Dataset Information

Guanine nucleotide-binding protein G?i2: a new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells.

ABSTRACT: The blood-brain barrier (BBB) selectively controls the exchanges between the blood and the brain: it is formed by tight junctions (TJs) between adjacent microvascular endothelial cells. The transmembrane protein claudin-5 is known as a key TJ protein at the BBB, although, the molecular mechanisms by which it regulates TJ tightness are poorly understood. To identify putative claudin-5 partners that contribute to TJ integrity, claudin-5-enriched membrane microdomains were prepared by cell fractionation, using the human brain endothelial cell line hCMEC/D3 and claudin-5 immunoprecipitates were submitted to tandem mass spectrometry. Because a high concentration of mannitol is known to transiently destabilize TJs, this analysis was performed in basal conditions, after mannitol treatment, and after recovery of TJ integrity. We here demonstrate that the G-protein subunit ?i2 (G?i2) interacts with claudin-5 and that association is correlated with TJ integrity in hCMEC/D3 cells; also, a selective expression of G?i2 is observed in human brain vasculature in situ. Moreover, small interfering RNA-mediated depletion of G?i2 or claudin-5 in hCMEC/D3 cells similarly increases their paracellular permeability and delays TJ recovery after mannitol treatment. Altogether, our results identify G?i2 as a novel claudin-5 partner required for TJ integrity in brain endothelial cells.

SUBMITTER: Luissint AC

PROVIDER: S-EPMC3345908 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Guanine nucleotide-binding protein Gαi2: a new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells.

Luissint Anny-Claude AC Federici Christian C Guillonneau François F Chrétien Fabrice F Camoin Luc L Glacial Fabienne F Ganeshamoorthy Kayathiri K Couraud Pierre-Olivier PO

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 20120215 5

The blood-brain barrier (BBB) selectively controls the exchanges between the blood and the brain: it is formed by tight junctions (TJs) between adjacent microvascular endothelial cells. The transmembrane protein claudin-5 is known as a key TJ protein at the BBB, although, the molecular mechanisms by which it regulates TJ tightness are poorly understood. To identify putative claudin-5 partners that contribute to TJ integrity, claudin-5-enriched membrane microdomains were prepared by cell fraction ...[more]

PMID: 22333621

Similar Datasets

Project description:BACKGROUND & AIMS:Inflammatory bowel diseases (IBD) increase risk for colorectal cancer. Mutations in the Mediterranean fever gene (MEFV or pyrin) are associated with hereditary autoinflammatory disease and severe IBD. Expression of MEFV, a sensor protein that the initiates assembly of the inflammasome complex, is increased in colon biopsies from patients with IBD. We investigated the role of pyrin in intestinal homeostasis in mice. METHODS:Mefv-/- mice and C57/BL6 mice (controls) were given azoxymethane followed by multiple rounds of dextran sodium sulfate (DSS) to induce colitis and tumorigenesis. In some experiments, Mefv-/- mice were given injections of recombinant interleukin 18 (rIL18) or saline (control) during DSS administration. Colon tissues were collected at different time points during colitis development and analyzed by histology, immunohistochemistry, immunoblots, or ELISAs (to measure cytokines). Spleen and mesenteric lymph node were collected, processed, and analyzed by flow cytometry. Colon epithelial permeability was measured in mice with colitis by gavage of fluorescent dextran and quantification of serum levels. RESULTS:MEFV was expressed in colons of control mice and expression increased during chronic and acute inflammation; high levels were detected in colon tumor and adjacent non-tumor tissues. Mefv-/- mice developed more severe colitis than control mice, with a greater extent of epithelial hyperplasia and a larger tumor burden. Levels of inflammatory cytokines (IL6) and chemokines were significantly higher in colons of Mefv-/- mice than control mice following colitis induction, whereas the level IL18, which depends on the inflammasome for maturation and release, was significantly lower in colons of Mefv-/- mice. Mefv-/- mice had increased epithelial permeability following administration of DSS than control mice, and loss of the tight junction proteins occludin and claudin-2 from intercellular junctions. STAT3 was activated (phosphorylated) in inflamed colon tissues from Mefv-/-, which also had increased expression of stem cell markers (OLFM4, BMI1, and MSI1) compared with colons from control mice. Administration of rIL18 to Mefv-/- mice reduced epithelial permeability, intestinal inflammation, the severity of colitis, and colon tumorigenesis. CONCLUSIONS:In studies with DSS-induced colitis, we found that pyrin (MEFV) is required for inflammasome activation and IL18 maturation, which promote intestinal barrier integrity and prevent colon inflammation and tumorigenesis. Strategies to increase activity of MEFV or IL18 might be developed for the treatment of IBD and prevention of colitis-associated tumorigenesis.

Project description:Brain microvascular endothelial cells form a highly selective blood brain barrier regulated by the endothelial tight junctions. Cerebral ischemia selectively targets tight junction protein complexes, which leads to significant damage to cerebral microvasculature. Short noncoding molecules called microRNAs are implicated in the regulation of various pathological states, including endothelial barrier dysfunction. In the present study, we investigated the influence of microRNA-155 (miR-155) on the barrier characteristics of human primary brain microvascular endothelial cells (HBMECs). Oxygen-glucose deprivation was used as an in vitro model of ischemic stroke. HBMECs were subjected to 3 hours of oxygen-glucose deprivation, followed by transfections with miR-155 inhibitor, mimic, or appropriate control oligonucleotides. Intact normoxia control HBMECs and 4 oxygen-glucose deprivation-treated groups of cells transfected with appropriate nucleotide were subjected to endothelial monolayer electrical resistance and permeability assays, cell viability assay, assessment of NO and human cytokine/chemokine release, immunofluorescence microscopy, Western blot, and polymerase chain reaction analyses. Assessment of endothelial resistance and permeability demonstrated that miR-155 inhibition improved HBMECs monolayer integrity. In addition, miR-155 inhibition significantly increased the levels of major tight junction proteins claudin-1 and zonula occludens protein-1, while its overexpression reduced these levels. Immunoprecipitation and colocalization analyses detected that miR-155 inhibition supported the association between zonula occludens protein-1 and claudin-1 and their stabilization at the HBMEC membrane. Luciferase reporter assay verified that claudin-1 is directly targeted by miR-155. Based on these results, we conclude that miR-155 inhibition-induced strengthening of endothelial tight junctions after oxygen-glucose deprivation is mediated via its direct target protein claudin-1.

Dataset Information

Guanine nucleotide-binding protein G?i2: a new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells.

Publications

Guanine nucleotide-binding protein Gαi2: a new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets