Identification and Characterization of Caveolins in Mouse Macrophages

Peter Gargalovic, Identification and Characterization of Caveolins in Mouse Macrophages. Doctor of Philosophy (Biochemistry and Molecular Biology), December 2002, 206 pp., 3 tables, 41 illustrations, references, 296 titles. The understanding of the mechanisms which control macrophage-lipid management, and their accumulation in atherosclerotic lesions, is of significant importance. Caveolins are proteins associated with cholesterol-rich membrane domains and are intimately linked to the regulation of lipid metabolism and transport. The expression and function of caveolin proteins in three macrophage cell types: thioglycollate-elicited mouse peritoneal macrophages, resident mouse peritoneal macrophages and the J774 macrophage cell line. Data in this work establish that the primary macrophages express caveolin-1 and -2, while J774 cells express only caveolin-2. Immunofluorescence microscopy studies indicate that caveolins in primary macrophages do not colocalize, with caveolin-1 being present on the cell surface and cavelon-2 in the Golgi compartment. Analysis of macrophages also showed that caveolin-1, but not caveolin-2, is present in detergent insoluble lipid raft membranes. While caveolin expression in macrophages is not regulated by sterols, both caveolin isoforms can be secreted from cholesterol-loaded macrophages in the presence of high-density lipoprotein (HDL). Secreted caveolins are part of the complex that has a density similar to HDL, which suggests their association with HDL and potentially a role in HDL-mediated reverse cholesterol transport. The examination of caveolin expression in macrophages shows that caveolin-1, but not caveolin-2 expression is highly upregulated by agents that induce apoptosis in these cells. Induction of caveolin-1 expression precedes DNA fragmentation, is independent of caspase activation, and correlates with the exposure of phosphatidylserine on the cell surface. Importantly, immunofluorescence analysis determined that caveolin-1 in lipid rafts colocalizes extensively with phosphatidylserine present on the surface of apoptotic cells. This study thus identifies caveolin-1 as a specific and early marker of the macrophage apoptotic phenotype. Findings here strongly implicate the involvement of caveolin-1 and lipid rafts in the changes of plasma membrane lipid composition as well as involvement in efficient clearance of apoptotic cells by a phosphatidylserine-mediated mechanism.