The Correlation of ATP-binding Cassette 1 mRNA Levels with Cholesterol Efflux from Various Cell Lines

Abstract

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages ؎ exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose-and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 ؊/؊ mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.

Effects of Apolipoprotein A-I on ATP-binding Cassette Transporter A1-mediated Efflux of Macrophage Phospholipid and Cholesterol

“…Removed from cAMP-stimulated J774 Cells-In agreement with previous studies (41,42), pretreatment of J774 cells with cAMP as described under "Experimental Procedures" increased the level of ABCA1 mRNA by ϳ6-fold; there was no change in the expression of ABCG1 (compare with Ref. 48) (data not shown).…”

“…Double labeling was achieved by incubation with 20 Ci/ml [ 3 H]choline chloride and 3 Ci/ml [ 14 C]cholesterol for 48 h. For preparation of cholesterol-enriched monolayers, the cells were labeled with the same medium that also contained 25 g/ml acetyl low density lipoprotein (LDL) and incubated for 48 h. All media were supplemented with 50 g/ml gentamycin. Following the 24 -48-h labeling period, the cells were washed and incubated with 0.2% (w/v) BSA and 2 g/ml CP-113,818 in RPMI 1640 medium with 0.3 mM CPT-cAMP for 12 h (41,42). Some wells were then washed with phosphate-buffered saline, dried, and extracted with isopropyl alcohol (43).…”

Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels

“…Functional activity of the ABCA1 transporter in the repopulated macrophages was assessed by the ability of pooled elicited peritoneal macrophages to mediate cholesterol efflux to human apoAI in response to cAMP analogues (14). As shown in Figure 2, a and c, macrophages isolated from mice transplanted with WT bone marrow exhibited WT response to a stable form of cAMP (cpt-cAMP).…”

The Recycling of Apolipoprotein E in Primary Cultures of Mouse Hepatocytes

“…It is interesting to speculate that it may be related to cholesterol efflux from the cell as both apoE and apoA-I have been shown to play crucial roles in this process. ApoE has been shown to promote cholesterol efflux from the cell (8), whereas apoA-I serves as an acceptor for cholesterol efflux (29,30) in a process probably mediated through the ATP-binding cassette A1 (ABCA1) pathway (31)(32)(33). In addition, there is evidence that binding of HDL/apoA-I to the cell surface mobilizes cholesterol from intracellular pools to the cell surface (34,35) where the lipid could be transported out of the cell via ABCA1.…”