Hypoxia-inducible microRNA-155 negatively regulates epithelial barrier in eosinophilic esophagitis by suppressing tight junction claudin-7.

MicroRNA (miRNA)-mediated mRNA regulation directs many homeostatic and pathological processes, but how miRNAs coordinate aberrant esophageal inflammation during eosinophilic esophagitis (EoE) is poorly understood. Here, we report a deregulatory axis where microRNA-155 (miR-155) regulates epithelial barrier dysfunction by selectively constraining tight junction CLDN7 (claudin-7). MiR-155 is elevated in the esophageal epithelium of biopsies from patients with active EoE and in cell culture models. MiR-155 localization using in situ hybridization (ISH) in patient biopsies and intra-epithelial compartmentalization of miR-155 show expression predominantly within the basal epithelia. Epithelial miR-155 activity was evident through diminished target gene expression in 3D organotypic cultures, particularly in relatively undifferentiated basal cell states. Mechanistically, generation of a novel cell line with enhanced epithelial miR-155 stable overexpression induced a functionally deficient epithelial barrier in 3D air-liquid interface epithelial cultures measured by transepithelial electrical resistance (TEER). Histological assessment of 3D esophageal organoid cultures overexpressing miR-155 showed notable dilated intra-epithelial spaces. Unbiased RNA-sequencing analysis and immunofluorescence determined a defect in epithelial barrier tight junctions and revealed a selective reduction in the expression of critical esophageal tight junction molecule, claudin-7. Together, our data reveal a previously unappreciated role for miR-155 in mediating epithelial barrier dysfunction in esophageal inflammation.

The Combination of Inositol Hexaphosphate and Inositol Inhibits Metastasis of Colorectal Cancer Cells by Upregulating Claudin 7.

Inositol hexaphosphate (IP6), a widely found natural bioactive substance in grains, effectively inhibits the progression of colorectal cancer (CRC) when used in combination with inositol (INS). We previously showed that supplementation of IP6 and INS upregulated the claudin 7 gene in orthotropic CRC xenografts in mice. The aim of this study was to elucidate the role of claudin 7 in the inhibition of CRC metastasis by IP6 and INS, and explore the underlying mechanisms. We found that IP6, INS and their combination inhibited the epithelial-mesenchymal transition (EMT) of colon cancer cell lines (SW480 and SW620), as indicated by upregulation of claudin 7 and E-cadherin, and downregulation of N-cadherin. The effect of IP6 and INS was stronger compared to either agent alone (combination index < 1). Furthermore, the silencing of the claudin 7 gene diminished the anti-metastatic effects of IP6 and INS on SW480 and SW620 cells. Consistent with in vitro findings, the combination of IP6 and INS suppressed CRC xenograft growth in a mouse model, which was neutralized by claudin 7. Taken together, the combination of IP6 and INS can inhibit CRC metastasis by blocking EMT of tumor cells through upregulation of claudin 7.

Modulation of microRNAs and claudin-7 in Caco-2 cell line treated with Blastocystis sp., subtype 3 soluble total antigen.

BACKGROUND: Blastocystis sp., is a eukaryote of the large intestine, which is reported from almost all countries. The pathogenesis of this protist is not clear. The current study aimed to analyze the effects of Blastocystis sp., ST3 soluble total antigen (B3STA) on the microRNAs (miRNAs) involved in the gut permeability and also pro-inflammatory cytokines, occludin, and claudin-7. METHODS: Blastocystis sp., ST3 isolated from stool sample was purified, and its soluble total antigen was extracted using freeze and thawing. The Caco-2 cell line was treated with B3STA for 24 h and the expression levels of mir-16, mir-21, mir-29a, mir-223, and mir-874 were analyzed. In addition, the expression levels of il-8, il-15, occludin, and claudin-7 genes were assessed. RESULTS: B3STA significantly upregulated the expression of mir-223, and mir-874, and downregulated mir-29a. The expression of mir-16 and mir-21 was not significant. In addition, the expression of il-8 and il-15 was not significant. B3STA significantly decreased the expression level of claudin-7 (P-value < 0.0001), but the expression of occludin was not significant. Our results showed significant correlation between all studied miRNAs, except mir-29a, with downregulation of claudin-7. CONCLUSIONS: This is the first study investigating the effects of Blastocystis sp., ST3 isolated from symptomatic subjects on the expression levels of miRNAs involved in the gut permeability. Our results demonstrated that B3STA may change miRNA expression, which are involved in the gut barrier integrity, and downregulates claudin-7, which is known as sealing factor.

Intestinal Claudin-7 deficiency impacts the intestinal microbiota in mice with colitis.

BACKGROUND: Intestinal epithelial cells form a physical barrier that protects the intestine against the intestinal microbiota through tight junctions (TJs) and adhesive junctions, while barrier disruption may lead to inflammatory bowel disease (IBD). Claudin-7 (Cldn7) has been implicated in this protection as an important member of TJs. Here, we experimentally study the effect of Cldn7 deletion on intestinal microbiota in colitis. METHODS: Colitis model was established based on inducible intestinal conditional Cldn7 gene knockout mice (Cldn7fl/fl; villin-CreERT2), by feeding with dextran sodium sulfate (DSS). AB-PAS staining and immunohistochemical staining of Muc2 mucin were used to detect the effect of Cldn7 deficiency on the mucus layer of mice with colitis, and fluorescence in situ hybridization was used to detect how Cldn7 promotes spatial separation of the gut microbiota from the host. The microbiota population was characterized by high-throughput 16S rRNA gene sequencing of DNA extracted from fecal samples. RESULTS: Compared with the controls, Cldn7 knockout increased susceptibility to colitis, including greater degree of weight loss, colon shortening, and a significantly higher disease activity index score. DSS-treated Cldn7 knockout mice promoted the migration of bacteria to the intestinal epithelium to some extent by damaging the intestinal mucus layer. Sequencing of 16S rRNA showed that DSS-treated Cldn7 knockout mice reduced the gut microbiota diversity and had greater relative abundance of Escherichia coli. LEfSe analysis indicated that Escherichia coli may be the key bacteria in Cldn7 knockout mice during DSS-induced colitis. Furthermore, the Tax4Fun analysis predicted that DSS-treated Cldn7 knockout mice enriched for microbiota impacting infectious diseases, immune system and metabolic functions. CONCLUSIONS: Our data suggests an association between intestinal Cldn7 knockout and microbiota dysbiosis during inflammatory events.

Claudin-7 deficiency promotes stemness properties in colorectal cancer through Sox9-mediated Wnt/ß-catenin signalling.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumour of the digestive tract that is characterized by high patient morbidity and mortality rates. Claudin-7 (Cldn7), a tight junction protein, was recently reported to function as a candidate tumour suppressor gene in CRC. Our previous study demonstrated that the large intestine of C57/BL6 mice showed intestinal adenomas and abnormal Ki67 expression and distribution in the intestinal crypt when Cldn7 was knocked out. The aim of this study was to further investigate whether Cldn7 deficiency has non-tight junction functions, affects intestinal stemness properties, promotes CRC and to determine the specific mechanism. METHODS: Cell proliferation assays, migration assays, apoptosis assays, tumour sphere formation assays in vitro, and subcutaneous xenograft models in vivo were used to determine the effects of Cldn7 knockdown on the biological characteristics of CRC stem cells. Western blotting, qPCR and immunofluorescence staining were performed to identify the epithelial-mesenchymal transition and the activation of Wnt/ß-catenin pathway in CRC stem cells. Cldn7 inducible conditional gene knockout mice and immunohistochemical staining further verified this hypothesis in vivo. The mechanism and target of Cldn7 were determined by performing a chromatin immunoprecipitation (ChIP) assay and coimmunoprecipitation (CoIP) assay. RESULTS: Cldn7 knock down in CRC stem cells promoted cell proliferation, migration, and globular growth in serum-free medium and the ability to form xenograft tumours; cell apoptosis was inhibited, while the cellular epithelial-mesenchymal transition was also observed. These changes in cell characteristics were achieved by activating the Wnt/ß-catenin pathway and promoting the expression of downstream target genes after ß-catenin entry into the nucleus, as observed in CRC cell lines and Cldn7 gene knockout mouse experiments. Using ChIP and CoIP experiments, we initially found that Cldn7 and Sox9 interacted at the protein level to activate the Wnt/ß-catenin pathway. CONCLUSIONS: Based on our research, Cldn7 deficiency confers stemness properties in CRC through Sox9-mediated Wnt/ß-catenin signalling. This result clarifies that Cldn7 plays an inhibitory role in CRC and reveals a possible molecular mechanism, which is conducive to further research on Cldn7 and cancer stem cells.

N-glycosylation status of Trop2 impacts its surface density, interaction with claudin-7 and exosomal release.

Trophoblast antigen 2 (Trop2) is a type I transmembrane protein post-translationally modified by N-linked glycosylation. It was originally detected in trophoblasts but was later shown to be frequently overexpressed in many epithelial cancers. Recently, anti-Trop2 antibody-drug conjugate has been FDA approved for the treatment of metastatic triple-negative breast and urothelial carcinomas, making it an important tumor antigen. The current study explored the significance of N-glycosylation of Trop2 by substituting specific N-glycan addition sites by site-directed mutagenesis. The mutant proteins were characterized in transiently transfected HEK293 cells. The N-glycosylation mutants did not affect protein expression, stability, dimerization ability and matriptase mediated cleavage. However, N120A and N208A mutants showed decreased interaction with its binding partner claudin-7. Our earlier reported Trop2 mutant V194A, which shows aberrant glycosylation, also displayed hampered interaction with claudin-7. To further characterize the mutants, stable clones expressing wild type and mutant Trop2 were generated in OVCAR3 cell line. Interestingly, surface biotinylation assay showed significantly higher surface expression of N120A and N208A mutants whereas surface localization was drastically reduced for V194A Trop2 mutant. Though overexpression of wild type Trop2 did not cause any change in fibronectin-mediated FAK (Focal adhesion kinase) signaling; expression of N120A mutant, surprisingly downregulated FAK signaling. Furthermore, exosomal release of Trop2 was also decreased in N120A and N208A mutants. This data suggests that site-specific N-glycan addition determines Trop2 surface density, claudin-7 interaction and exosomal release.

Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility.

Dysfunction of tight junctions is a critical step during the initial stage of tumor progression. Trophoblast cell surface antigen 2 (Trop-2) belongs to the family of tumor-associated calcium signal transducer (TACSTD) and is required for the stability of claudin-7 and claudin-1, which are often dysregulated or lost in carcinogenesis. Here, we investigated the effects of Trop-2 phosphorylation on cell motility. Analyses using HCT116 cells expressing WT Trop-2 (HCT116/WT) or Trop-2 alanine-substituted at Ser-303 (HCT116/S303A) or Ser-322 (HCT116/S322A) revealed that Trop-2 is phosphorylated at Ser-322. Furthermore, coimmunoprecipitation and Transwell assays indicated that Trop-2 S322A interacted with claudin-7 the strongest, and a phosphomimetic variant, Trop-2 S322E, the weakest and that HCT116/S322E cells have the highest motility and HCT116/S322A cells the lowest. All cell lines had similar levels of claudin-7 mRNA, but levels of claudin-7 protein were markedly decreased in the HCT116/S322E cells, suggesting posttranscriptional control of claudin-7. Moreover, claudin-7 was clearly localized to cell-cell borders in HCT116/S322A cells but was diffusely distributed on the membrane and partially localized in the cytoplasm of HCT116/S322E and HCT116/WT cells. These observations suggested that Trop-2 phosphorylation plays a role in the decrease or mislocalization of claudin-7. Using protein kinase C (PKC) inhibitors and PKC-specific siRNAs, we found that PKCα and PKCδ are responsible for Trop-2 phosphorylation. Of note, chemical PKC inhibition and PKCα- and PKCδ-specific siRNAs reduced motility. In summary, our findings provide evidence that Trop-2 is phosphorylated at Ser-322 by PKCα/δ and that this phosphorylation enhances cell motility and decreases claudin-7 localization to cellular borders.

