing to the downregulation of the vessel formation-related genes of hlx1 and myl7. In conclusion, cldnh was required for the normal development of the neural retina and its vessels in zebrafish, providing a basis for elucidating its role in the pathogenesis of retinal vascular or inflammatory diseases.

Claudin-7 Inhibits Proliferation and Metastasis in Salivary Adenoid Cystic Carcinoma Through Wnt/ß-Catenin Signaling.

The aim of this study was to investigate claudin-7 (CLDN7) expression in salivary adenoid cystic carcinoma (SACC) and its function in SACC cells. We determined CLDN7 expression in SACC tumors via immunohistochemistry and western blotting and evaluated the association between CLDN7 expression and clinicopathologic variables. Besides this, we constructed a stably transfected CLDN7 knockdown SACC-LM cell line via RNAi and assessed its biological behavior changes (cell viability, migration, and invasion). The correlation between CLDN7 and epithelial-mesenchymal transition (EMT) was analyzed. Additionally, a subcutaneous tumor formation model was used to assess SACC-LM cells tumorigenicity after the CLDN7 knockdown. In the present study, we found the CLDN7 expression of tumor group was lower than that in normal salivary glands and was significantly correlated with lymph node metastasis, recurrence, and gender. CLDN7 knockdown could add the proliferation and metastasis ability of SACC by regulating EMT through Wnt/ß-catenin signaling pathway. In addition, CLDN7 knockdown in SACC promoted tumor growth in nude mice. CLDN7 inhibits cell proliferation and metastasis by inactivating the Wnt/ß-catenin signaling in SACC. Thus, CLDN7 expression might be a useful marker to identify the potential for progression in SACC.

Claudins regulate gene and protein expression of the retinal pigment epithelium independent of their association with tight junctions.

Claudin-19 is the major claudin in the tight junctions of the retinal pigment epithelium (RPE). Claudin-3 is also uniformly expressed albeit in lesser amounts. Besides modulating transepithelial diffusion, claudins modulate gene expression. The absence of claudin-19 and claudin-3 in the RPE cell lines, ARPE-19 and hTERT-RPE-1, provide an opportunity to examine whether exogenous claudins regulate gene expression in the absence of tight junctions. Quantitative RT-PCR was used to compare gene expression in ARPE-19 and hTERT-RPE-1 with that of highly differentiated, human fetal RPE. Claudin-19 and claudin-3 were exogenously expressed using an adenoviral vector. The transepithelial electrical resistance (TER) was measured using Endohm electrodes, and the effects of claudin on the actin cytoskeleton were determined by immunocytochemistry. The effect of claudin on gene expression was examined by quantitative RT-PCR and western blotting. Aside from claudin-19 and claudin-3, ARPE-19 and hTERT-RPE-1 expressed most junction-associated mRNAs in amounts comparable to human fetal RPE, but some RPE signature and maturation genes were under-expressed. Unlike ARPE-19, hTERT-RPE-1 failed to form tight junctions or develop a TER. Claudins exogenously expressed in hTERT-RPE-1 failed to crystalize an apical junctional complex. Actin filaments were not redistributed from stress fibers to cortical bands, and a TER was not established. In hTERT-RPE-1, claudins were found only in internal vesicular-like structures. Nonetheless, claudins increased the expression of the mRNAs for a collection of RPE-enriched proteins. Claudin-19 and claudin-3 had different effects on gene and protein expression indicating activation of overlapping, but distinct, signaling pathways. A major difference was the ability of claudin-19 to affect steady-state levels of ADAM9 and tyrosinase in ARPE-19. In conclusion, claudins can increase the barrier function of a pre-existing apical junctional complex, but on its own it cannot recruit tight junction proteins to form a complex de novo. Many effects of claudin on gene expression did not require an association with the apical junctional complex. Although claudin-19 shared many effects with claudin-3, claudin-19 exerted unique effects on the maturation of RPE.

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability.

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (PCa). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with ß-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and PCa was measured. PCa in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of PCa in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Upregulation of Claudin-7 Expression by Angiotensin II in Colonic Epithelial Cells of Mice Fed with NaCl-Depleted Diets.

Dietary NaCl depletion increases Na+ and Cl- absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1- without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl- mediated by the elevation of CLDN7 expression in the colon.

The Significance of Relative Claudin Expression in Odontogenic Tumors.

Claudins are integral to the structure and function of tight junctions. Altered claudin expression has been shown to affect disease behavior and patient prognosis in various neoplasms. The objectives of this study were to analyze the claudin-1, -4 and -7 expression in odontogenic tumors and characterize their expression pattern in distinct tumor cell types in relation to the recurrence potential. Sixty-nine cases of odontogenic tumors, including 43 ameloblastomas (AM), 17 adenomatoid odontogenic tumors (AOT), 6 ameloblastic fibromas (AF) and 3 ameloblastic carcinomas (AC) were investigated for claudin-1, -4 and -7 expression immunohistochemically. The staining was analyzed semi-quantitatively and categorized into 4 levels, based on the percentage of positively stained neoplastic epithelial cells. Claudin-1 was expressed in all AOT and AF cases, whereas most AC (66.7%) showed no expression. The claudin-1 staining was moderate-to-intense in the odontogenic epithelium of AF. In contrast, its staining of ameloblast-like cells and stellate reticulum-like cells in AM was weak. Claudin-7 expression was noted in all tumor types studied, while the expression of claudin-4 was limited and mainly localized in the squamous differentiated cells of AM and AC. AM showed significantly higher claudin-4, but lower claudin-7 expression than AOT. In addition, AC showed diminished claudin-1 immunoreactivity, compared to AOT. Low claudin-1 expression in AM was significantly associated with the increased clinical recurrence. The loss of claudin-1 may underlie the locally invasive nature of AM.

Sox2 maintains epithelial cell proliferation in the successional dental lamina.

OBJECTIVES: The successional dental lamina is the distinctive structure on the lingual side of the vertebrate tooth germ. The aim of this study was to investigate the relationship among Sox2, Claudin10 and laminin5 and the role of Sox2 in successional dental lamina proliferation during vertebrate tooth development. MATERIALS AND METHODS: To understand the successional dental lamina, two types of successional tooth formation, that in geckos (with multiple rounds of tooth generation) and that in mice (with only one round of tooth generation), were analysed. RESULTS: Unique coexpression patterns of Sox2 and Claudin10 expression were compared in the successional dental lamina from the cap stage to the late bell stage in the mouse tooth germ and in juvenile gecko teeth to support continuous tooth replacement. Furthermore, Laminin5 expression was shown in the cap stage and decreased after the bell stage. Upon comparing the epithelial cell cycles and cell proliferation in successional dental lamina regions between mouse and gecko molars using BrdU and IdU staining and pulse-chase methods, distinctive patterns of continuous expression were revealed. Moreover, Sox2 overexpression with a lentiviral system resulted in hyperplastic dental epithelium in mouse molars. CONCLUSIONS: Our findings indicate that the regulation of Sox2 in dental lamina proliferation is fundamental to the successional dental lamina in both species.

In vitro effects of glyphosate and Roundup on Sertoli cell physiology.

Roundup (R), a formulation that contains glyphosate (G) as the active ingredient, is a commonly used nonselective herbicide that has been proposed to affect male fertility. It is well known that an adequate Sertoli cell function is essential to maintain germ cell development. The aim of the present study was to analyze whether G and R are able to affect Sertoli cell functions, such as energy metabolism and blood-testis barrier (BTB) integrity. Sertoli cell cultures from 20-day-old rats were exposed to 10 and 100 ppm of G or R, doses which do not decrease cell viability. Neither G nor R caused impairment in lactate production or fatty acid oxidation. G and R decreased Transepithelial Electrical Resistance, which indicates the establishment of a Sertoli cell junction barrier. However, neither G nor R modified the expression of claudin11, ZO1 and occludin, proteins that constitute the BTB. Analysis of cellular distribution of claudin11 by immunofluorescence showed that G and R induced a delocalization of the signal from membrane to the cytoplasm. The results suggest that G and R could alter an important function of Sertoli cell such as BTB integrity and thus they could compromise the normal development of spermatogenesis.

Claudin-2: Roles beyond Permeability Functions.

