Acyl-coenzyme A:cholesterol acyltransferase promotes oxidized LDL/oxysterol-induced apoptosis in macrophages

Abstract

7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of cPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388D1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.

Fig. 1

An ACAT inhibitor prevents 7-ketocholesterol (7KC)-induced apoptosis in P388D1 cells. P388D1 cells were preincubated for 1 h with an ACAT inhibitor, 58-035 (10 μg/ml), before a 16 h incubation with and without 7KC (10 μg/ml), as indicated. The cells were then assayed for caspase-3 activity as described in Materials and Methods. FLU, fluorescence light units. The data represent means ± SD of triplicate treatments. * P < 0.05 versus 7KC-treated controls.

Fig. 2

An ACAT inhibitor reduces the induction of apoptosis by 7KC and oxidized low density lipoprotein (OxLDL) in isolated peritoneal macrophages. A: The effect of 58-035 on the induction of terminal transferase-mediated dUTP nick end labeling (TUNEL)-positive cells after a 24 h treatment with 7KC treatment. Resident peritoneal macrophages were isolated, plated on glass cover slips, and allowed to attach. The media was changed and supplemented with or without 58-035 (10 μg/ml) for 1 h before supplementation with and without 7KC (10 μg/ml), as indicated. The incubations were continued for 24 h. Apoptosis was then assayed by in situ TUNEL analysis as described in Materials and Methods. B: The effect of 58-035 on the induction of TUNEL-positive cells after 24 and 48 h incubations with OxLDL. Isolated resident peritoneal macrophages were supplemented with and without 58-035 (10 μg/ml) for 1 h before the addition of OxLDL (50 μg/ml). The incubation was continued for 24 and 48 h, as indicated. The induction of apoptosis was determined by in situ TUNEL analysis. The results are presented as mean percentages of TUNEL-positive cells ± SD in 10 random fields for each condition. The results shown are representative of three independent experiments. * P < 0.01 and ** P < 0.001 versus 7KC- or OxLDL-treated controls.

Fig. 3

The induction of apoptosis by OxLDL and 7KC is suppressed in ACAT-1-deficient (ACAT-1^-/-) peritoneal macrophages. Resident peritoneal macrophages from wild-type and ACAT-1^-/- mice were isolated and treated for 24 h with OxLDL (50 μg/ml), 7KC (10 μg/ml), or vehicle, as indicated. MPMs were then subjected to in situ TUNEL analysis as described for Fig. 2. The results shown are representative of three independent experiments. * P < 0.001 and ** P < 0.0005 versus OxLDL- and 7KC-treated controls, respectively. Error bars represent standard deviation.

Fig. 4

Treatment of P388D1 cells with an inhibitor of cholesterol trafficking, U18666A, preferentially inhibits cholesteryl ester accumulation, but does not inhibit 7KC-induced apoptosis. A, B: P388D1 cells were supplemented with or without U18666A (5 μM) for 1 h before supplementation with and without 7KC (10 μg/ml), as indicated. At this time, either [¹⁴C]cholesterol (0.5 μCi/ml) or [³H]7KC (1.0 °Ci/ml) was added to each well. After a 16 h incubation, total lipids were extracted, and the radioactivity incorporated into cholesteryl esters (A) and 7-ketocholesteryl esters (7KC esters; B) was determined. C: The effect of U18666A on the induction of caspase-3 activity in P388D1 cells. P388D1 cells were preincubated with U18666A (5 μM) for 1 h before a 16 h treatment with and without 7KC (10 μg/ml), as indicated. The cells were then harvested and caspase-3 activity was determined as described above. FLU, fluorescence light units. * P < 0.05 and ** P = 0.048 versus oxysterol-treated controls. Error bars represent standard deviation.

Fig. 5

58-035, 5,8,11,14-eicosatetraynoic acid (ETYA), and arachidonyl trifluoromethyl ketone (AACOCF₃) inhibit the formation of 7KC esters in P388D1 cells. A: The effects of 58-035, ETYA, and AACOCF₃ on the accumulation of [³H]7KC esters in P388D1 cells. P338D1 cells were cultured in the presence of [³H]7KC (1.0 μCi/ml) in media supplemented with 58-035 (10 μg/ml), ETYA (20 μM), or AACOCF₃ (20 μM), as indicated. Unlabeled 7KC (10 μg/ml) was added and the cells were incubated for 16 h. Total lipids were extracted, and the radioactivity incorporated into 7KC esters was determined. B: The effects of a cyclooxygenase-1 inhibitor, FR-122047 (10 μM), and a cyclooxygenase-2 inhibitor, NS-398 (5 μM), on the accumulation of [³H]7KC esters in P388D1 cells were compared with treatment with 58-035 and ETYA. P388D1 cells were cultured in the presence or absence of the indicated inhibitors for 1 h before supplementation with [³H]7KC or unlabeled 7KC for 16 h, as described for A. * P < 0.05, ** P = 0.12, and *** P = 0.33 versus oxysterol-treated controls. Error bars represent standard deviation.

Fig. 6

Selected reaction monitoring chromatographic traces of a standard mixture (A) containing 16:0, 18:0, 18:1, 18:2, and 20:4 7KC esters, as described in Materials and Methods. Selected reaction monitoring traces of a macrophage total lipid extract (B) represent analysis of ∼1.4% of the total extract. The top panel in each set contains overlaid selected reaction monitoring traces for 16:0 and 16:1; the middle panel contains traces for 18:0, 18:1, 18:2, and 18:3; and the bottom panel contains traces for 20:0, 20:1, and 20:4. The amplification factors for each trace relative to 7KC-18:0 are shown in parentheses; the same factor was applied to each selected reaction monitoring trace in each panel.

Fig. 7

The effect of supplementing arachidonic acid (AA) and oleic acid in the presence of AACOCF₃ on the formation of [³H]7KC esters and the induction of caspase-3 activity by 7KC. A: The effect of AA or oleic acid supplementation on AACOCF₃ inhibition of [³H]7KC ester formation. P388D1 cells were cultured in the presence or absence of AACOCF₃ (20 μM) or 58-035 (10 μg/ml) for 1 h and then supplemented with or without 7KC (10 μg/ml) and either oleic acid or AA (50 μM) complexed to fatty acid-free BSA, as indicated. [³H]7KC (1.0 μCi/ml) was added to the cells, and after a 6 h incubation, the lipids were extracted and the radioactivity incorporated into [³H]7KC esters was determined as described for Fig. 5. B: The effect of AA or oleic acid supplementation on AACOCF₃ inhibition of 7KC-induced apoptosis. P388D1 cells were supplemented with or without oleic acid or AA (50 μM) in the presence of either AACOCF₃ (20 μM) or 58-035 (10 μg/ml) for 1 h before the addition of unlabeled 7KC (10 μg/ml), as indicated. After a 16 h incubation, the cells were harvested, and caspase-3 activity was determined as described above. FLU, fluorescence light units. Error bars represent standard deviation.