Claudin-2 Overexpression Research Articles

Claudin-2 Mediates the Proximal Tubular Epithelial Cell-Fibroblast Crosstalk via Paracrine CTGF.

Proximal tubular epithelial cell (PTEC) is vulnerable to injury in diabetic kidney disease (DKD) due to high energy expenditure. The injured PTECs-derived profibrotic factors are thought to be driving forces in tubulointerstitial fibrosis (TIF) as they activate surrounding fibroblasts. However, the mechanisms remain unclear. The diabetes with uninephrectomy (DKD) rats were used to evaluated renal histological changes and the expression of Claudin-2 by immunofluorescence staining. Then, Claudin-2 expression in PTECs were modulated and subsequently determined the proliferation and activation of fibroblasts by building a transwell co-culture system in normal glucose (NG)or high glucose (HG) condition. Decreased expression of Claudin-2 in PTECs accompanied by tight junction disruption and increased interstitial fibrosis, were detected in DKD rats. In vitro, downregulated Claudin-2 in PTECs promoted proliferation and activation of fibroblasts, which coincided with elevated expression of profibrotic connective tissue growth factor (CTGF) in PTECs. Silenced CTGF inhibited the profibrotic of PTECs via Claudin-2 inhibition. Fibroblasts co-cultured with PTECs transitioned more to myofibroblasts and generated extracellular matrix (ECM) significantly in response to high glucose (HG) stimulation whereas overexpression of Claudin-2 in PTECs reversed the above results. Upregulating CTGF disrupted the beneficial anti-fibrosis effects obtained by overexpression of Claudin-2 in HG condition. Our study suggested that Claudin-2 in PTECs, a key mediator of paracellular cation and water transport, promotes the activation and proliferation of surrounding fibroblasts significantly via CTGF in a paracrine manner.

Chronic Ouabain Targets Pore-Forming Claudin-2 and Ameliorates Radiation-Induced Damage to the Rat Intestinal Tissue Barrier.

Ionizing radiation (IR) causes disturbances in the functions of the gastrointestinal tract. Given the therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against IR-induced disturbances in the barrier and transport properties of the jejunum and colon of rats. Male Wistar rats were subjected to 6-day intraperitoneal injections of vehicle or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to total-body X-ray irradiation (10 Gy) or a sham irradiation. Isolated tissues were examined 72 h post-irradiation. Electrophysiological characteristics and paracellular permeability for sodium fluorescein were measured in an Ussing chamber. Histological analysis and Western blotting were also performed. In the jejunum tissue, ouabain exposure did not prevent disturbances in transepithelial resistance, paracellular permeability, histological characteristics, as well as changes in the expression of claudin-1, -3, -4, tricellulin, and caspase-3 induced by IR. However, ouabain prevented overexpression of occludin and the pore-forming claudin-2. In the colon tissue, ouabain prevented electrophysiological disturbances and claudin-2 overexpression. These observations may reveal a mechanism by which circulating ouabain maintains tight junction integrity under IR-induced intestinal dysfunction.

Modulation of Intestinal Corticotropin-Releasing Hormone Signaling by the Herbal Preparation STW 5-II: Possible Mechanisms for Irritable Bowel Syndrome Management.

Corticotropin-releasing factor (CRF) mediates stress responses and alters the gut-brain axis, contributing to the pathogenesis of irritable bowel syndrome (IBS), which is recognized by abdominal pain accompanied by bowel habit disturbance. STW 5-II, a mixture of six herbal extracts, is clinically effective in functional dyspepsia and IBS. Here we aimed to establish an organoid-based stress-induced IBS-like model to investigate the mechanisms of action of STW 5-II. STW 5-II (10, 20, and 30 g/mL) was applied to intestinal organoids for 24 h before being treated with CRF (100 nM) for 48 h. The effects of STW 5-II on CRF signaling were investigated using several in vitro and in silico approaches. STW 5-II activities were further explored by in silico PyRx screening followed by molecular docking of the main 52 identified compounds in STW 5-II with both CRF receptors CRFR1 and CRFR2. CRF exposure stimulated inflammation and increased proinflammatory mediators, while STW 5-II dose-dependently counteracted these effects. STW 5-II inhibited CRF-induced claudin-2 overexpression and serotonin release. Docking of the STW 5-II constituents oleanolic acid and licorice saponin G2 to CRFR1 and CRFR2, respectively, showed a good affinity. These multi-target activities support and elucidate the clinically proven efficacy of STW 5-II in disorders of gut-brain interaction.

