Oxidized Low Density Lipoprotein Inhibits Macrophage Apoptosis by Blocking Ceramide Generation, Thereby Maintaining Protein Kinase B Activation and Bcl-XL Levels

Abstract

Macrophages play a central role in the development and progression of atherosclerotic lesions. It is well known that oxidized low density lipoprotein (ox-LDL) promotes the recruitment of monocytes (which differentiate to macrophages) into the intima. We reported recently that ox-LDL blocks apoptosis in bone marrow-derived macrophages deprived of macrophage colonystimulating factor (M-CSF) by a mechanism involving protein kinase B (PKB) (Hundal, R., Salh, B., Schrader, J., Gó mez-Muñ oz, A., Duronio, V., and Steinbrecher, U. (2001) J. Lipid Res. 42, 1483-1491). The aims of the present study were 1) to define the apoptotic pathway involved in the pro-survival effect of ox-LDL; 2) to determine which PKB target mediated this effect; and 3) to identify mechanisms responsible for PKB activation by ox-LDL. Apoptosis following M-CSF withdrawal was accompanied by activation of the caspase 9-caspase 3 cascade and cytochrome c release from mitochondria, but the caspase 8 pathway was unaffected. M-CSF withdrawal resulted in a marked and selective reduction in Bcl-X L protein and mRNA levels, and this decrease was prevented by ox-LDL. The ability of ox-LDL to preserve Bcl-X L levels was blocked by NFB antagonists, thereby implicating IB kinase as a key PKB target. M-CSF deprivation resulted in activation of acid sphingomyelinase and an increase in ceramide levels. Desipramine (a sphingomyelinase inhibitor) prevented the increase in ceramide and inhibited apoptosis after M-CSF deprivation. Ox-LDL completely blocked the increase in acid sphingomyelinase activity as well as the increase in ceramide after M-CSF deprivation. Pretreatment of macrophages with C 2 -ceramide reversed the effect of ox-LDL on PKB and macrophage survival. These results indicate that ox-LDL prevents apoptosis in M-CSFdeprived macrophages at least in part by inhibiting acid sphingomyelinase. This in turn prevents ceramide-induced down-regulation of PKB, the activity of which is required to maintain production of Bcl-X L .

Differential targeting of protein kinase B in cell death induced by sulindac and its metabolite sulindac sulfide

“…Equally, the observation that PKB can prevent sulindac-induced cell death implies that PKB must influence mitochondrial integrity. This is in agreement with evidence indicating PKB can prevent cytochrome c release (38,39).…”

Ceramide‐1‐phosphate promotes cell survival through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway

“…BMDMs were isolated from femurs of 6-to 8-week-old female CD-1 mice as described [19]. Cells were plated in RPMI 1640 medium containing 10% FBS and 10% L-cell conditioned medium as the source of macrophage colony stimulating factor (M-CSF) [19].…”

“…Cells were plated in RPMI 1640 medium containing 10% FBS and 10% L-cell conditioned medium as the source of macrophage colony stimulating factor (M-CSF) [19]. After 24 h, non-adherent cells were harvested and cultured in the above medium until about 80% confluence was reached (4-6 days) prior to use in the experiments.…”

“…The medium was then replaced by fresh RPMI 1640 medium in the presence or absence of agonists and/or inhibitors as appropriate. Cell viability was estimated by measuring the rate of reduction of the tetrazolium dye MTS as described [19].…”

“…Radioactivity in PI (3,4,5) P3 was determined essentially as described [20] after labeling BMDM with 200 lCi/ml of [ 32 P]orthophosphate overnight in phosphate-free RPMI 1640 with 10% FBS and 10% L-cell conditioned medium as the source for M-CSF [19]. Cells were then washed twice with RPMI 1640 without label or M-CSF.…”