Members Of Claudin Family Research Articles

The Different Gene Expression Profile in the Eutopic and Ectopic Endometrium Sheds New Light on the Endometrial Seed in Endometriosis.

The changes in endometrial cells, both in the eutopic endometrium of patients with and without endometriosis and in lesions at ectopic sites, are frequently described and often compared to tumorigenesis. In tumorigenesis, the concept of "seed and soil" is well established. The seed refers to tumor cells with metastatic potential, and the soil is any organ or tissue that provides a suitable environment for the seed to grow. In this systematic review (PRISMA-S), we specifically compared the development of endometriosis with the "seed and soil" hypothesis. To determine changes in the endometrial seed, we re-analyzed the mRNA expression data of the eutopic and ectopic endometrium, paying special attention to the epithelial-mesenchymal transition (EMT). We found that the similarity between eutopic endometrium without and with endometriosis is extremely high (~99.1%). In contrast, the eutopic endometrium of patients with endometriosis has a similarity of only 95.3% with the ectopic endometrium. An analysis of EMT-associated genes revealed only minor differences in the mRNA expression levels of claudin family members without the loss of other cell-cell junctions that are critical for the epithelial phenotype. The array data suggest that the changes in the eutopic endometrium (=seed) are quite subtle at the beginning of the disease and that most of the differences occur after implantation into ectopic locations (=soil).

A loss of function mutation in CLDN25 causing Pelizaeus-Merzbacher-like leukodystrophy.

Claudin-25 (CLDN-25), also known as Claudin containing domain 1, is an uncharacterized claudin family member. It has less conserved amino acid sequences when compared to other claudins. It also has a very broad tissue expression profile and there is currently a lack of functional information from murine knockout models. Here, we report a de novo missense heterozygous variant in CLDN25 (c. 745G>C, p. A249P) found in a patient diagnosed with Pelizaeus-Merzbacher-like leukodystrophy and presenting with symptoms such as delayed motor development, several episodes of tonic absent seizures and generalized dystonia. The variant protein does not localize to the cell-cell borders where it would normally be expected to be expressed. Amino acid position 249 is located 4 amino acids from the C-terminal end of the protein where most claudin family members have a conserved binding motif for the key scaffolding protein ZO-1. However, CLDN-25 does not contain this motif. Here, we show that the C-terminal end of CLDN-25 is required for its junctional localization in a ZO-1 independent manner. The A249P mutant protein as well as a deletion mutant lacking its last 5 C-terminal amino acids also failed to localize to the cell-cell border in vitro. Intriguingly, cellular knockout of CLDN25, in vitro, appeared to increase the integrity of the tight junction between 2 contacting cells, while driving highly unusual increased movement of solutes between cells. We propose that the barrier function of CLDN-25 is akin to a decoy claudin, whereby decreasing its expression in "leaky" epithelial cells and endothelial cells will drive dynamic changes in the adhesion and interaction capacity of cell-cell contact points. While it remains unclear how this de novo CLDN-25 mutant induces leukodystrophy, our findings strongly suggest that this mutation induces haploinsufficiency of CLDN-25. Elucidating the function of this uncharacterized claudin protein will lead to a better understanding of the role of claudin proteins in health and disease.

Comprehensive analysis of the expression and prognosis value of claudin family members in clear cell renal cell carcinoma