Increase in Anticancer Drug-Induced Toxicity by Fisetin in Lung Adenocarcinoma A549 Spheroid Cells Mediated by the Reduction of Claudin-2 Expression.

Claudin-2 (CLDN2), a component of tight junction, is involved in the reduction of anticancer drug-induced toxicity in spheroids of A549 cells derived from human lung adenocarcinoma. Fisetin, a dietary flavonoid, inhibits cancer cell growth, but its effect on chemosensitivity in spheroids is unknown. Here, we found that fisetin (20 μM) decreases the protein level of CLDN2 to 22.3%. Therefore, the expression mechanisms were investigated by real-time polymerase chain reaction and Western blotting. Spheroids were formed in round-bottom plates, and anticancer drug-induced toxicity was measured by ATP content. Fisetin decreased the phosphorylated-Akt level, and CLDN2 expression was decreased by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting the inhibition of PI3K/Akt signal is involved in the reduction of CLDN2 expression. Hypoxia level, one of the hallmarks of tumor microenvironment, was reduced by fisetin. Although fisetin did not change hypoxia inducible factor-1α level, it decreased the protein level of nuclear factor erythroid 2-related factor 2, a stress response factor, by 25.4% in the spheroids. The toxicity of doxorubicin (20 μM) was enhanced by fisetin from 62.8% to 40.9%, which was rescued by CLDN2 overexpression (51.7%). These results suggest that fisetin can enhance anticancer drug toxicity in A549 spheroids mediated by the reduction of CLDN2 expression.

NOVEL CONTRIBUTIONS OF CLAUDIN-4 TO EPITHELIAL BARRIER REGULATION AND COLITIS

Abstract Intestinal barrier dysfunction is linked to human IBD and contributes to progression of experimental, immune-mediated colitis. Claudin family tight junction proteins are critical determinants of epithelial paracellular permeability and barrier function. Expression of one of these, claudin-2, a pore-forming claudin, is specifically upregulated in IBD. We have shown that claudin-2 overexpression or, conversely, channel inhibition, augment or attenuate, respectively, immune-mediated colitis severity. We sought to understand claudin contributions to barrier function using in vitro and in vivo models. Claudin-4 overexpression has been shown to reduce cation permeability in vitro, leading to the conclusion that this protein is a barrier-forming claudin. We found, however, that, in the absence of claudin-2 expression, neither knockout nor overexpression of claudin-4 affected barrier function. Instead, claudin-4 specifically suppressed claudin-2 pore function by destabilizing claudin-2 polymers and disrupting claudin-2 anchoring at the tight junction. Thus, claudin-4 is not barrier-forming but is the first pore-regulating claudin. Together with observations that claudin-2 exacerbates colitis in vivo and that claudin-4 antagonizes claudin-2 function in vitro led us to hypothesize that intestinal epithelial claudin-4 knockout would exacerbate colitis. Knockout mice were, however, protected from both immune-mediated and DSS-induced chemical colitis. Conversely, transgenic claudin-4 overexpression exacerbated colitis. Preliminary in vitro studies suggest that claudin-4 may limit collective migration and epithelial wound repair. As a whole, our studies require a reconsideration of claudin functional classifications and reveal unanticipated contributions of claudin-4 to barrier regulation and epithelial wound repair. The relationship between these functions and claudin-4 downregulation in human IBD remains to be explored.

NOVEL CONTRIBUTIONS OF CLAUDIN-4 TO EPITHELIAL BARRIER REGULATION AND COLITIS

Intestinal barrier dysfunction is linked to human IBD and contributes to progression of experimental, immune-mediated colitis. Claudin family tight junction proteins are critical determinants of epithelial paracellular permeability and barrier function. Expression of one of these, claudin-2, a pore-forming claudin, is specifically upregulated in IBD. We have shown that claudin-2 overexpression or, conversely, channel inhibition, augment or attenuate, respectively, immune-mediated colitis severity.

MiRNA-182-5p aggravates experimental ulcerative colitis via sponging Claudin-2

Tight junction proteins play crucial roles in maintaining the integrity of intestinal mucosal barrier. MiRNA-182-5p is capable of targeting claudin-2 which is one of the vital tight junction proteins and the effect and mechanism of miRNA-182-5p was explored here in the DSS-induced colitis model. The pathological conditions were evaluated via hematoxylin and eosin staining. The gene expression level was assessed via PCR. Quantitative immunohistochemistry analysis was performed for the measurement of claudin-2. microRNA.org online tool was used for target gene prediction. Luciferase reporter assay and RNA pull-down assay were performed to detect the target of miRNA-182-5p. The inflammatory and oxidative stress level were measured using corresponding kits. MiRNA-182-5p was highly expressed in colitis model and miRNA-182-5p inhibitor exerted protective effects on colitis induced by DSS in mice. The protective effects includded improvement of pathological changes, increases in anti-inflammation and anti-oxidative genes, and up-regulation of TGF-β1. Claudin-2 mRNA was predicted as the target of miRNA-182-5p, which was validated via luciferase reporter assay and RNA pull-down assay. Claudin-2 overexpression was found in miRNA-182-5p inhibitor group. Consistent with the role of miRNA-182-5p, claudin-2 overexpression also exerted protective effects on DSS-induced colitis in mice. Inhibition of miRNA-182-5p exerted protective effects on colitis via targeting and upregulating claudin-2. The findings in study provide a new therapeutic strategy for colitis treatment and lay the foundation for future study.

Claudin-2 inhibits renal clear cell carcinoma progression by inhibiting YAP-activation

BackgroundClaudin-2 expression is upregulated in multiple cancers and promotes cancer malignancy. Remarkably, the regulation of claudin-2 expression in kidney cell lines contrasts its reported regulation in other organs. However, claudin-2 role in renal clear cell carcinoma (RCC) remains unknown despite its predominant expression in the proximal tubular epithelium (PTE), the site of RCC origin.MethodsPublicly available and independent patient databases were examined for claudin-2 association with RCC. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by Mass spectroscopy, immunoprecipitation and mutational studies, and functional evaluations.ResultsWe show that the significant decrease in claudin-2 expression characterized PTE cells and Ex-vivo cultured mouse kidney subjected to dedifferentiation. Inhibition of claudin-2 was enough to induce mesenchymal plasticity and invasive mobility in these models. Further, a progressive loss of claudin-2 expression associated with the RCC progression and poor patient survival. Overexpression of claudin-2 in RCC-derived cancer cells inhibited tumorigenic abilities and xenograft tumor growth. These data supported a novel tumor-suppressive role of claudin-2 in RCC. Mechanistic insights further revealed that claudin-2 associates with YAP-protein and modulates its phosphorylation (S127) and nuclear expression. The tumor suppressive effects of claudin-2 expression were lost upon deletion of its PDZ-binding motif emphasizing the critical role of the PDZ-domain in claudin-2 interaction with YAP in regulating RCC malignancy.ConclusionsOur results demonstrate a novel kidney specific tumor suppressive role for claudin-2 protein and further demonstrate that claudin-2 co-operates with the YAP signaling in regulating the RCC malignancy.

Intestinal Barrier, Permeability and Nonspecific Inflammation in Functional Gastrointestinal Disorders

Aim of review . Description of intestinal mucosal barrier permeability disruption causes as morbid factors in gastrointestinal tract (GIT) functional disorders. General provisions . Intestinal mucosal barrier is composed of several elements: pre-epithelial barrier, a mucosal lining of epithelium; epithelial barrier that leverages tight junctional structures and high regenerative capacity of epithelium; post-epithelial barrier established by immune cells and blood vessels. Gut microbiota profile may also be constituent of the intestinal mucosal barrier. Functional dyspepsia (FD) and irritable bowel syndrome (IBS) leave distinct morphological imprint. For instance, FD patients exhibit variant expression of cell junction proteins (claudin-1, zonula occludens, occludin, E-cadherin, β -catenin and desmoglein-2), and IBS patients — overexpression of claudin-2. Permeability disruption leads to inflammatory changes in intestinal wall resulting in visceral and central sensitisation, as well as intestinal motor dysfunctions causal of IBS and FD. Conclusion . Disruption of intestinal mucosal barrier may be considered a pathogenetic factor of PD and IBS progression.

Inactivation of paracellular cation-selective claudin-2 channels attenuates immune-mediated experimental colitis in mice.

The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.

Abstract 4029: The Wnt target gene CLAUDIN-2 regulates tumorigenesis and stemness in human liver cancer cells

Abstract Background: In our previous studies on T-cell factor (TCF)-4 isoforms, we identified a unique target gene CLAUDIN-2, which was upregulated by lack of SxxSS motif in exon 9 of TCF-4 (Exp Cell Res 2011, Liver Int 2013). This type of TCF-4 (TCF-4J) conferred high tumorigenic ability and hypoxia resistance to hepatocellular carcinoma (HCC) cells (PLoS One 2012). Of interest, the TCF-4J isoform-overexpressing HCC cells (J cells) strongly expressed the leaky tight junction protein CLAUDIN-2 under sphere-forming conditions, suggesting that J cells have cancer stem cell (CSC)-like features involving CLAUDIN-2 upregulation. Thus, the Aim of this study was to assess expression profile of CLAUDIN-2 and the CSC-relevant function of the molecule, such as tumorigenicity, in liver cancer cells. Methods: The human liver cancer cell lines used in this study were as follows: HAK-1A, HAK-1B (Hepatology 1993), Huh-7, HepG2, Hep3B, HLF, KMCH-1 (Hepatology 1987), KMCH-2 (J Hepatol 1996), KYN-1, KYN-2, and HAK-5. KMCH-1 and -2 are unique combined hepatocellular-cholangiocarcinoma cell lines, and HAK-5 is a sarcomatous HCC cell line. Lentivirus-mediated delivery of siRNA for CLAUDIN-2 (Santa Cruz) and plasmid-mediated CLAUDIN-2 cDNA (Origene) were utilized to generate stable knock-down (KD) and overexpressing cells, respectively. Results: CLAUDIN-2 was exclusively expressed in both KMCH-1 and KMCH-2 cells, showing 240~370-fold mRNA level of that in the well-differentiated HCC line HAK-1A. The protein expression was strongly observed at cytoplasmic membrane and cytoplasm in KMCH-1 and KMCH-2 cells, but not in the others examined. Gene silencing of CLAUDIN-2 resulted in slower cell proliferation. In xenograft experiment in nude mice, CLAUDIN-2-KD KMCH-2 cells formed much smaller tumors in size than the control cells. Of interest, overexpression of CLAUDIN-2 in constitutively low expressing cell lines, such as Hep3B and HLF cells, lead to high expression of Nanog, a stemness gene product. Conclusion: As previously shown in transformed HCC cells by the Wnt transcription factor TCF-4J, CLAUDIN-2 was a key molecule regulating cell proliferation and tumorigenesis in non-gene-engineered human combined hepatocellular-cholangiocarcinoma cells. To elucidate precise mechanism of exclusively high expression of this molecule in such difficult-to-treat type of liver cancer may provide insights into generating novel therapeutic approaches. Citation Format: Hironori Koga, Fumitaka Wada, Mitsuhiko Abe, Hideki Iwamoto, Toru Nakamura, Takahiko Sakaue, Atsutaka Masuda, Toshimitsu Tanaka, Hirohisa Yano, Takuji Torimura. The Wnt target gene CLAUDIN-2 regulates tumorigenesis and stemness in human liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4029.

Tight Junction Protein Claudin-2 Promotes Self-Renewal of Human Colorectal Cancer Stem-like Cells.

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil-based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer.Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925-38. ©2018 AACR.

IL-22 Upregulates Epithelial Claudin-2 to Drive Diarrhea and Enteric Pathogen Clearance

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na+ channel formed byclaudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C.rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C.rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.

Are alterations of tight junctions at molecular and ultrastructural level different in duodenal biopsies of patients with celiac disease and Crohn's disease?

Abnormalities of transmembrane and cytoplasmic proteins of tight junctions (TJ) have been implicated in pathogenesis of both celiac (CeD) and Crohn's diseases (CD). Since disease pathogenesis in CeD and CD are different, we planned to study if there is any differential expression pattern of TJ marker proteins and ultrastructural changes, respectively, in duodenal villi vs crypts. Endoscopic duodenal biopsies from treatment naïve patients with CeD (n = 24), active CD (n = 28), and functional dyspepsia (as controls, n = 15), both at baseline and 6 months after treatment, were subjected to light microscopic analysis (modified Marsh grading); immune-histochemical staining and Western blot analysis to see the expression of key TJ proteins [trans-membrane proteins (claudin-2, claudin-3, claudin-4, occludin, and JAM) and cytoplasmic protein (ZO-1)]. Transmission electron microscopy and image analysis of the TJs were also performed. There was significant overexpression of claudin-2 (pore-forming) and occludin (protein maintaining cell polarity) with under-expression of claudin-3 and claudin-4 (pore-sealing proteins) in treatment naïve CeD and active CD with simultaneous alteration in ultrastructure of TJs such as loss of penta-laminar structure and TJ dilatation. Normalization of some of these TJ proteins was noted 6 months after treatment. These changes were not disease specific and were not different in duodenal villi and crypts. Overexpression of pore-forming and under-expression of pore-sealing TJ proteins lead to dilatation of TJ. These changes are neither disease specific nor site specific and the end result of mucosal inflammation.

Significant water absorption goes paracellular in kidney proximal tubules

it had been a fundamental dispute for several decades, whether water passes epithelia such as kidney proximal tubule or small intestine along the transcellular, paracellular, or both pathways. By discovery of the transmembranal aquaporin water channels (AQP), the molecular basis of the

Claudin-4 forms a paracellular barrier, revealing the interdependence of claudin expression in the loose epithelial cell culture model opossum kidney cells.

The effect of claudins on paracellular fluxes has been predominantly studied in either Madin-Darby canine kidney (MDCK) or LLCPK cells. Neither model system has a very low transepithelial resistance (TER) as observed in leaky epithelia. Moreover, results from one model system are not always consistent with another. Opossum kidney (OK) cells form tight junctions yet have a very low TER. We therefore set out to characterize the paracellular transport properties of this cell culture model. Ussing chamber dilution potential measurements revealed that OK cells exhibit a very low TER (11.7 ± 1.4 Ω·cm(2)), slight cation selectivity (P(Na)/P(Cl) = 1.10 ± 0.01), and the Eisenman permeability sequence IV; the permeability of monovalent cations ranking K(+) > Cs(+) > Rb(+) > Na(+) > Li(+). Quantitative real-time PCR studies found that OK cells endogenously express claudin-4 > -1 > -6 > -20 > -9 > -12 > -11 > -15. Overexpression of claudin-4 significantly increased TER, decreased Na(+) and Cl(-) permeability, and increased levels of claudin-1, -6, and -9 mRNA. Knockdown of claudin-4 in the overexpressing cells significantly decreased TER without altering claudin expression; thus claudin-4 forms a barrier in OK cells. Knockdown of endogenous claudin-4 decreased claudin-1, -9, and -12 expression without altering TER. Claudin-2 overexpression decreased TER, significantly increased Na(+) and Cl(-) permeability, and decreased claudin-12 and -6 expression. Together these results demonstrate that claudin expression is tightly coupled in OK cells.

Mo1733 Targeted Claudin-2 Overexpression Protects From Experimental Colitis and Colitis-Induced Colon Cancer

G A A b st ra ct s 2011). Although classical Th1 cells via IL-12 stimulation are thought to be pathogenic in colitic conditions, the exact developmental mechanism still remains unknown. We here clarify how classical Th1 cells participate in development of colitis. METHODS: RAG-2-/mice were transferred with CD4+CD45RBhigh T cells obtained from RORγtgfp/+ reporter (RORγt-R) or RORγt-/mice. 2) RAG-2-/mice were transferred with In Vitro-manipulated Th1 cells under Th1 polarized condition (in the presence of IL-12 and anti-IL-4 mAb) from CD4+CD45RBhigh T cells obtained from RORγt-R or RORγt-/mice. 3) RAG-2-/mice were transferred with LP RORγt (GFP)-expressing CD4+ T cells from colitic RAG-2-/mice previously transferred with RORγt-R CD4+CD45RBhigh T cells. RESULTS: 1) RAG-2-/mice transferred with RORγt-R CD4+CD45RBhigh T cells developed colitis with the accumulation of Th17, Th17/Th1, and Th1 cells in inflamed mucosa, while those with RORγt/CD4+CD45RBhigh T cells did not. 2) Surprisingly, both RAG-2-/mice transferred with In Vitro-manipulated RORγt-R Th1 cells and RAG-2-/mice with In Vitro-manipulated RORγt-/Th1 cells did similarly develop colitis with the accumulation of Th1 cells, but not Th17 and Th17/Th1 cells, in inflamed mucosa. 3) RAG-2-/mice were transferred with LP RORγtexpressing CD4+ T cells developed colitis with the marked accumulation of RORγt-losing Th1 cells in the inflamed mucosa. CONCLUSIONS: Our series of transfer experiments clearly explain why both Th1 and Th17 cells are mandatory for the development of colitis; 1) classical Th1 cells require the help of Th17 and/or Th17/Th1 cells for their developmental pathway In Vivo and 2) alternative Th1 cells are differentiated from Th17 and/or Th17/ Th1 cells.

