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The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.

Background & aimsThere is evidence from clinical studies that compromised intestinal epithelial permeability contributes to the development of nonalcoholic steatohepatitis (NASH), but the exact mechanisms are not clear. Mice with disruption of the gene (F11r) encoding junctional adhesion molecule A (JAM-A) have defects in intestinal epithelial permeability. We used these mice to study how disruption of the intestinal epithelial barrier contributes to NASH.MethodsMale C57BL/6 (control) or F11r(-/-) mice were fed a normal diet or a diet high in saturated fat, fructose, and cholesterol (HFCD) for 8 weeks. Liver and intestinal tissues were collected and analyzed by histology, quantitative reverse-transcription polymerase chain reaction, and flow cytometry. Intestinal epithelial permeability was assessed in mice by measuring permeability to fluorescently labeled dextran. The intestinal microbiota were analyzed using 16S ribosomal RNA sequencing. We also analyzed biopsy specimens from proximal colons of 30 patients with nonalcoholic fatty liver disease (NAFLD) and 19 subjects without NAFLD (controls) undergoing surveillance colonoscopy.ResultsF11r(-/-) mice fed a HFCD, but not a normal diet, developed histologic and pathologic features of severe NASH including steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis, whereas control mice fed a HFCD developed only modest steatosis. Interestingly, there were no differences in body weight, ratio of liver weight:body weight, or glucose homeostasis between control and F11r(-/-) mice fed a HFCD. In these mice, liver injury was associated with significant increases in mucosal inflammation, tight junction disruption, and intestinal epithelial permeability to bacterial endotoxins, compared with control mice or F11r(-/-) mice fed a normal diet. The HFCD led to a significant increase in inflammatory microbial taxa in F11r(-/-) mice, compared with control mice. Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r(-/-) mice fed a HFCD. Protein and transcript levels of JAM-A were significantly lower in the intestinal mucosa of patients with NAFLD than without NAFLD; decreased expression of JAM-A correlated with increased mucosal inflammation.ConclusionsMice with defects in intestinal epithelial permeability develop more severe steatohepatitis after a HFCD than control mice, and colon tissues from patients with NAFLD have lower levels of JAM-A and higher levels of inflammation than subjects without NAFLD. These findings indicate that intestinal epithelial barrier function and microbial dysbiosis contribute to the development of NASH. Restoration of intestinal barrier integrity and manipulation of gut microbiota might be developed as therapeutic strategies for patients with NASH.

BackgroundThe EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased β-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2.ResultsWe found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and β-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-β-catenin upstream of PI3K and AKT.ConclusionsWe conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of β-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

BackgroundThe EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased β-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2.ResultsWe found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and β-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-β-catenin upstream of PI3K and AKT.ConclusionsWe conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of β-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.

The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling.