期刊
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
卷 20, 期 11, 页码 2465-2470出版社
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.ATV.20.11.2465
关键词
caveolin-1; ras; cholesterol; LDL; ECs
资金
- NHLBI NIH HHS [HL-43023] Funding Source: Medline
To explore the role of LDL in caveolin-Ras regulation in human endothelial cells (ECs), we incubated confluent human umbilical vein endothelial cells (HUVECs) with LDL. This resulted in a high steady-state caveolin-1 (Cav-1) expression at both the mRNA and protein levels. LDL exposure appeared not to regulate the abundance of Cav-1. Immunofluorescence staining showed that Cav-1 protein migrated from the cytoplasm to the cell membrane after LDL exposure. Cav-1 protein and cholesterol partitioned mainly into the caveola fractions, and LDL increased both Cav-1 and cholesterol in these fractions. Ras protein in caveola fractions was also increased by LDL. Increased Ras was detected in Cav-1 immunoprecipitated samples, and conversely, increased Cav-1 was found in Ras-immunoprecipitated samples. We also demonstrated LDL-increased Ras activity in HUVEC's by measuring the GTP/GTP + GDP ratio of Ras with [P-32]orthophosphate labeling in the cells. Finally, we determined the binding of [H-3]-labeled free cholesterol and recombinant H-Ras to Cav-1 fusion proteins in vitro. Both cholesterol and Ras bound to full-length GST-Cav-1, scaffolding domain (61-101), and C-terminal (135-178) Cav-1 fusion peptides. Addition of cholesterol enhanced Ras binding to the full-length and scaffolding domain of Cav-1 but not to the C-terminal Cav-1. These findings strongly suggest a role for Cav-1 in cholesterol trafficking and cholesterol-mediated intracellular signaling, which may mediate EC activation by LDL.
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