Background & Aims Integrity of the intestinal epithelium is required for nutrition absorption and defense against pathogens. Claudins are cell adhesion molecules that localize at tight junctions (TJs); many are expressed in the intestinal tract, but little is known about their functions. Claudin-7 is unique in that it has a stronger basolateral membrane distribution than other claudins, which localize primarily to apical TJs in the intestinal epithelium. We investigated the basolateral functions of claudin-7 and assessed the effects of disruption of Cldn7 in intestines of mice. Methods We generated Cldn7−/− mice and examined their intestines by histology, molecular and cellular biology, and biochemistry approaches. We carried out gene silencing experiments in epithelial cell lines using small interfering (si)RNAs. Results The Cldn7−/− mice had severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing, and inflammation. Intestines of Cldn7−/− mice produced significantly higher levels of cytokines, the NF-κB p65 subunit, and COX-2; they also upregulated expression of matrix metalloproteinases (MMPs)-3 and -7. siRNA in epithelial cell lines demonstrated that the increased expression of MMP-3 resulted directly from claudin-7 depletion, whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that intestines of Cldn7−/− mice had intercellular gaps below TJs and cell-matrix loosening. Deletion of Cldn7 reduced expression and altered localization of the integrin α2 subunit; disrupted formation of complexes of claudin-7, integrin α2, and claudin-1 that normally form in epithelial basolateral compartments of intestines. Conclusion In mice, claudin-7 has non-TJ functions, including maintenance of epithelial cell–matrix interactions and intestinal homeostasis.
Claudin-7, a member of the claudin family, is highly expressed in distal nephrons of kidneys and has been reported to be involved in the regulation of paracellular Cl(-) permeability in cell cultures. To investigate the role of claudin-7 in vivo, we generated claudin-7 knockout mice (Cln7(-/-)) by the gene-targeting deletion method. Here we report that Cln7(-/-) mice were born viable, but died within 12 days after birth. Cln7(-/-) mice showed severe salt wasting, chronic dehydration, and growth retardation. We found that urine Na(+), Cl(-), and K(+) were significantly increased in Cln7(-/-) mice compared with that of Cln7(+/+) mice. Blood urea nitrogen and hematocrit were also significantly higher in Cln7(-/-) mice. The wrinkled skin was evident when Cln7(-/-) mice were approximately 1 wk old, indicating that they suffered from chronic fluid loss. Transepidermal water loss measurements showed no difference between Cln7(+/+) and Cln7(-/-) skin, suggesting that there was no transepidermal water barrier defect in Cln7(-/-) mice. Claudin-7 deletion resulted in the dramatic increase of aldosterone synthase mRNA level as early as 2 days after birth. The significant increases of epithelial Na(+) channel alpha, Na(+)-Cl(-) cotransporter, and aquaporin 2 mRNA levels revealed a compensatory response to the loss of electrolytes and fluid in Cln7(-/-) mice. Na(+)-K(+)-ATPase alpha(1) expression level was also greatly increased in distal convoluted tubules and collecting ducts where claudin-7 is normally expressed. Our study demonstrates that claudin-7 is essential for NaCl homeostasis in distal nephrons, and the paracellular ion transport pathway plays indispensable roles in keeping ionic balance in kidneys.
Mutations in WNK4 have been linked to hypertension in PHAII. Paracellular ion transport has been reported to be involved in this disease process; however, the specific molecular target has not been identified. In this study, we found that TJ protein claudin-7 and WNK4 were partially co-localized in renal tubules of rat kidney and co-immunoprecipitated in kidney epithelial cells. The wild-type and PHAII-causing mutant, but not the kinase-dead mutant, phosphorylated claudin-7. We have identified ser 206 in the COOH-terminus of claudin-7 as a putative phosphorylation site for WNK4. More importantly, disease-causing mutant enhanced claudin-7 phosphorylation and significantly increased paracellular permeability to Cl À .
Although δ-catenin/neural plakophilin-related armadillo protein (NPRAP) was reported to interact with presenilin-1 (PS-1), the effects of PS-1 on δ-catenin have not been established. In this study, we report that overexpression of PS-1 inhibits the δ-catenin-induced dendrite-like morphological changes in NIH 3T3 cells and promotes δ-catenin processing and turnover. The effects of PS-1 on endogenous δ-catenin processing were confirmed in hippocampal neurons overexpressing PS-1, as well as in the transgenic mice expressing the disease-causing mutant PS-1 (M146V). In addition, disease-causing mutant PS-1 (M146V and L286V) enhanced δ-catenin processing, whereas PS-1/γ-secretase inhibitors could block the formation of processed forms of δ-catenin. Together, our findings suggest that PS-1 can affect δ-catenin-induced morphogenesis possibly through the regulation of its processing and stability. KeywordsAlzheimer's disease; δ-Catenin/NPRAP; Presenilin/γ-secretase Alzheimer's disease (AD) is the most common type of senile dementia and is characterized by progressive loss of memory and cognitive dysfunction [16]. In familial Alzheimer's disease (FAD), over 70 mutations have been significantly associated with presenilin-1 (PS-1) and presenilin-2 (PS-2) [7]. Multiple lines of evidence indicate that PS contributes directly to the intramembranous "γ-secretase" processing of many proteins of diverse functions, including APP [3], the developmental signaling receptor Notch1 [4], and cell adhesion molecules N-and E-cadherin [15]. Interestingly, it is reported that PS-1 interacts with a member of the armadillo/ β-catenin family termed δ-catenin, a protein expressed mostly in the brain and encoded on chromosome 5 [21]. δ-Catenin, or neural plakophilin-related armadillo protein (NPRAP), is a member of the p120 ctn subfamily of armadillo/β-catenin proteins, which are defined as proteins with 10 armadillo (ARM) repeats in characteristic spacing and with diverse NH 2 -and COOH-terminal sequences that flank the ARM repeats [18]. δ-Catenin is a nervous system-specific adherens junction protein involved in cell motility and expressed early in neuronal development [14]. The overexpression of δ-catenin can induce the branching of cellular processes in 3T3 cells In this report, we investigated the effects of PS-1 on δ-catenin. The co-overexpressed PS-1 significantly impaired δ-catenin-induced cellular branching in NIH 3T3 fibroblasts. We also found that PS-1 expression promoted δ-catenin processing when they were co-transfected in 3T3 cells and when PS-1 was overexpressed in hippocampal neurons, which was sensitive to PS-1/γ-secretase inhibition and was facilitated by the Alzheimer's disease-causing PS-1 mutation. Our findings suggest that one function of PS-1 expression is to promote δ-catenin processing and, thereby, affect the function of δ-catenin in cells. Experimental procedures Plasmid constructionConstruction of full-length-δ-catenin constructs in pEGFP-C1 or pRFP-C1 has been described previously [11]. The wild-type PS-1 and...
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