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Cerebrospinal fluid (CSF) levels of amyloid-β 42 (Aβ42) and tau have been evaluated as endophenotypes in Alzheimer's disease (AD) genetic studies. Although there are sex differences in AD risk, sex differences have not been evaluated in genetic studies of AD endophenotypes. We performed sex-stratified and sex interaction genetic analyses of CSF biomarkers to identify sex-specific associations. Data came from a previous genome-wide association study (GWAS) of CSF Aβ42 and tau (1527 males, 1509 females). We evaluated sex interactions at previous loci, performed sex-stratified GWAS to identify sex-specific associations, and evaluated sex interactions at sex-specific GWAS loci. We then evaluated sex-specific associations between prefrontal cortex (PFC) gene expression at relevant loci and autopsy measures of plaques and tangles using data from the Religious Orders Study and Rush Memory and Aging Project. In Aβ42, we observed sex interactions at one previous and one novel locus: rs316341 within SERPINB1 (p = 0.04) and rs13115400 near LINC00290 (p = 0.002). These loci showed stronger associations among females (β = - 0.03, p = 4.25 × 10; β = 0.03, p = 3.97 × 10) than males (β = - 0.02, p = 0.009; β = 0.01, p = 0.20). Higher levels of expression of SERPINB1, SERPINB6, and SERPINB9 in PFC was associated with higher levels of amyloidosis among females (corrected p values < 0.02) but not males (p > 0.38). In total tau, we observed a sex interaction at a previous locus, rs1393060 proximal to GMNC (p = 0.004), driven by a stronger association among females (β = 0.05, p = 4.57 × 10) compared to males (β = 0.02, p = 0.03). There was also a sex-specific association between rs1393060 and tangle density at autopsy (p = 0.047; p = 0.96), and higher levels of expression of two genes within this locus were associated with lower tangle density among females (OSTN p = 0.006; CLDN16 p = 0.002) but not males (p ≥ 0.32). Results suggest a female-specific role for SERPINB1 in amyloidosis and for OSTN and CLDN16 in tau pathology. Sex-specific genetic analyses may improve understanding of AD's genetic architecture.
Our recent study has shown that cellular junctions in myelin and in the epi-/perineruium that encase nerve fibers regulate the permeability of the peripheral nerves. This permeability may affect propagation of the action potential. Direct interactions between the PDZ₁ domain of zonula occludens (ZO₁ or ZO₂) and the C-termini of claudins are known to be crucial for the formation of tight junctions. Using the purified PDZ₁ domain of ZO₂ and a variety of C-terminal mutants of peripheral nerve claudins (claudin-1, claudin-2, claudin-3, claudin-5 in epi-/perineurium; claudin-19 in myelin), we have utilized NMR spectroscopy to determine specific roles of the 3 C-terminal claudin residues (position -2, -1, 0) for their interactions with PDZ₁ of ZO₂. In contrast to the canonical model that emphasizes the importance of residues at the -2 and 0 positions, our results demonstrate that, for peripheral nerve claudins, the residue at position -1 plays a critical role in association with PDZ₁, while the side-chain of residue 0 plays a significant but lesser role. Surprisingly, claudin-19, the most abundant claudin in myelin, exhibited no binding to ZO₂. These findings reveal that the binding mechanism of claudin/ZO in epi-/perineurium is distinct from the canonical interactions between non-ZO PDZ-containing proteins with their ligands. This observation provides the molecular basis for a strategy to develop drugs that target tight junctions in the epi-/perineurium of peripheral nerves.
Published by Elsevier Inc.
In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest that loss of claudin-7 potentiates EMT to promote colon cancer, in a manner dependent on Rab25.
Peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein strongly expressed in myelinating Schwann cells of the peripheral nervous system. Myriad missense mutations in PMP22 result in varying degrees of peripheral neuropathy. We used Rosetta 3.5 to generate a homology model of PMP22 based on the recently published crystal structure of claudin-15. The model suggests that several mutations known to result in neuropathy act by disrupting transmembrane helix packing interactions. Our model also supports suggestions from previous studies that the first transmembrane helix is not tightly associated with the rest of the helical bundle.
OBJECTIVE - Helicobacter pylori strains that express the oncoprotein CagA augment risk for gastric cancer. However, the precise mechanisms through which cag(+) strains heighten cancer risk have not been fully delineated and model systems that recapitulate the gastric niche are critical for understanding pathogenesis. Gastroids are three-dimensional organ-like structures that provide unique opportunities to study host-H. pylori interactions in a preclinical model. We used gastroids to inform and direct in vitro studies to define mechanisms through which H. pylori modulates expression of the cancer-associated tight junction protein claudin-7.
DESIGN - Gastroids were infected by luminal microinjection, and MKN28 gastric epithelial cells were cocultured with H. pylori wild-type cag(+) strains or isogenic mutants. β-catenin, claudin-7 and snail localisation was determined by immunocytochemistry. Proliferation was assessed using 5-ethynyl-2'-deoxyuridine, and levels of claudin-7 and snail were determined by western blot and flow cytometry.
RESULTS - Gastroids developed into a self-organising differentiation axis and H. pylori induced mislocalisation of claudin-7 and increased proliferation in a CagA- and β-catenin-dependent manner. In MKN28 cells, H pylori-induced suppression of claudin-7 was regulated by β-catenin and snail. Similarly, snail expression was increased and claudin-7 levels were decreased among H. pylori-infected individuals.
CONCLUSIONS - H. pylori increase proliferation in a strain-specific manner in a novel gastroid system. H. pylori also alter expression and localisation of claudin-7 in gastroids and human epithelial cells, which is mediated by β-catenin and snail activation. These data provide new insights into molecular interactions with carcinogenic potential that occur between H. pylori and epithelial cells within the gastric niche.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naive Cl-2TG mice harbored significantly increased numbers of regulatory (CD4(+)Foxp3(+)) T cells than WT littermates. Furthermore, macrophages isolated from Cl-2TG mouse colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.
AMPA receptor (AMPA-R) complexes consist of channel-forming subunits, GluA1-4, and auxiliary proteins, including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. The combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors, we compared the proteins that copurify with each receptor type in the rat brain. This interactome study identified the majority of known interacting proteins and, more importantly, provides candidates for additional studies. We validate the claudin homolog GSG1L as a newly identified binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes, and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function.
Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.
The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.
BACKGROUND - The assembly of distinct proteins into tight junctions results in the formation of a continuous barrier that regulates the paracellular flux of water, ions, and small molecules across epithelia. The claudin protein family encompasses numerous major structural components of tight junctions. These proteins specify the permeability characteristics of tight junctions and consequently, some of the physiological properties of epithelia. Furthermore, defective claudin expression has been found to correlate with some diseases, tumor progression, and defective morphogenesis. Investigating the pattern of claudin expression during embryogenesis or in certain pathological conditions is necessary to begin disclosing the role of these proteins in health and disease.
RESULTS - This study analyzed the expression of several claudins during mouse pancreas organogenesis and in pancreatic intraepithelial neoplasias of mouse and human origin.
CONCLUSIONS - Our results underscored a distinctive, dynamic distribution of certain claudins in both the developing pancreas and the pancreatic epithelium undergoing neoplastic transformation.
Copyright © 2012 Wiley Periodicals, Inc.
Claudin-2 is a unique member of the claudin family of transmembrane proteins, as its expression is restricted to the leaky epithelium in vivo and correlates with epithelial leakiness in vitro. However, recent evidence suggests potential functions of claudin-2 that are relevant to neoplastic transformation and growth. In accordance, here we report, on the basis of analysis of mRNA and protein expression using a total of 309 patient samples that claudin-2 expression is significantly increased in colorectal cancer and correlates with cancer progression. We also report similar increases in claudin-2 expression in inflammatory bowel disease-associated colorectal cancer. Most importantly, we demonstrate that the increased claudin-2 expression in colorectal cancer is causally associated with tumor growth as forced claudin-2 expression in colon cancer cells that do not express claudin-2 resulted in significant increases in cell proliferation, anchorage-independent growth and tumor growth in vivo. We further show that the colonic microenvironment regulates claudin-2 expression in a manner dependent on signaling through the EGF receptor (EGFR), a key regulator of colon tumorigenesis. In addition, claudin-2 expression is specifically decreased in the colon of waved-2 mice, naturally deficient in EGFR activation. Furthermore, genetic silencing of claudin-2 expression in Caco-2, a colon cancer cell line, prevents the EGF-induced increase in cell proliferation. Taken together, these results uncover a novel role for claudin-2 in promoting colon cancer, potentially via EGFR transactivation.