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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2465.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Lipoprotein Promotes Caveolin-1 and Ras Translocation to Caveolae

Role of Cholesterol in Endothelial Signaling

Yi Zhu; Hai-Ling Liao; Nanping Wang; Yuan Yuan; Kuo-Sheng Ma; Lynne Verna; Michael B. Stemerman

From the Division of Biomedical Sciences, University of California, Riverside.

Correspondence to Yi Zhu, MD, Division of Biomedical Sciences, University of California, Riverside, CA 92521. E-mail yi.zhu{at}ucr.edu

Abstract—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 HUVECs by measuring the GTP/GTP+GDP ratio of Ras with [³²P]orthophosphate labeling in the cells. Finally, we determined the binding of [³H]-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.

Key Words: caveolin-1 • ras • cholesterol • LDL • ECs

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