Objective: Clear cell renal cell carcinoma (ccRCC) accounts for 80% of all renal cancers and has a poor prognosis. Studies have found that the abnormal expression of claudins (CLNDs) can influence the functions of tight junctions (TJs) and plays an important role in the occurrence, development, and metastasis of malignant tumours in a variety of epithelial malignant tumours. However, the roles of the different CLNDs in ccRCC are poorly understood. Methods: Used the GEPIA, UALCAN, HPA, Kaplan-Meier Plotter, cBioPortal, String-DB, GeneMANIA, Metascape, and TIMER databases to collect data. Results: The expression levels of CLDN7, 8, 10, 11, 14, 16, and 19 were significantly reduced and promoters were hypermethylated in ccRCC-affected tissues. ccRCC patients with low expression levels are associated highly with the clinical cancer stages, tumour grades, and poor overall survival. Protein interaction and enrichment analysis showed the differential expressions of CLDNs were primarily associated with epithelial cell differentiation and regulation of cell adhesion. Moreover, differential expressions of CLDNs were significantly correlated with the infiltration of diverse immune cells, including six types of CD4+ T cells, macrophages, neutrophils, B cells, CD8+ T cells, and dendritic cells in ccRCC. Conclusion: They are expected to serve as biomarkers for predicting the prognosis of ccRCC patients.

Claudin-10 is a new candidate prognostic marker in metastatic high-grade serous carcinoma

The objective of this study was to analyze the expression and prognostic role of the tight junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p < 0.001), and was higher in HGSC surgical specimens compared to effusions (p < 0.001). qRT-PCR confirmed the presence of CLDN10 mRNA in HGSC effusions. High (> 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease.

Abstract 1865: Development of CTIM-76, a highly specific Claudin 6 bispecific antibody for treatment of ovarian cancer

Abstract Solid tumors lead to 580,000 deaths annually in the US, and safe and effective therapeutics for many late-stage solid tumors are lacking. Ovarian cancer alone kills 14,000 people each year, and many patients do not respond to currently available treatments. Here we introduce CTIM-76, a CLDN6 T-cell engager antibody as a potential treatment of ovarian, endometrial, and other solid tumors. The tight junction protein Claudin 6 (CLDN6) is a validated therapeutic target for many solid tumor types, including ovarian, endometrial, testicular, and gastric. It is differentially expressed on cancer cells with no reported expression in normal, healthy tissue. Despite being an attractive target, therapeutic monoclonal antibodies (MAbs) targeting CLDN6 are difficult to discover due to an abundance of closely related family members and an absolute need for high specificity. The extracellular region of CLDN6 closely resembles the widely expressed family member CLDN9 (3 amino acids different). The few CLDN6 MAbs in clinical development have demonstrated significant binding to other CLDN family members and most have now been halted from further development. Starting with a panel of highly specific CLDN6 antibodies, we engineered a large set (&amp;gt; 50) of CLDN6xCD3 bispecific antibodies (CLDN6 bispecifics) using multiple formats and CD3 arms that encompass different geometries and binding stoichiometries, as these factors are expected to play a critical role in the potency of these molecules both in vitro and in vivo. In vitro T cell cytotoxicity and cytokine release assays as well as developability studies were used to select a small panel of antibodies. These were tested in vivo and demonstrated efficacy in xenograft studies in PBMC-engrafted mice. From this smaller panel, we chose a lead molecule, CTIM-76 for IND-enabling studies. CTIM-76 shows a combination of potent killing of CLDN6-expressing cells, no killing of cells expressing other closely related claudin family members, and excellent productivity and developability. The exquisite specificity of CTIM-76 suggests the potential to address the need for potent therapeutic modalities for ovarian and other cancers without compromising patient safety. Citation Format: Joseph Rucker, Ileine Sanchez, Kyle Doolan, Breanna Tyrell, Anna Lobley, Nicholas Molino, Kristen Shema, Kyle Guldner, Hayley Roth, Alyssa Cunningham, Kate Slovik, Riley Payne, Eric Butz, Evan Dick, Edward Calamai, Ross Chambers. Development of CTIM-76, a highly specific Claudin 6 bispecific antibody for treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1865.

Double mutation of claudin-1 and claudin-3 causes alopecia in infant mice.

Hair follicles (HFs) undergo cyclic phases of growth, regression, and rest in association with hair shafts to maintain the hair coat. Nonsense mutations in the tight junction protein claudin (CLDN)-1 cause hair loss in humans. Therefore, we evaluated the roles of CLDNs in hair retention. Among the 27 CLDN family members, CLDN1, CLDN3, CLDN4, CLDN6, and CLDN7 were expressed in the inner bulge layer, isthmus, and sebaceous gland of murine HFs. Hair phenotypes were observed in Cldn1 weaker knockdown and Cldn3-knockout (Cldn1Δ/Δ Cldn3-/- ) mice. Although hair growth was normal, Cldn1Δ/Δ Cldn3-/- mice showed striking hair loss in the first telogen. Simultaneous deficiencies in CLDN1 and CLDN3 caused abnormalities in telogen HFs, such as an aberrantly layered architecture of epithelial cell sheets in bulges with multiple cell layers, mislocalization of bulges adjacent to sebaceous glands, and dilated hair canals. Along with the telogen HF abnormalities, which shortened the hair retention period, there was an enhanced proliferation of the epithelium surrounding HFs in Cldn1Δ/Δ Cldn3-/- mice, causing accelerated hair regrowth in adults. Our findings suggested that CLDN1 and CLDN3 may regulate hair retention in infant mice by maintaining the appropriate layered architecture of HFs, a deficiency of which can lead to alopecia.

Abstract P2-26-20: Marvel D3 and its associated junctional proteins in breast cancer

Abstract Marvel D3 and its associated junctional proteins in breast cancer Wenxiao Ji, Wen G. Jiang, Tracey A. Martin CCMRC, Cardiff University School of Medicine, Cardiff, Wales, UK Introduction. Marvel (Membrane Associated Domain Containing) proteins are a small family of proteins that are concentrated at the tight junction (TJ) region of epithelial and endothelial cells. They are important players in the formation and regulation of TJs. The Marvel protein family has three members, Marvel-D1, Marvel-D2 and Marvel-D3, all known to be TJ components. Marvel-D3 is more prominent in TJ regulation, by interacting widely with other proteins that co-localised at TJs. In the present study, we have examined the expression of Marvel-D3 and its splicing variants, their relationship with other known interactive TJ partners in breast cancer and in disease progression and clinical outcome. Methods. Marvel-D3 and its variants were quantified in a breast cancer cohort that contained both normal and tumour tissues. The expression was analysed together with the known Marvel-D3 interactive molecules available in our database of the same cohort. The profile of the expression of the molecules was also integrated to search for a pattern of the expression that may have clinical significance including the survival of the patients. Results. Marvel-D3 had significantly high levels in breast cancer tissues than normal tissues (p=0.011). The difference between normal and tumour tissues for two Marvel-D3 variants were not statistically significant. Of all the known Marvel-D3 interactive proteins that were available for analyses, we identified thirteen with viability in integrated analyses; they include the JAM family members, Marvel-D2, occludin, the zonula occluden family members, small number of the claudin family members and cingulin, all being important TJ components. The integrated expression of these Marvel-D3 partners presented a highly significant predictive power for the overall survival of the patients (RUC=0.821, p&amp;lt; 0.0001). Stratifying the patients based on the profile has a significant value in evaluating survival (survival rate 95.7% with favourable expression versus 57.1% unfavourable, during the followup period). This predictive power is independent of other clinical and hormonal receptor status (p&amp;lt; 0.001, HR=1.226 (95% CI 1.094-1.373)). We have also noted that this predictive value is applicable to subtypes of the breast cancer and hormone receptor status with similar predictive power. Conclusion. Expression of Marvel-D3, a TJ component, together with its interactive TJ partners, including both transmembrane and intracellular subcoat proteins, form an important clinical indicator for clinical progression of breast cancer. Citation Format: WENXIAO JI, Wen G. Jiang, Tracey A. Martin. Marvel D3 and its associated junctional proteins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-20.

Abstract 2892: Development of claudin 6 bispecific antibodies for treatment of ovarian cancer

Abstract There is growing interest in applying antibody modalities including bispecifics, antibody-drug conjugates, and CART to solid tumors. However, identifying appropriate tumor-specific targets that avoid adverse effects in healthy tissue has been challenging. The tight junction protein Claudin 6 (CLDN6) is a validated therapeutic target for many solid tumor types, including ovarian, endometrial, testicular, and gastric. It is differentially expressed on cancer cells with no reported expression in normal, healthy tissue. Despite being an attractive target, therapeutic monoclonal antibodies (MAbs) targeting CLDN6 are difficult to discover due to an abundance of closely related family members and an absolute need for high specificity. There are 26 human CLDN family members, and most are broadly expressed and highly conserved. The extracellular region of CLDN6 closely resembles the widely expressed CLDN9 (3 amino acids different). The few CLDN6 MAbs in clinical development have demonstrated significant binding to other CLDN family members and most have now been halted from development. Using Integral Molecular’s MPS antibody discovery platform, we have been able to isolate, and optimize rare antibodies against CLDN6 that do not cross-react with other CLDN family members. The exquisite specificity of our CLDN6 antibodies makes them amenable to use as the tumor-targeting arm of bispecific T-Cell Engagers. Starting with highly specific CLDN6 antibodies with a range of affinities, we engineered a large set (&amp;gt; 50) of CLDN6xCD3 bispecific antibodies (CLDN6 bispecifics) using multiple formats and CD3 arms that encompass different valencies and geometries. We designed the CLND6 arms to include antibody moieties with different affinities, epitopes, and stoichiometries, as these factors are expected to play a critical role in the potency of these molecules both in in vitro and in vivo. The full panel of bispecifics has been functionally tested in in vitro T cell cytotoxicity assays cells and has demonstrated potent killing of CLDN6-expressing cells with minimal killing of cells expressing other closely related claudin family members. We have also extensively characterized this panel of bispecific antibodies for detailed binding to both CD3 and CLDN6, selectivity against closely related claudin family members, and developability. The bispecifics were also screened for specificity against ~6,000 membrane proteins, representing &amp;gt; 95% of the entire human membrane proteome. Solid tumors lead to 580,000 deaths annually in the US, and safe and effective therapeutics for many late-stage solid tumors are lacking. Ovarian cancer alone kills 14,000 people each year, and many patients do not respond to currently available treatments. The exquisite specificity of our CLDN6xCD3 bispecifics suggests their potential to address the need for potent therapeutic modalities for ovarian and other cancers without compromising patient safety. Citation Format: Joseph Rucker, Kyle Doolan, Breanna Tyrell, Anna Lobley, Nick Molino, Kristen Shema, Kyle Guldner, Alyssa Cunningham, Ross Chambers, Riley Payne. Development of claudin 6 bispecific antibodies for treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2892.

Claudin‐23 Strengthens the Colonic Epithelial Barrier by Regulating Claudin‐3 and ‐4 proteins in the Tight Junction Plasma Membrane

Colonic epithelial barrier function is controlled by differential expression of Claudin (CLDN) proteins in the crypt‐luminal axis. We recently identified the expression of a CLDN family member, CLDN‐23, in intestinal epithelial cells (IECs). However, the role of CLDN‐23 in regulating epithelial barrier function has not been identified. Analysis of the human and murine intestinal crypt‐luminal axis revealed increased CLDN‐23 expression in differentiated IECs facing the lumen compared to the proliferative crypt‐base IECs. Similarly, CLDN‐23 expression was higher in differentiated human and murine IECs cultured as a monolayer on permeable supports compared to proliferative IECs cultured in a 3D matrigel matrix. To investigate the contribution of CLDN‐23 in regulating IEC barrier function in vivo, we generated inducible intestinal‐epithelia specific (Villin‐CreERT2) Cldn23 knockout mice (Cldn23ERΔIEC). Functional analysis displayed increased intestinal mucosal permeability to 4kDa FITC dextran, indicating compromised epithelial barrier function. To confirm this observation, we created human IECs in which CLDN‐23 expression was either silenced or enhanced in vitro. CLDN‐23 loss increased paracellular permeability to 4kDa FITC dextran and reduced transepithelial electrical resistance (TEER) consistent with a leaky barrier. Conversely, CLDN‐23 overexpression resulted in improved barrier function. Altogether, these data suggest that CLDN‐23 controls the IEC barrier function.Evaluation of CLDN protein interactions employing co‐culture of HeLa cells expressing CLDN‐23 with those expressing either CLDN‐2, CLDN‐3, or CLDN‐4 showed that CLDN‐23 interacts in trans with barrier‐forming CLDN‐3 and CLDN‐4, but not with pore‐forming CLDN‐2. Using in situ proximity ligation assay, we observed close association in cis between CLDN‐23 and barrier‐forming CLDN‐3 and CLDN‐4 in the TJ plasma membrane, but not with CLDN‐2. These findings suggest that CLDN‐23 interacts in both cis and trans with barrier‐forming CLDN‐3 and CLDN‐4 in differentiated IECs. Interestingly, the overexpression of CLDN‐23 in human IECs revealed up‐regulation of barrier‐forming CLDN‐3 protein expression in the TJ while decreasing the pore‐forming CLDN‐2 protein at this site. In conclusion, we identified that CLDN‐23 strengthens the colonic barrier properties by orchestrating the composition of CLDN protein interactions in epithelial TJs.

Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

Abstract 1860: Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells

Abstract There is a large unmet need for targeted therapies to treat ovarian cancer and other solid tumors. A promising strategy is the use of T cell engaging bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to tumor-associated antigens (TAA) on cancer cells and CD3 on T cells. Effective and safe use of these therapeutics depends on selective targeting of cancer cells over normal tissue, feasible when a TAA is more highly expressed on cancer cells versus normal tissues. Selectivity can also be improved by using a mixed valency “2 + 1” bispecific format, coupled with target affinity tuning, to achieve avid binding to the TAA. Claudin-6 (CLDN6) is a tetraspan membrane protein, a member of the claudin family of tight junction proteins, and has been identified as a promising TAA for ovarian cancer due to its high expression on ovarian and other cancer tissues and low expression on normal tissue. However, a complicating factor is that many proteins in the claudin family have high sequence identity. Most similar to CLDN6 is CLDN9, and the two proteins differ at only 3/76 residues in their extracellular loops, creating a challenge for the development of highly selective CLDN6 antibodies. We analyzed CLDN6 and CLDN9 expression using a combination of normal tissue gene expression data (GTEx), cancer cell genomics data (TCGA), and immunohistochemistry (IHC) on a panel of cancer and normal tissues. Data confirmed the tumor-specific expression pattern of CLDN6 but indicated high CLDN9 expression in normal tissues, suggesting that strong antibody selectivity for CLDN6 versus CLDN9 is critical. We created CLDN6-selective T cell engaging bispecific antibodies by starting with a highly selective monoclonal antibody humanized from a mouse hybridoma. Selectivity was further improved by engineering the CDR regions. Variant antibodies were screened for binding to transiently transfected HEK293E cells that highly expressed either CLDN6, CLDN9, or other homologous claudin family members. Selective variants were converted into the XmAb® 2 + 1 bispecific antibody format and tested in T cell-dependent cellular cytotoxicity (TDCC) assays using cancer cell lines expressing CLDN6, or HEK293E cells transfected with claudin homologs. A set of lead CLDN6 x CD3 bispecifics displaying a range of potencies on CLDN6+ cancer cell lines and high selectivity for CLDN6 over CLDN9 and other homologous claudins was identified. Tolerability and pharmacokinetics of these molecules are currently being assessed in non-human primates, and activity will be evaluated in humanized mouse models of ovarian cancer. Citation Format: Matthew S. Faber, Sung-Hyung Lee, Yoon Kyung Kim, Jing Qi, Kendra N. Avery, Duc-Hanh T. Nguyen, Rumana Rashid, Araz Eivazi, Seung Y. Chu, Juan E. Diaz, Connie Ardila, Ruschelle Love, Alex Nisthal, Norman J. Barlow, Christine Bonzon, Umesh S. Muchhal, Matthew J. Bernett, John R. Desjarlais. Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1860.

Claudin-2 promotes colorectal cancer liver metastasis and is a biomarker of the replacement type growth pattern

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases.

Expression Patterns of Claudin Family Members During Tooth Development and the Role of Claudin-10 (Cldn10) in Cytodifferentiation of Stratum Intermedium.

There is growing evidence showing that tight junctions play an important role in developing enamel. Claudins are one of the main components of tight junctions and may have pivotal functions in modulating various cellular events, such as regulating cell differentiation and proliferation. Mutations in CLDN10 of humans are associated with HELIX syndrome and cause enamel defects. However, current knowledge regarding the expression patterns of claudins and the function of Cldn10 during tooth development remains fragmented. In this study, we aimed to analyze the expression patterns of claudin family members during tooth development and to investigate the role of Cldn10 in amelogenesis. Using cap analysis gene expression of developing mouse tooth germs compared with that of the whole body, we found that Cldn1 and Cldn10 were highly expressed in the tooth. Furthermore, single-cell RNA-sequence analysis using 7-day postnatal Krt14-RFP mouse incisors revealed Cldn1 and Cldn10 exhibited distinct expression patterns. Cldn10 has two isoforms, Cldn10a and Cldn10b, but only Cldn10b was expressed in the tooth. Immunostaining of developing tooth germs revealed claudin-10 was highly expressed in the inner enamel epithelium and stratum intermedium. We also found that overexpression of Cldn10 in the dental epithelial cell line, SF2, induced alkaline phosphatase (Alpl) expression, a marker of maturated stratum intermedium. Our findings suggest that Cldn10 may be a novel stratum intermedium marker and might play a role in cytodifferentiation of stratum intermedium.

Role of Claudins in Renal Branching Morphogenesis.

Claudins are a family of tight junction proteins that are expressed during mouse kidney development. They regulate paracellular transport of solutes along the nephron and contribute to the final composition of the urinary filtrate. To understand their roles during development, we used a protein reagent, a truncated version of the Clostridium perfringens enterotoxin (C‐CPE), to specifically remove a subset of claudin family members from mouse embryonic kidney explants at embryonic day 12. We observed that treatment with C‐CPE decreased the number and the complexity of ureteric bud tips that formed: there were more single and less bifid ureteric bud tips when compared to control‐treated explants. In addition, C‐CPE‐treated explants exhibited ureteric bud tips with larger lumens when compared to control explants (p < .05). Immunofluorescent analysis revealed decreased expression and localization of Claudin‐3, −4, −6, and −8 to tight junctions of ureteric bud tips following treatment with C‐CPE. Interestingly, Claudin‐7 showed higher expression in the basolateral membrane of the ureteric bud lineage and poor localization to the tight junctions of the ureteric bud lineage both in controls and in C‐CPE‐treated explants. Taken together, it appears that claudin proteins may play a role in ureteric bud branching morphogenesis through changes in lumen formation that may affect the efficiency by which ureteric buds emerge and branch.

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple-negative breast cancer.

The up‐regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple‐negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down‐regulated in human TNBC, and Claudin15 down‐regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down‐regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non‐TNBC MCF‐7 cells. Conversely, an up‐regulation of Claudin15 remarkably reduced TNBC MDA‐MB‐231 cell migration, invasion and VM formation. We also showed that down‐regulation of Claudin15 was Twist1‐dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15‐deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1‐Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.

Temporal Effects of Quercetin on Tight Junction Barrier Properties and Claudin Expression and Localization in MDCK II Cells.

Kidney stones affect 10% of the population. Yet, there is relatively little known about how they form or how to prevent and treat them. The claudin family of tight junction proteins has been linked to the formation of kidney stones. The flavonoid quercetin has been shown to prevent kidney stone formation and to modify claudin expression in different models. Here we investigate the effect of quercetin on claudin expression and localization in MDCK II cells, a cation-selective cell line, derived from the proximal tubule. For this study, we focused our analyses on claudin family members that confer different tight junction properties: barrier-sealing (Cldn1, -3, and -7), cation-selective (Cldn2) or anion-selective (Cldn4). Our data revealed that quercetin’s effects on the expression and localization of different claudins over time corresponded with changes in transepithelial resistance, which was measured continuously throughout the treatment. In addition, these effects appear to be independent of PI3K/AKT signaling, one of the pathways that is known to act downstream of quercetin. In conclusion, our data suggest that quercetin’s effects on claudins result in a tighter epithelial barrier, which may reduce the reabsorption of sodium, calcium and water, thereby preventing the formation of a kidney stone.

Dichotomous roles of claudins as tumor promoters or suppressors: lessons from knockout mice.

Claudins are a family of integral tight junction proteins that regulate paracellular permeability in polarized epithelia. Overexpression or reduction of claudins can both promote and limit cancer progression, revealing complex dichotomous roles for claudins depending on cellular context. In contrast, recent studies demonstrating tumor formation in claudin knockout mouse models indicate a role for several claudin family members in suppressing tumor initiation. For example, intestine-specific claudin-7 knockout mice spontaneously develop atypical hyperplasia and intestinal adenomas, while claudin-18 knockout mice develop carcinomas in the lung and stomach. Claudin-4, -11, and -15 knockout mice show increased cell proliferation and/or hyperplasia in urothelium, Sertoli cells, and small intestinal crypts, respectively, possibly a precursor to cancer development. Pathways implicated in both cell proliferation and tumorigenesis include Yap/Taz and insulin-like growth factor-1 receptor (IGF-1R)/Akt pathways, among others. Consistent with the tumor suppressive role of claudins shown in mice, in humans, claudin-low breast cancer has been described as a distinct entity with a poor prognosis, and claudin-18-Rho GTPase activating protein 26 (CLDN18-ARHGAP26) fusion protein as a driver gene aberration in diffuse-type gastric cancer due to effects on RhoA. Paradoxically, claudins have also garnered interest as targets for therapy, as they are sometimes aberrantly expressed in cancer cells, which may or may not promote cancer progression. For example, a chimeric monoclonal antibody which targets cells expressing claudin-18.2 through antibody-dependent cell-mediated cytotoxicity has shown promise in multiple phase II studies. In this review, we focus on new findings supporting a tumor suppressive role for claudins during cancer initiation.

Peripheral Nerve Injury Induces Dynamic Changes of Tight Junction Components

Tight junctions seal off physical barriers, regulate fluid and solute flow, and protect the endoneurial microenvironment of the peripheral nervous system. Physical barriers in the peripheral nervous system were disrupted after nerve injury. However, the dynamic changes of tight junction components after peripheral nerve injury have not been fully determined yet. In the current study, by using previously obtained deep sequencing outcomes and bioinformatic tools, we found that tight junction signaling pathway was activated after peripheral nerve injury. The investigation of the temporal expression patterns of components in tight junction signaling pathway suggested that many claudin family members were down-regulated after nerve injury. Moreover, we examined the effects of matrix metalloproteinases 7 and 9 (MMP7 and MMP9) on tight junction genes both in vitro and in vivo and found that MMP7 and MMP9 modulated the expressions of genes coding for claudin 1, claudin 10, and claudin 22. Our study revealed the dynamic changes of tight junction components after peripheral nerve injury and thus might contribute to the understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.

Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

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

ABSTRACTClaudins 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.