Claudin-2 is expressed in the tight junctions of leaky epithelia, where it forms cation-selective and water permeable paracellular channels. Its abundance is under fine control by a complex signaling network that affects both its synthesis and turnover in response to various environmental inputs. Claudin-2 expression is dysregulated in many pathologies including cancer, inflammation, and fibrosis. Claudin-2 has a key role in energy-efficient ion and water transport in the proximal tubules of the kidneys and in the gut. Importantly, strong evidence now also supports a role for this protein as a modulator of vital cellular events relevant to diseases. Signaling pathways that are overactivated in diseases can alter claudin-2 expression, and a good correlation exists between disease stage and claudin-2 abundance. Further, loss- and gain-of-function studies showed that primary changes in claudin-2 expression impact vital cellular processes such as proliferation, migration, and cell fate determination. These effects appear to be mediated by alterations in key signaling pathways. The specific mechanisms linking claudin-2 to these changes remain poorly understood, but adapters binding to the intracellular portion of claudin-2 may play a key role. Thus, dysregulation of claudin-2 may contribute to the generation, maintenance, and/or progression of diseases through both permeability-dependent and -independent mechanisms. The aim of this review is to provide an overview of the properties, regulation, and functions of claudin-2, with a special emphasis on its signal-modulating effects and possible role in diseases.

Novel Role for Claudin-11 in the Regulation of Osteoblasts via Modulation of ADAM10-Mediated Notch Signaling.

The claudin (Cldn) family comprises 27 members of 20 to 34 kDa transmembrane tight junction proteins. In addition to Cldns' established canonical role as barriers controlling paracellular flow of molecules, a distinct noncanonical role for them as mediators of cell signaling is now emerging. In our studies evaluating Cldn family expression levels during osteoblast differentiation, Cldn-11 showed the largest increase (60-fold). Immunohistochemistry studies revealed high Cldn-11 expression in trabecular (Tb) bone lining cells. Micro-CT analysis of femurs and vertebrae of Cldn-11 knock-out (KO) mice at 12 weeks of age exhibited a 40% (p < 0.01) reduction in Tb bone volume adjusted for tissue volume compared with control mice, a change caused by significant reductions in Tb number and thickness and increase in Tb separation. Histomorphometry and serum biomarker studies revealed that reduced bone formation, not increased resorption, is the cause for reduced Tb bone volume in the Cldn-11 KO mice. Cldn-11 KO osteoblasts expressed reduced ALP and BSP, whereas Cldn-11 overexpression in MC3T3-E1 cells increased expression of ALP and BSP. Mechanistically, Cldn-11 interacted with tetraspanin (Tspan)3 in osteoblasts, and Tspan3 knockdown reduced osteoblast differentiation. Because members of the Tspan family regulate cell functions via Notch signaling, we evaluated whether Cldn-11/Tspan3 regulates Notch signaling in osteoblasts. Accordingly, Notch targets Hey1 and Hey2 were significantly upregulated in Cldn-11 overexpressing cultures but downregulated in both Cldn-11 KO and Tspan3 knockdown osteoblasts. Because ADAM10 has been shown to interact with Tspan family members to regulate Notch signaling, we evaluated whether Cldn-11 regulates ADAM10 expression. Cldn-11 overexpressing cells express more mature ADAM10, and an ADAM10 inhibitor blocked the Cldn-11 effect on osteoblast differentiation. Based on these data, we propose Cldn-11 as a novel component of an osteoblast cell surface protein complex, comprising Tspan3 and ADAM10, which regulates Notch signaling and cell differentiation. © 2019 American Society for Bone and Mineral Research.

Newly synthesized claudins but not occludin are added to the basal side of the tight junction.

A network of claudin strands creates continuous cell-cell contacts to form the intercellular tight junction barrier; a second protein, occludin, is associated along these strands. The physiological barrier remains stable despite protein turnover, which involves removal and replacement of claudins both in the steady state and during junction remodeling. Here we use a pulse-block-pulse labeling protocol with fluorescent ligands to label SNAP/CLIP-tags fused to claudins and occludin to identify their spatial trafficking pathways and kinetics in Madin-Darby canine kidney monolayers. We find that claudins are first delivered to the lateral membrane and, over time, enter the junction strand network from the basal side; this is followed by slow replacement of older claudins in the strands. In contrast, even at early times, newly synthesized occludin is found throughout the network. Taking the results together with our previous documentation of the mechanism for claudin strand assembly in a fibroblast model, we speculate that newly synthesized claudins are added at strand breaks and free ends; these are most common in the basalmost edge of the junction. In contrast, occludin can be added directly within the strand network. We further demonstrate that claudin trafficking and half-life depend on carboxy-terminal sequences and that different claudins compete for tight junction localization.

Anisomycin, a JNK and p38 activator, suppresses cell-cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures.

Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.

Mucin 5B, carbonic anhydrase 9 and claudin 18 are potential theranostic markers of gallbladder carcinoma.

