| Summary |
Tight junctions (TJs), the most apical cell-cell junction, seal adjacent epithelial cells and regulate the passage of ions and small molecules through the paracellular pathway. TJs are made up of claudins, occludins, and junctional adhesion molecules. Claudin-7 specifically is highly expressed in the intestine at both the apical and basolateral compartments. Claudin-7 is essential for barrier function and intestinal homeostasis as deletion of claudin-7 in mouse models induces an inflammatory bowel disease (IBD)-like phenotype, exhibiting severe inflammation, intestinal epithelial damage, mucosal ulcerations, weight loss, and bloody-loose stools. Additionally, claudin-7 is involved in many cellular processes including cell proliferation, cell-matrix interactions, and epithelial-to-mesenchymal transition (EMT). We hypothesize that claudin-7 exhibits other roles in addition to its traditional function in barrier function to regulate large intestine stem cell functions, as well as interaction with and alteration of other cell junction proteins as viewed at the nanoscale level.In this study, we demonstrate the role of TJ protein claudin-7 in the regulation of large intestine stem cell survival, self-renewal, proliferation, and differentiation, vital processes for intestinal regeneration. Conditional knockout of claudin-7 in the intestines led to altered gene expression profiles, disrupted epithelial cell differentiation, epithelial hyperplasia, downregulated Notch signaling, and loss of the active stem cell pool. Additionally, isolated claudin-7 deficient crypts were unable to survive and form fully closed spheroids in ex vivo organoid culture, demonstrating the essential role of claudin-7 in stem cell survival. Stem cell survival was rescued through activation of the Notch signaling cascade, as well as inhibition of Hippo signaling. Delayed knockout of claudin-7 in established organoids also demonstrated disrupted differentiation ability with loss of claudin-7. Additionally, claudin-7 has been shown to interact with other junctional complexes to regulate barrier function. In this study, we utilized super-resolution Stochastic Optical Reconstruction Microscopy (STORM) to examine the interaction between junctional proteins at the nanoscale level and highlighted the role of claudin-7 in modulating their interactions. Deletion of claudin-7 led to disorganized cell-cell junctions, as well as disorganized nanoarchitecture and decreased interaction of junctional proteins including TJ proteins ZO-1 and claudin-1, adherens junction (AJ) proteins [beta]-catenin and p120ctn, and focal adhesion proteins integrin [alpha]2 and focal adhesion kinase. This demonstrated the importance of claudin-7 in maintaining the functional interaction of junctional proteins.Overall, our study demonstrated additional non-TJ functions of claudin-7 in regulating large intestinal epithelial stem cell functions including survival, self-renewal, and differentiation, as well as modulation of junctional protein localization and nanoarchitecture. Taken together, these results provide novel information that may be used in the study of intestinal regeneration for IBD and colorectal cancer (CRC). |
