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Rigidity and inflammatory responses of interconnected endothelial cells are stimulated by rhodocetin-αβ via the neuropilin-1-MET-axis

ABSTRACT: Using the snake venom component rhodocetin-αβ (RCαβ), we elucidated how neuropilin-1 (NRP1)-dependent signaling of the hepatocyte growth factor/scatter factor (HGF/SF) receptor, MET, modulates endothelial cell-cell contacts, cell tension, and consequently nuclear factor kappa B (NFκB)-triggered proinflammatory response. We show that RCαβ in confluent ECs, also if exposed to fluid shear forces, induces loss of force-transmitting focal adhesions and a change of vinculin localization from cell-matrix contacts to cell-cell contacts. Together with RhoA activation, this enhances force transmission via intercellular contacts and raises cell rigidity within the EC monolayer. Presumably due to the combined effects of redirecting forces and NRP1-MET-signaling, RCαβ, in contrast to HGF, activates NFκB and thus a distinct set of genes. Transcriptome analysis revealed an RCαβ-induced activation of the intercellular adhesion molecule-1 (ICAM1), of other markers of EC activation and of several inflammatory cytokines, resulting in increased pericellular permeability and enhanced leukocyte attachment and transmigration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE121297 | GEO | 2019/03/04

REPOSITORIES: GEO

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Project description:MET, the receptor for hepatocyte growth factor (HGF), is involved in wide variety of biological events and its aberrant activation is implicated in the pathogenesis of cancer. Especially, MET signaling is associated with resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), key drugs in therapy of non-small cell lung cancer. MET has 11 potential N-glycosylation sites, however, the site-specific roles of N-glycans have not been elucidated. Here, we report that N-glycans regulate proteolytic processing of MET and HGF induced MET signaling, site specifically. N-glycosylation inhibitors suppressed processing and trafficking of endogenous MET in H1975 and EBC-1 lung cancer cells and exogenous MET in CHO cells. We purified the recombinant extracellular domain of human MET and determined the site-specific N-glycan structures and occupancy using nanoflow liquid chromatography-electrospray ionization mass spectrometry. The results indicated that most sites were fully glycosylated and the dominant population were complex-type which were rich in sialic acids and core fucoses. To examine the effects of N-glycan deletion of MET, we prepared endogenous-MET-knockout Flp-In CHO cells and transfected with series of N-glycan deletion mutants of MET. It was found that several N-glycans are implicated in processing of MET. It was also suggested that the N-glycans of -subunit of MET positively regulate HGF signaling, and the N-glycans of β-subunit negatively regulate HGF signaling. In all-N-glycan deletion mutant, processing and signaling were significantly suppressed. It was observed that the cell proliferation rate was suppressed in all-N-glycan deletion mutant, although the cell surface expression levels of MET were not completely diminished. We examined the HGF biding affinity for the recombinant extracellular domain of MET and found that peptide N-glycosidase F treatment did not affect the ligand binding affinity. It was suggested that N-glycans of MET affect the status and the function of the receptor site-specifically.

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Rigidity and inflammatory responses of interconnected endothelial cells are stimulated by rhodocetin-αβ via the neuropilin-1-MET-axis

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