Macrophage-specific Abca1 knock-out (Abca1؊M/؊M ) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1 ؊M/؊M and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1 ؊M/؊M macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1 ABCA1 (ATP-binding cassette transporter A1) is a plasma membrane protein that is widely expressed throughout the body (1, 2) and functions as a primary gatekeeper for eliminating excess free cholesterol (FC) 2 from tissues by effluxing cellular FC and phospholipid (PL) to lipid-free apoA-I, resulting in the formation of nascent high density lipoprotein (HDL) particles (3, 4). The nascent discoid-shaped HDL then undergoes a maturation process that involves additional lipid acquisition and conversion of FC to cholesteryl ester (CE) by lecithin:cholesterol acyltransferase to become mature spherical plasma HDL. Mutations that inactivate the human ABCA1 gene result in Tangier disease, which is characterized by extremely low HDL cholesterol concentrations, mildly elevated plasma trigelyceride levels, and accumulation of cholesterol in macrophages (5-10). Targeted deletion of Abca1 in mice and a natural mutation of Abca1 in the Wisconsin hypoalpha mutant chicken recapitulate the Tangier plasma lipid phenotype, supporting the essential role of ABCA1 in HDL formation (11-15). Although ABCA1 is expressed in many cells in the body, recent studies in hepatocyte-and intestinal epithelium-specific Abca1 knock-out mice suggest that the liver contributes 70 -80% of the plasma HDL pool, whereas the intestine contributes 20 -30% (16, 17). Although mobilization of excess FC from macrophages is dependent on ABCA1 and results in the formation of nascent HDL particles, transplantation of bone marrow from Abca1 knock-out (KO) mice into wild-type (WT) mice or transplantation of WT marrow into Abca1 KO recipients has little effect on plasma HDL concentrations, suggesting that macrophage ABCA1 expression has minimal impact on plasma HDL concentrations (18,19).Macrophages are a primary cell type involved in innate immunity. Although macrophage ABCA1 has a minimal impact on plasma lipid levels, there is evidence that its activity modulates the inflammatory response of macrophages to pathogen-associated molecules such as lipopolysaccharide

Patients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1 -L/-L ). Abca1 -L/-L mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1 -L/-L mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time.

Patients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1 -L/-L ). Abca1 -L/-L mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1 -L/-L mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time.

Cholesterol is a key component of cell membranes and is essential for cell growth and proliferation. How the accumulation of cellular cholesterol affects lymphocyte development and function is not well understood. We demonstrate that ATP-binding cassette transporter G1 (ABCG1) regulates cholesterol homeostasis in thymocytes and peripheral CD4 T cells. Our work is the first to describe a cell type in Abcg1-deficient mice with such a robust change in cholesterol content and the expression of cholesterol metabolism genes. Abcg1-deficient mice display increased thymocyte cellularity and enhanced proliferation of thymocytes and peripheral T lymphocytes in vivo. The absence of ABCG1 in CD4 T cells results in hyperproliferation in vitro, but only when cells are stimulated through the TCR. We hypothesize that cholesterol accumulation in Abcg1−/− T cells alters the plasma membrane structure, resulting in enhanced TCR signaling for proliferation. Supporting this idea, we demonstrate that B6 T cells pretreated with soluble cholesterol have a significant increase in proliferation. Cholesterol accumulation in Abcg1−/− CD4 T cells results in enhanced basal phosphorylation levels of ZAP70 and ERK1/2. Furthermore, inhibition of ERK phosphorylation in TCR-stimulated Abcg1−/− T cells rescues the hyperproliferative phenotype. We describe a novel mechanism by which cholesterol can alter signaling from the plasma membrane to affect downstream signaling pathways and proliferation. These results implicate ABCG1 as an important negative regulator of lymphocyte proliferation through the maintenance of cellular cholesterol homeostasis.

Objectives-The aim of this study was to determine the role of ATP binding cassette transporter A1 (ABCA1) on generation of different-sized nascent HDLs. Methods and Results-HEK293 cells stably-transfected with ABCA1 (HEK293-ABCA1) or non-transfected (control) cells were incubated with lipid free 125 I-apoA-I for 24 hours. Incubation of apoA-I with HEK293-ABCA1 cells, but not control cells, led to the formation of heterogeneous-sized, pre-␤ migrating nascent HDL subpopulations (pre-␤1 to -4) that varied in size (7.1 to 15.7 nm), lipid, and apoA-I content. Kinetic studies suggested that all subpopulations were formed simultaneously, with no evidence for a precursor-product relationship between smaller and larger-sized particles. When isolated nascent pre-␤ HDLs (pre-␤1 to -4) were added back to HEK293-ABCA1 cells, their ability to bind to ABCA1 and efflux lipid was severely compromised. Heat-denaturation of pre-␤1 HDL resulted in partial recovery of ABCA1 binding, suggesting that initial interaction of apoA-I with ABCA1 results in a constrained conformation of apoA-I that decreases subsequent binding. Key Words: ATP binding cassette transporter A1 Ⅲ apolipoprotein AI Ⅲ high density lipoprotein T he inverse relationship between plasma high-density lipoprotein (HDL) cholesterol concentration and the risk of premature atherosclerotic vascular diseases has generated interest in understanding the steps involved in HDL assembly and catabolism. HDLs are proposed to be antiatherogenic because of their ability to accept excess cellular cholesterol in peripheral tissues and transport it to the liver in a process denoted as reverse cholesterol transport (RCT). 1 HDLs are classified into two subpopulations based on electrophoretic mobility on agarose gels: ␣-HDL (90% to 95% total HDL in plasma) and pre-␤ HDL (5% to 10%). 2 Heterogeneity is found among pre-␤ and ␣-HDLs. Using 2-dimensional gel electrophoresis (2D-gel), several pre-␤ and ␣-HDL subpopulations have been identified as a function of increasing size. 3 Although heterogeneity among pre-␤ and ␣-HDLs is well-documented, little is known about the mechanism of formation and metabolism of these pre-␤ and ␣-HDL subpopulations. Conclusions-InteractionABCA1 is critical for maintaining normal plasma HDL levels and nascent HDL biogenesis. A critical role for ABCA1 in HDL metabolism was demonstrated in patients with Tangier disease, a genetic disorder in which the ABCA1 gene is mutated. 4 -6 These patients have plasma HDL-cholesterol and apoA-I concentrations Ͻ5% of normal, accumulation of CE in macrophages, and increased risk of atherosclerosis. 7,8 Fibroblasts from these patients also have a significant reduction in lipid efflux to apoA-I compared with controls, suggesting ABCA1 was essential for mediating transport of intracellular lipid to apoA-I. 9 Additionally, ABCA1 knockout mice recapitulate the Tangier disease phenotype, verifying the role of ABCA1 in cellular lipid homeostasis and plasma HDL maintenance. 10 Plasma pre-␤ HDLs exist as several subpopulations...