Decrease in claudin‐2 expression enhances cell migration in renal epithelial madin–darby canine kidney cells

Migration of renal epithelial cells increases after renal tubular damage, but its mechanism has not been clarified in detail. Hyperosmotic stress increased a cellular injury concomitant with a decrease in mRNA and protein expression of claudin-2 in renal tubular epithelial Madin-Darby canine kidney cells. We hypothesized that claudin-2 is involved in the regulation of cell migration. To knockdown claudin-2 expression, we made the cells expressing doxycycline-inducible claudin-2 shRNA vector. Claudin-2 knockdown affected neither the endogenous expression levels of claudin-1, -3, -4, and -7 nor the Triton X-100 solubility of these claudins. Transepithelial electrical resistance was increased by claudin-2 knockdown without affecting permeability to FITC-dextran (4,000 Da). BrdU incorporation assay and cell counting revealed that cell proliferation and viability are unaffected by claudin-2 knockdown. In the wound-healing assay, the recovery rate of wound area was increased by claudin-2 knockdown. The mRNA expression and activity of matrix metalloproteinase-9 (MMP-9) were increased by claudin-2 knockdown. A selective MMP-9 inhibitor suppressed cell migration in the claudin-2 knockdown cells. Hyperosmotic stress increased the expression and activity of MMP-9, which were inhibited by claudin-2 overexpression. These results suggest that the decrease in claudin-2 expression enhances cell migration mediated by the increase in the expression and activity of MMP-9.

Claudin-2 is selectively enriched in and promotes the formation of breast cancer liver metastases through engagement of integrin complexes

The liver represents the third most frequent site of metastasis in patients with breast cancer. We performed in vivo selection using 4T1 breast cancer cells to identify genes associated with the liver metastatic phenotype. Coincident with the loss of numerous tight-junctional proteins, we observe claudin-2 overexpression, specifically in liver-aggressive breast cancer cells. We further demonstrate that claudin-2 is both necessary and sufficient for the ability of 4T1 breast cancer cells to colonize and grow in the liver. The liver-aggressive breast cancer cells display a claudin-2-mediated increase in their ability to adhere to extracellular matrix (ECM) components, such as fibronectin and type IV collagen. Claudin-2 facilitates these cell/matrix interactions by increasing the cell surface expression of α(2)β(1)- and α(5)β(1)-integrin complexes in breast cancer cells. Indeed, claudin-2-mediated adhesion to fibronectin and type IV collagen can be blocked with neutralizing antibodies that target α(5)β(1) and α(2)β(1) complexes, respectively. Immunohistochemical analyses reveal that claudin-2, although weakly expressed in primary human breast cancers, is readily detected in all liver metastasis samples examined to date. Together, these results uncover novel roles for claudin-2 in promoting breast cancer adhesion to the ECM and define its importance during breast cancer metastasis to the liver.

Effect of Claudin Expression on Paracellular Permeability, Migration and Invasion of Colonic Cancer Cells

Alteration in the expression of claudins, consisting of tight junctions (TJs), has been reported in various clinically isolated tumors. Claudins play an important role not only in the intercellular barrier function of TJs but also in migration and invasiveness of cancer cells. However, the use of different types of cells and different claudins in these studies has complicated the picture. In this study, we systematically examined the effect of claudin (claudin-1, -2, -3, -4 and -15) overexpression on the paracellular permeability, migration and invasiveness of Caco-2 colonic cancer cells. Overexpression of claudin-4 or claudin-2 increased or decreased, respectively, paracellular permeability. Overexpression of claudin-4 specifically stimulated the invasive activity of the Caco-2 cells. Furthermore, activation of matrix metalloproteinase (MMP)-2 and MMP-9 were observed in the claudin-4-overexpressing cells, suggesting that the invasive activity was stimulated through an increase in MMP activity. Overexpression of claudin-2 or claudin-3 and -4 stimulated or inhibited, respectively, the migration activity of the Caco-2 cells. Immunostaining analysis revealed that each of the overexpressed claudins localized at TJs under the conditions used to evaluate paracellular permeability. In contrast, they localized mainly in intracellular compartments under experimental conditions designed to assess cell invasion and migration. Overall, the results of this study show that the effect exerted by the claudins on the intercellular barrier function of TJs, as well as on cell migration and invasive activity, differs depending on the particular claudin species. Furthermore, the subcellular localization of the claudins varies according to the culture conditions.