AIMS: Gallbladder cancer (GBC) is an aggressive tumour that is usually diagnosed at advanced stages and is characterised by a poor prognosis. Using public data of normal human tissues, we found that mRNA and protein levels of mucin 5B (MUC5B) and carbonic anhydrase 9 (CA9) were highly increased in gallbladder tissues. In addition, previous evidence has shown that claudin 18 (CLDN18) protein expression is higher in GBC. The aim of this study was to perform an analysis of these cell surface proteins during the histological progression of GBC in order to identify their theranostic potential. METHODS AND RESULTS: MUC5B expression, CA9 expression and CLDN18 expression were examined by immunohistochemistry in a series of 179 chronic cholecystitis (including 16 metaplastic tissues), 15 dysplasia and 217 GBC samples by the use of tissue microarray analysis. A composite staining score was calculated from staining intensity and percentage of positive cells. Immunohistochemical analysis showed high expression of MUC5B and CA9 among normal epithelium, metaplastic tissues, and dysplastic tissues. However, expression of both proteins was observed in roughly 50% of GBC samples. In contrast, CLDN18 was absent in normal epithelium, but its expression was higher in metaplastic cells. Among GBC cases, approximately half showed high CLDN18 expression. No associations were found between MUC5B, CA9 and CLDN18 expression and any clinicopathological features. CONCLUSIONS: CLDN18 is a new metaplasia marker in gallbladder tissues, and is conserved in approximately half of GBC cases. MUC5B and CA9 are highly conserved during GBC histological progression. The three markers are potential theranostic markers, in particular CA9 and CLDN18, for which there are already targeted therapies available.

Differences in the expression profiles of claudin proteins in human gastric carcinoma compared with non­neoplastic mucosa.

Numerous genetic alterations associated with cancer progression have the potential to serve as biomarkers for the early diagnosis of cancer. Numerous studies have suggested that claudin proteins, which are the primary components of tight junction structures, are associated with the regulation of cell polarity and cell differentiation. To investigate the expression profiles of the tight junction proteins claudin­2, ­5, ­7 and ­8 in gastric carcinoma, immunohistochemical analysis, western blotting and reverse transcription­quantitative polymerase chain reaction analysis was used to detect the expression profiles of these claudin proteins in gastric carcinoma tissues and in homologous non­neoplastic mucosal tissues. According to the present study, the expression levels of claudin­7 and claudin­8 were downregulated, while the expression of claudin­5 was upregulated in gastric carcinoma tissues compared with in non­neoplastic mucosal tissues. Additionally, no notable difference was observed between claudin­2 expression in gastric carcinoma tissues and non­neoplastic mucosae. Correlations between claudin­7 and ­8 expression and lymphatic metastasis in gastric carcinoma tissues were additionally reported. In summary, the present study revealed the distinct expression profiles of claudin­5, ­7 and ­8 in non­neoplastic mucosal tissues and gastric carcinoma tissues. Furthermore, the expression of these claudin proteins was highly associated with metastatic progression and prognosis in patients with gastric carcinoma, and had predictive value for the metastasis and survival of patients with gastric carcinoma.

The expression profile of Claudin family members in the developing mouse lung and expression alterations resulting from exposure to secondhand smoke (SHS).

Claudins are tight junctional proteins implicated in cell polarity and epithelial barrier maintenance. Claudin misregulation adversely impacts developmental aspects of cell differentiation and proliferation. The current research evaluated transcriptional expression for Claudins 1-11 and 18 in the developing murine lung at embryonic days (E) 14.5, 16.5, and 18.5 and at post-natal day (PN) 3 and PN15. Mouse lungs were also assessed by immunohistochemical analysis to qualitatively evaluate Claudin protein expression. Pregnant dams were further exposed to secondhand smoke (SHS) from embryonic day (E)15.5 to 18.5 and Claudin mRNA was immediately screened in pup lungs. Other than Claudin-6, mRNA expression patterns for Claudin family members tended to decrease at E16.5, increase at E18.5, and decrease again at PN3 before reaching a peak of expression at PN15. Claudin-6 mRNA expression decreased through gestation and into post-natal periods. Immunohistochemical profiling implicated a subset of Claudins as plausible orchestrators of proximal vs. distal lung barrier establishment. Assessment of Claudin mRNA expression at E18.5 following SHS exposure revealed a significant reduction in transcription for all Claudins except Claudin-18 (no change). These data support the need for further studies using gene targeted mice that knock-in/out specific Claudins so that precise functions in the normal and diseased lung can be determined.