Abstract 1435: LOX-1 Upregulation Shifts Endocytosis of Cholera Toxin from Caveolae-mediated to Noncaveolae-mediated Pathway in Endothelial Cells
Background and Objectives Endocytosis via specific pathways is critical for endothelial functions such as eNOS activation. Caveolae and coated pits are two major portals for endocytosis, but characteristics of their plasticity are unknown. Cholera toxin (CT) and transferrin (Tf) are representative substances endocytosed via caveolae and coated pits, respectively. We examined spatiotemporal dynamics of CT and Tf endocytosis into endothelial cells following upregulation of Lectin-like oxidized LDL receptor-1 (LOX-1).
Methods and Results Immunofluorescence and tracking of caveolin-1-GFP confirmed that fluorescence-labeled CT was endocytosed via caveolae in normal endothelial cells, but via a noncaveolae pathway in endothelial cells treated with TNFα (10 ng/ml, 12 hr). Although endocytosed CT was transferred to EEA-1-positive early endosomes irrespective of TNFα treatment, time course analysis indicated that TNFα enhanced endocytotic efficiency for CT, but not for Tf. Optiprep ultracentrifugation showed LOX-1 in normal endothelial cells to be confined to the lightest buoyant membrane fraction, but LOX-1 was shown not to be colocalized with caveolin-1 and CT, suggesting that LOX-1 was present in a caveolin-1-negative lipid raft without involvement in CT endocytosis. When endothelial cells were treated with TNFα, however, upregulated LOX-1 overflowed from the light fraction to a heavier fraction, and most of the CT became endocytosed via a non-caveolar pathway with high colocalization of clustered LOX-1 or Tf. Live cell imaging confirmed this using LOX-1-GFP, which was observed to spatially behave like endogenously upregulated LOX-1, and a large fraction of CT was colocalized with LOX-1-GFP. Dominant negative dynamin-2 blocked endocytosis of CT and Tf, with or without TNFα treatment.
Conclusion CT endocytosis shifts from a caveolae-mediated to a noncaveolae-mediated pathway as LOX-1 expression increases in endothelial cells. The pathway shared by LOX-1 trafficking may include one that is clathrin-mediated, since it was found to be highly colocalized with Tf and was still dynamin-2-dependent. Such shifting of endocytotic pathway may affect endothelial function in pathophysiology of vascular inflammation and atherosclerosis.