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.

Claudin7-dependent exosome-promoted reprogramming of nonmetastasizing tumor cells.

Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEXs were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX.

Claudin-7 downregulation induces metastasis and invasion in colorectal cancer via the promotion of epithelial-mesenchymal transition.

The dysregulation of the tight junctions (TJs) protein claudin-7 is closely related to the development and metastasis of colorectal cancer (CRC). The aim of this study was to investigate the expression of claudin-7 and characterize the relationship between claudin-7 expression and epithelial-mesenchymal transition (EMT) in CRC. In this study, the expression of claudin-7, E-cadherin, vimentin and snail-1 was detected by immunohistochemistry (IHC) in a set of 80 CRC specimens comprising 20 specimens each of well-differentiated, moderately differentiated, poorly differentiated and liver metastases tissues. The correlation between claudin-7 and EMT-related proteins in the stably transfected claudin-7 knockdown HCT116 cell line was analyzed by IHC, immunofluorescence (IF), Western blotting (WB) and nude mouse xenograft models. The results revealed that the expression of claudin-7 was downregulated as CRC tissue differentiation grade decreased, and that low claudin-7 expression corresponded to the downregulation of E-cadherin (r = 0.725, p < 0.001) and upregulation of vimentin (r = -0.376, p = 0.001) and snail-1 (r = -0.599, p < 0.001). Additionally, in the claudin-7 knockdown HCT116 cell line, the staining intensity and expression of E-cadherin was decreased, while the immunoreactivity and expression of vimentin and snail-1 was increased. Futhermore, the result of tumor formation experiment was consistent with CRC tissues. In conclusion, the expression of claudin-7 in CRC is downregulated as differentiation grade decreases. Claudin-7 downregulation may promote the invasion and metastasis of CRC by regulating EMT. Our results provide new perspectives for a potential therapeutic target for CRC.

Claudin 7 as a possible novel molecular target for the treatment of pancreatic cancer.

BACKGROUND/OBJECTIVES: Pancreatic cancer consists of various subpopulations of cells, some of which have aggressive proliferative properties. The molecules responsible for the aggressive proliferation of pancreatic cancer may become molecular targets for the therapies against pancreatic cancer. METHODS: From a human pancreatic cancer cell line, MIA PaCa-2, MIA PaCa-2-A cells with an epithelial morphology and MIA PaCa-2-R cells with a non-epithelial morphology were clonogenically isolated by the limiting dilution method. Gene expression of these subpopulations was analyzed by DNA microarray. Gene knockdown was performed using siRNA. RESULTS: Although the MIA PaCa-2-A and MIA PaCa-2-R cells displayed the same DNA short tandem repeat (STR) pattern identical to that of the parental MIA PaCa-2 cells, the MIA PaCa-2-A cells were more proliferative than the MIA PaCa-2-R cells both in culture and in tumor xenografts generated in immunodeficient mice. Furthermore, the MIA PaCa-2-A cells were more resistant to gemcitabine than the MIA PaCa-2-R cells. DNA microarray analysis revealed a high expression of claudin (CLDN) 7 in the MIA PaCa-2-A cells, as opposed to a low expression in the MIA PaCa-2-R cells. The knockdown of CLDN7 in the MIA PaCa-2-A cells induced a marked inhibition of proliferation. The MIA PaCa-2-A cells in which CLDN7 was knocked down exhibited a decreased expression of phosphorylated extracellular signal-regulated kinase (p-Erk)1/2 and G1 cell cycle arrest. CONCLUSIONS: CLDN7 may be expressed in the rapidly proliferating and dominant cell population in human pancreatic cancer tissues and may be a novel molecular target for the treatment of pancreatic cancer.

Claudin-7 Modulates Cl- and Na+ Homeostasis and WNK4 Expression in Renal Collecting Duct Cells.

Claudin-7 knockout (CLDN7-/-) mice display renal salt wasting and dehydration phenotypes. To address the role of CLDN7 in kidneys, we established collecting duct (CD) cell lines from CLDN7+/+ and CLDN7-/- mouse kidneys. We found that deletion of CLDN7 increased the transepithelial resistance (TER) and decreased the paracellular permeability for Cl- and Na+ in CLDN7-/- CD cells. Inhibition of transcellular Cl- and Na+ channels has no significant effect on TER or dilution potentials. Current-voltage curves were linear in both CLDN7+/+ and CLDN7-/- CD cells, indicating that the ion flux was through the paracellular pathway. The impairment of Cl- and Na+ permeability phenotype can be rescued by CLDN7 re-expression. We also found that WNK4 (its mutations lead to hypertension) expression, but not WNK1, was significantly increased in CLDN7-/- CD cell lines as well as in primary CLDN7-/- CD cells, suggesting that the expression of WNK4 was modulated by CLDN7. In addition, deletion of CLDN7 upregulated the expression level of the apical epithelial sodium channel (ENaC), indicating a potential cross-talk between paracellular and transcellular transport systems. This study demonstrates that CLDN7 plays an important role in salt balance in renal CD cells and modulating WNK4 and ENaC expression levels that are vital in controlling salt-sensitive hypertension.

Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors.

The expression pattern of tight junction proteins (TJPs) varies among organs and tumor types. In this study, we examined the immunoreactivity of claudin (CLDN)-1, -4, and -7, and JAM-A in salivary gland tumors (SGTs) by histological types and cell types to estimate their usefulness as differential diagnostic markers. Immunoreactivity of CLDN1 was higher in ductal epithelium cells of SGTs than in non-tumor tissues. Conversely, immunoreactivity of CLDN1 was significantly decreased in basal/myoepithelium cells of SGTs compared with that in non-tumor tissues. There was no significant difference between the immunoreactivity of CLDN1 in benign tumors and that in malignant tumors. Immunoreactivity of CLDN4, CLDN7, and JAM-A in ductal epithelium cells was higher in many SGTs than in non-tumor tissues. There was a difference depending on the histological type of SGT in immunoreactivity of CLDN4, CLDN7, and JAM-A in basaloid/myoepithelial cells. It was possible to classify SGTs by a hierarchical clustering using immunoreactivity of TJPs. The results suggest that an immunohistochemical marker panel including these TJPs may be useful for differential diagnosis of SGTs and that CLDN1 is associated with tumorigenesis of SGTs.

Claudin7b is required for the formation and function of inner ear in zebrafish.

Zebrafish has become an excellent model for studying the development and function of inner ear. We report here a zebrafish line in which claudin 7b (cldn7b) locus is interrupted by a Tol2 transposon at its first intron. The homozygous mutants have enlarged otocysts, smaller or no otoliths, slowly formed semicircular canals, and insensitiveness to sound stimulation. These abnormal phenotypes and hearing loss of inner ear could be mostly rescued by injection of cldn7b-mRNA into one-cell stage homozygous mutant embryos. Mechanistically, cldn7b-deficiency interrupted the formation of apical junction complexes (AJCs) in otic epithelial cells of inner ear and the ion-homeostasis of endolymph, which then led to the loss of proper contact between otoliths and normally developed hair cells in utricle and saccule or aberrant mechanosensory transduction. Thus, Cldn7b is essential for the formation and proper function of inner ear through its unique role in keeping an initial integrity of otic epithelia during zebrafish embryogenesis.

Developmental Expression of Claudins in the Mammary Gland.

Claudins are a large family of membrane proteins whose classic function is to regulate the permeability of tight junctions in epithelia. They are tetraspanins, with four alpha-helices crossing the membrane, two extracellular loops, a short cytoplasmic N-terminus and a longer and more variable C-terminus. The extracellular ends of the helices are known to undergo side-to-side (cis) interactions that allow the formation of claudin polymers in the plane of the membrane. The extracellular loops also engage in head-to-head (trans) interactions thought to mediate the formation of tight junctions. However, claudins are also present in intracellular structures, thought to be vesicles, with less well-characterized functions. Here, we briefly review our current understanding of claudin structure and function followed by an examination of changes in claudin mRNA and protein expression and localization through mammary gland development. Claudins-1, 3, 4, 7, and 8 are the five most prominent members of the claudin family in the mouse mammary gland, with varied abundance and intracellular localization during the different stages of post-pubertal development. Claudin-1 is clearly localized to tight junctions in mammary ducts in non-pregnant non-lactating animals. Cytoplasmic puncta that stain for claudin-7 are present throughout development. During pregnancy claudin-3 is localized both to the tight junction and basolaterally while claudin-4 is found only in sparse puncta. In the lactating mouse both claudin-3 and claudin-8 are localized at the tight junction where they may be important in forming the paracellular barrier. At involution and under challenge by lipopolysaccharide claudins -1, -3, and -4 are significantly upregulated. Claudin-3 is still colocalized with tight junction molecules but is also distributed through the cytoplasm as is claudin-4. These largely descriptive data provide the essential framework for future mechanistic studies of the function and regulation of mammary epithelial cell claudins.

Intestinal epithelial Cldn-7 regulates intestinal inflammation by altering the gut microbiota.

BACKGROUND AND AIM: Tight junctions maintain gut homeostasis by forming a physical barrier that protects the gut from invasion by microbiota. Cldn-7 is an important component involved in this protection, but the relationship between Cldn-7, intestinal inflammation, and gut microbiota has not been clarified. Here, we hypothesize that Cldn-7 depletion affects intestinal inflammation by altering the gut microbiota. METHODS: Based on the induced intestinal condition of Cldn-7 knockout mice (Cldn7fl/fl;villin-CreaERT2), we established the intestinal flora depletion model and colitis model by antibiotic drinking and feeding with dextran sodium sulfate (DSS). The environment of Cldn-7 gene deletion mice was changed by co-housing experiment. AB-PAS staining and Muc2 were used to detect the effect of co-housing and Cldn-7 deficiency on the mucus layer after flora depletion. qRT-PCR was used to detect the expression of intestinal inflammatory factors and AMPs in mice. Feces were collected and proportions of microbiota were analyzed by 16 S rRNA amplicon sequencing. RESULTS: Mice in the co-housing experiment had altered intestinal microbiota, including diversity, composition, and functional prediction, compared to controls. Intestinal inflammation was restored to some extent following altered intestinal microbiota. The intestinal inflammation caused by Cldn-7 deficiency and susceptibility to DSS could be reduced after antibiotic administration compared to controls, in terms of phenotype, pathological changes, inflammatory factors, mucus barrier, and expression of AMPs. CONCLUSIONS: In analyses of intestinal tissues, colitis induction, and gut microbiota in mice with intestinal disruption of Cldn-7, we found this protein to prevent intestinal inflammation by regulating the gut microbiota. Cldn-7might therefore be an important mediator of host-microbiome interactions. Our research has revealed that Cldn-7 plays an indispensable role in maintaining intestinal homeostasis by regulating the gut microbiota and impacting intestinal inflammation. These findings provide new insights into the pathogenesis of ulcerative colitis.

Matrix metalloproteinase-7 and claudin-7 as novel identified therapeutic targets for restoration of intestinal epithelial barrier in inflammatory bowel diseases.

Intestinal tight junction disruption and mucosal immune dysregulation contribute to pathogenesis and progression of inflammatory bowel diseases (IBD). A proteolytic enzyme matrix metalloproteinase 7 (MMP-7), which is highly expressed in intestinal tissue, is implicated to IBD and other immune overactivation-associated diseases. In the issue of the Frontiers in Immunology, Ying Xiao and colleagues demonstrate that MMP-7-mediated claudin-7 degradation promotes IBD pathogenesis and disease progression. Therefore, inhibition of MMP-7 enzymatic activity can be a therapeutic strategy for the treatment of IBD.

Rab25 suppresses colon cancer cell invasion through upregulating claudin­7 expression.

Ras­related protein 25 (Rab25) is a member of small GTPase and is implicated in cancer cell progression of various types of cancer. Growing evidence suggests the context­dependent role of Rab25 in cancer invasiveness. Claudin­7 is a tight junction protein and has been known to suppress cancer cell invasion. Although Rab25 was reported to repress cancer aggressiveness through recycling ß1 integrin to the plasma membrane, the detailed underlying mechanism remains to be elucidated. The present study identified the critical role of claudin­7 in Rab25­induced suppression of colon cancer invasion. 3D Matrigel system and modified Boyden chamber analysis showed that enforced expression of Rab25 attenuated colon cancer cell invasion. In addition, Rab25 inactivated epidermal growth factor receptor (EGFR) and increased E­cadherin expression. Unexpectedly, it was observed that Rab25 induces claudin­7 expression through protein stabilization. In addition, ectopic claudin­7 expression reduced EGFR activity and Snail expression as well as colon cancer cell invasion. However, silencing of claudin­7 expression reversed the tumor suppressive role of Rab25, thereby increasing colon cancer cell invasiveness. Collectively, the present data indicated that Rab25 inactivates EGFR and colon cancer cell invasion by upregulating claudin­7 expression.

Colonic crypt stem cell functions are controlled by tight junction protein claudin-7 through Notch/Hippo signaling.

The tight junction protein claudin-7 is essential for tight junction function and intestinal homeostasis. Cldn7 deletion in mice leads to an inflammatory bowel disease-like phenotype exhibiting severe intestinal epithelial damage, weight loss, inflammation, mucosal ulcerations, and epithelial hyperplasia. Claudin-7 has also been shown to be involved in cancer metastasis and invasion. Here, we test our hypothesis that claudin-7 plays an important role in regulating colonic intestinal stem cell function. Conditional knockout of Cldn7 in the colon led to impaired epithelial cell differentiation, hyperproliferative epithelium, a decrease in active stem cells, and dramatically altered gene expression profiles. In 3D colonoid culture, claudin-7-deficient crypts were unable to survive and form spheroids, emphasizing the importance of claudin-7 in stem cell survival. Inhibition of the Hippo pathway or activation of Notch signaling partially rescued the defective stem cell behavior. Concurrent Notch activation and Hippo inhibition resulted in restored colonoid survival, growth, and differentiation to the level comparable to those of wild-type derived crypts. In this study, we highlight the essential role of claudin-7 in regulating Notch and Hippo signaling-dependent colonic stem cell functions, including survival, self-renewal, and differentiation. These new findings may shed light on potential avenues to explore for drug development in colorectal cancer.

Molecular Characteristics and Therapeutic Vulnerabilities of Claudin-low Breast Cancers Derived from Cell Line Models.

BACKGROUND/AIM: Breast cancers constitute heterogeneous tumor groups and their categorization in subtypes based on the expression of the estrogen (ER), progesterone (PR) and HER2 receptors has advanced therapeutics. Claudin-low breast cancer has been proposed as an additional subtype which is mostly ER, PR and HER2 negative, but its identification has not led to corresponding specific treatments yet. MATERIALS AND METHODS: Breast cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were assessed for mRNA suppression of claudins and mRNA expression of ER and ERBB2 (the gene encoding HER2). The set of identified claudin-low cell lines were compared with representative ER-/ERBB2- cell lines for associated molecular alterations, gene dependencies through CRISPR and microRNA arrays and in vitro drug sensitivities using the Genomics of Drug Sensitivity in Cancer (GDSC) project. RESULTS: Claudin-low cell lines display up-regulation of mRNA expression of epithelial to mesenchymal transition (EMT) regulators. Methylation sensitive genes are down-regulated in claudin-low lines compared with other cell lines, without associated up-regulation of DNA methyltransferases. Dependency screen microarrays reveal dependencies of claudin-low cell lines on components of the cytoskeleton but no consistent dependencies in known oncogenes or tumor suppressors. Potential drug sensitivities revealed in the drug screens included sensitivities to WNT pathway modulators, tyrosine kinase cascade inhibitors and BET inhibitors. On the other hand, claudin-low cell lines showed resistance to deacetylase inhibitors. CONCLUSION: Claudin-low cell line models duplicate features of claudin-low breast cancers and may serve as guides for identification of drugs worth exploring for further development.