AKT/Protein Kinase B Regulation of BCL Family Members during Oxysterol-induced Apoptosis

Antonio E Rusiñol,Douglas Thewke,June Liu,Natalie Freeman,Sankhavaram R Panini,Michael S Sinensky

doi:10.1074/jbc.m308619200

Antonio E Rusiñol, Douglas Thewke + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m308619200

Copy DOI

Abstract

Cells of the vasculature, including macrophages, smooth muscle cells, and endothelial cells, exhibit apoptosis in culture upon treatment with oxidized low density lipoprotein, as do vascular cells of atherosclerotic plaque. Several lines of evidence support the hypothesis that the apoptotic component of oxidized low density lipoprotein is one or more oxysterols, which have been shown to induce apoptosis through the mitochondrial pathway. Activation of the mitochondrial pathway of apoptosis is regulated by members of the BCL family of proteins. In this study, we demonstrate that, in the murine macrophage-like cell line P388D1, oxysterols (25-hydroxycholesterol and 7-ketocholesterol) induced the degradation of the prosurvival protein kinase AKT (protein kinase B). This led, in turn, to the activation of the BCL-2 homology-3 domain-only proteins BIM and BAD and down-regulation of the anti-apoptotic multi-BCL homology domain protein BCL-xL. These responses would be expected to activate the pro-apoptotic multi-BCL homology domain proteins BAX and BAK, leading to the previously reported release of cytochrome c observed during oxysterol-induced apoptosis. Somewhat surprisingly, small interfering RNA knockdown of BAX resulted in a complete block of the induction of apoptosis by 25-hydroxycholesterol.

Highlights

Many of the pathological events associated with the development of atherosclerosis are believed [1] to be mediated by oxidized low density lipoprotein.1 The constitutive uptake by macrophages of ox-LDL is through specialized scavenger receptors, resulting in these cells becoming lipid-laden foam cells [2]
Several lines of evidence support the hypothesis that the apoptotic component of oxidized low density lipoprotein is one or more oxysterols, which have been shown to induce apoptosis through the mitochondrial pathway
We demonstrate that, in the murine macrophage-like cell line P388D1, oxysterols (25hydroxycholesterol and 7-ketocholesterol) induced the degradation of the prosurvival protein kinase AKT

Summary

EXPERIMENTAL PROCEDURES

Materials—RAW 264.7 cells were purchased from American Type Culture Collection (Manassas, VA). RAW 264.7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM Hepes buffer (pH 7.4), 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 ␮g/ml streptomycin, and 50 ␮M 2-mercaptoethanol. Following an 18-h incubation, the cells were harvested, and caspase-3 and “caspase-3-like” activities were measured as described under “Experimental Procedures.”. D, total AKT levels were determined by immunoblotting after treatment with 10 ␮g/ml 25-OHC for 12 h in the presence or absence of 15 ␮M proteasome inhibitor I (PI-I)

Oxysterols Increases AKT Degradation

RESULTS

DISCUSSION