Macrophage ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), and ABCG1 have been shown to promote cholesterol efflux to extracellular acceptors in vitro and influence atherosclerosis in mice, but their roles in mediating reverse cholesterol transport (RCT) from macrophages in vivo are unknown. Using an assay of macrophage RCT in mice, we found that primary macrophages lacking ABCA1 had a significant reduction in macrophage RCT in vivo, demonstrating the importance of ABCA1 in promoting macrophage RCT, however substantial residual RCT exists in the absence of macrophage ABCA1. Using primary macrophages deficient in SR-BI expression, we found that macrophage SR-BI, which was shown to promote cholesterol efflux in vitro, does not contribute to macrophage RCT in vivo. To investigate whether macrophage ABCG1 is involved in macrophage RCT in vivo, we used ABCG1-overexpressing, -knockdown, and -knockout macrophages. We show that increased macrophage ABCG1 expression significantly promoted while knockdown or knockout of macrophage ABCG1 expression significantly reduced macrophage RCT in vivo. Finally, we show that there was a greater decrease in macrophage RCT from cells where both ABCA1 and ABCG1 expression were knocked down than from ABCG1-knockdown cells. These results demonstrate that ABCA1 and ABCG1, but not SR-BI, promote macrophage RCT in vivo and are additive in their effects.

Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family.

Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound125 I-LDL was destined for internalization and degradation, whereas about 70% of the surfacebound 125 I-HDL 3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL 3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.

Plasma lipoprotein metabolism is tightly regulated by several members of the triglyceride lipase family, including endothelial lipase (EL) and lipoprotein lipase (LPL). Our previous work suggested that EL is proteolytically processed. In this report, we have used a combination of epitope tagging, mutagenesis, and N-terminal sequencing to determine the precise location of the cleavage site within EL. The cleavage occurs immediately after the sequence RNKR, a known recognition sequence for the proprotein convertase (PC) family. We demonstrate that some PCs, but not all, can proteolytically cleave EL at this site and thereby directly regulate EL enzymatic activity through modulating EL cleavage. Furthermore, specific knockdown of individual PCs proves that PCs are the proteases that cleave EL in human endothelial cells. Interestingly, a homologous site in LPL is also cleaved by PCs. This action is unusual for PCs, which are traditionally known as activators of pro-proteins, and highlights a potential role of PCs in lipid metabolism through their proteolytic processing of lipases.Site-specific proteolysis is crucial in regulating many fundamental biological pathways, including the sequential initiation of activation of blood coagulation factors and activation of caspases and digestive enzymes (1-3). The proprotein convertase (PC) 5 family is composed of at least nine members and belongs to the subtilisin superfamily of serine endoproteases (4). The basic amino acid-specific PCs catalyze the proteolytic maturation of a strikingly diverse collection of substrates at paired basic amino acid processing sites to generate biologically active molecules in the secretory pathway (5). Some PC substrates are proteins involved in lipid metabolism. Furin, the first discovered and best characterized PC, was shown to cleave the low density lipoprotein receptorrelated protein (6), and a mutation of the processing site of chicken low density lipoprotein receptor-related protein impaired efficient exit from the endoplasmic reticulum (7). Subtilisin-kexin isozyme-1/Site-1 protease (SKI-1/S1P), an early Golgi-localized PC (8), cleaves membranebound sterol regulatory element-binding proteins and releases the active subunit of sterol regulatory element-binding proteins to maintain cholesterol homeostasis (9).PCs were considered to be redundant because a large body of in vitro work had shown that several PCs could cleave the same substrate. Recently, lessons from the studies of neural apoptosis-regulated convertase 1 (NARC-1), a newly discovered PC (10, 11), reinforced the importance of individual PCs in regulating plasma lipid metabolism. However, the substrate of NARC-1 is not yet identified.Endothelial lipase (EL) is a recently described modulator of lipoprotein metabolism that belongs to the triglyceride lipase family, which also includes lipoprotein lipase (LPL) and hepatic lipase (HL) (12). Like LPL and HL, the EL protein is secreted and binds to heparan sulfate proteoglycans on the endothelial cell surface, where it interacts wit...

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