IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling

Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1-/- mice and noted that ∼ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1-/- mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1 -/- MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process. © 2013 Li et al

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock