The concept that selective transfer of high density lipoprotein (HDL)-derived cholesteryl esters (CE) does not require lipoprotein internalization has been challenged recently by evidence that implicates HDL recycling during the selective uptake process. This has prompted us to examine the role of the low density lipoprotein receptor-related protein (LRP) in selective uptake. LRP is an endocytic receptor for lipoprotein lipase (LpL) and apolipoprotein E (apoE) ligands that are able to mediate selective uptake. We report that molecules that interfere with ligand binding to LRP, such as the receptor-associated protein (RAP), suramin, ␣ 2 -macroglobulin, or lactoferrin, inhibit HDL-CE selective uptake by human primary adipocytes and SW872 liposarcoma cells by 35-50%. This partial inhibition of selective uptake from total HDL was not due to preferential inhibition of the HDL 2 or HDL 3 subfractions. Selective uptake by the scavenger receptor BI was not inhibited by RAP, excluding its involvement. Furthermore, in SW872 cells in which LRP was reduced to 14% of control levels by stable antisense expression, selective uptake was attenuated by at least 33%, confirming a role for LRP in this process. RAP, ␣ 2 -macroglobulin, lactoferrin, and suramin (individually or in paired combinations) also attenuated selective uptake of HDL-CE by primary human adipocytes by about 40%. On the other hand, human skin fibroblasts express LRP abundantly but lack the capacity for selective uptake, demonstrating that other molecules are required. In SW872 cells, exogenous apoE or LpL can facilitate selective uptake but only the apoE-enhanced uptake can be inhibited by RAP, implicating apoE as a likely co-mediator. We discuss the possible mechanisms by which the endocytic receptor, LRP, can mediate selective uptake.

The ␣ 2 -macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that interacts with a variety of molecules. It is implicated in biologically important processes such as lipoprotein metabolism, neurological function, tissue remodeling, protease complex clearance, and cell signal transduction. However, the regulation of LRP gene expression remains largely unknown. In this study, we have analyzed 2 kb of the 5-flanking region of the LRP gene and identified a predicted peroxisome proliferator response element (PPRE) from ؊1185 to ؊1173. Peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands such as fatty acids and rosiglitazone increased functional cell surface LRP by 1.5-2.0-fold in primary human adipocytes and in the SW872 human liposarcoma cell line as assessed by activated ␣ 2 -macroglobulin binding and degradation. These agents were found to increase LRP transcription. Gel shift analysis of the putative PPRE demonstrated direct binding of PPAR␥/retinoid X receptor ␣ heterodimers to the PPRE in the LRP gene. Furthermore, these heterodimers could no longer interact with a mutated PPRE probe. The isolated promoter was functional in SW872 cells, and its activity was increased by 1.5-fold with the addition of rosiglitazone. Furthermore, the isolated response element was similarly responsive to rosiglitazone when placed upstream of an ideal promoter. Mutagenesis of the predicted PPRE abolished the ability of this construct to respond to rosiglitazone. These data demonstrate that fatty acids and rosiglitazone directly stimulate transcription of the LRP gene through activation of PPAR␥ and increase functional LRP expression.The ␣ 2 -macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) 1 is a 600-kDa multifunctional endocytic receptor that belongs to the low density lipoprotein receptor gene family (1). LRP binds and internalizes a broad range of biologically diverse ligands. These include proteases of the fibrinolytic pathway (2) and serpin-enzyme complexes (3) as well as proteins important in lipoprotein metabolism such as lipoprotein lipase, hepatic lipase, lipoprotein(a), and apoE-rich lipoproteins (4 -9). Targeted deletion of LRP in the mouse results in early embryonic death, demonstrating a critical function for LRP in prenatal development (10). LRP has also been shown to have a dual role in ␤-amyloid metabolism by enhancing ␤-amyloid precursor protein conversion to ␤-amyloid (11) and mediating the clearance of ␤-amyloid (12, 13). These data support a potentially complex role for LRP in the pathogenesis of Alzheimer's disease (14). In addition, LRP mediates signal transduction by interacting with cytosolic adaptor and scaffold proteins including DAB-1, JIP-2, and PSD-95 (15). A 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulumresident protein that functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity (16 -18). RAP is required for the proper folding and export...

One of the conserved functional pathways linked to engulfment of apoptotic corpses involves two membrane proteins low density lipoprotein receptor-related protein-1 (LRP) and ABCA1 and the LRP adapter protein GULP. Because LRP and ABCA1 play roles in cellular lipid trafficking and efflux, here we addressed whether the third member, the LRP adapter protein GULP, also affects cellular lipid transport. Several lines of evidence show that overexpression of GULP causes glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment that is accompanied by down-regulation of ABCA1 and decreased efflux. Conversely, knockdown of endogenous GULP expression promoted cholesterol flux through the late endosomes and up-regulation of ABCA1, even in the context of a disease state such as Niemann-Pick Type C disease. Mechanistically, we were able to show that trafficking of the LRP ligands ␣ 2 -macroglobulin and prosaposin, a protein cofactor necessary for glycosphingolipid degradation, are impaired in cells expressing full-length GULP protein, resulting in glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment. On the other hand, knockdown of endogenous GULP results in enhanced targeting of prosaposin and enhanced clearance of glycosphingolipids and cholesterol from the late endosomes. Taken together, these data reveal that GULP/LRP/ ABCA1 represents a triad of molecules involved in engulfment and cellular lipid homeostasis.Engulfment of apoptotic cells is an important process that plays a critical role in development, organogenesis, cellular homeostasis, wound healing, and autoimmunity. Recently, studies from worms to mammals have identified the existence of a dedicated machinery to clear apoptotic cells and have identified several players involved in this process (reviewed in Refs. 1 and 2). Among the eight genes identified to date in Caenorhabditis elegans, the transmembrane proteins CED-1 and CED-7 together with the cytoplasmic adapter protein CED-6 comprise a signaling pathway leading to recognition and engulfment of apoptotic corpses. The mammalian functional orthologue of CED-1 has been identified as LRP1 (the low density lipoprotein-receptor related protein, also known as CD91) (3). LRP1 is a multiligand receptor and has been shown to play a key role in signaling, lipoprotein uptake, and atherosclerosis (4). The CED-7 homologue ABCA1 has been demonstrated to be involved in phospholipid translocation to the outer leaflet of the cell membrane (5) and in engulfment (6). Hamon et al. (5) have shown that overexpression of ABCA1 in cell lines can promote engulfment and that loss of ABCA1 in mice can cause a partial defect in engulfment, supporting it as a member of the triad. Genetic mutations in ABCA1 have been shown to cause Tangier disease (7-10), which manifests in very low concentrations of plasma high density lipoprotein and accumulation of cholesteryl ester in macrophagic cells in various tissues (reviewed in Ref. 11). The mammalian functional homol...

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energydependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.-Harder, C. J., G. HDL has a functional role in the protection against atherosclerosis, and its plasma concentration is inversely correlated with the risk of cardiovascular disease. One of the protective actions of HDL involves cholesterol removal from peripheral cells for transport to the liver for biliary secretion (reviewed in 1). This process, termed ''reverse cholesterol transport,'' is mediated, in part, by the well-established HDL receptor, scavenger receptor class B type I (SR-BI) (2). In contrast to the holoparticle uptake of the LDL pathway (3), SR-BI mediates cholesterol, cholesteryl ester (CE), and phospholipid uptake via a pathway that does not involve significant degradation of the HDL particle, a process known as ''selective uptake.'' This process was originally described to consist of two phases. First, the lipoprotein binds to SR-BI on the cell surface, and second, the lipids in the lipoprotein are transferred to a membrane (reviewed in 1).Despite intense interest in SR-BI, the cellular mechanism by which SR-BI contributes to the selective uptake and intracellular trafficking of HDL-derived CE is still not fully understood. Early kinetic studies demonstrated that the rate of CE transfer is proportional to the amount of CE in HDL, suggesting that SR-BI forms a hydrophobic channel that facilitates the movement of sterol down a concentration gradient into the plasma membrane (4). Experiments involving reconstitution of SR-BI into liposomes showed that SR-BI mediates CE selective uptake from HDL into multilamellar vesicles independent of...

The genetic modification of cells to develop cell-based vaccines and to modulate immune responses in vivo can be risky and inconvenient to perform in clinical situations. A novel chelator lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA) that, via the NTA group has high affinity for 6His peptide, was used to directly anchor recombinant forms of T cell costimulatory molecules containing a C-terminal 6-His sequence onto tumor cell surfaces. Initial experiments using murine P815 tumor cells established the optimum conditions for incorporating NTA-DTDA onto the membranes of cells. P815 cells with incorporated NTA-DTDAbound hexahistidine-(6His)-tagged forms of the extracellular domains of murine B7.1 and CD40 (B7.1-6H and CD40-6H) at very high levels (fluorescence 200–300-fold above background), and both proteins could be anchored onto the cells simultaneously. Significant loss of the anchored or “engrafted” protein occurred through membrane internalization following culture of the cells under physiological conditions, but P815 cells with engrafted B7.1-6H and/or CD40-6H stimulated the proliferation of allogenic and syngeneic splenic T cells in vitro, and generated cytotoxic T cells when used as vaccines in syngeneic animals. Furthermore, the immunization of syngeneic mice with P815 cells engrafted with B7.1-6H or with B7.1-6H and CD40-6H induced protection against challenge with the native P815 tumor. The results indicate that the use of chelator lipids like NTD-DTDA to engraft costimulatory and/or other molecules onto cell membranes could provide a convenient alternative to transfection in the development of cell-based vaccines and for modulation of immune function.