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

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. 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. Many of the pathological events associated with the development of atherosclerosis are believed (1.Witztum J.L. Steinberg D. Trends Cardiovasc. Med. 2001; 11: 93-102Crossref PubMed Scopus (387) Google Scholar) to be mediated by oxidized low density lipoprotein (ox-LDL). 1The abbreviations used are: ox-LDL, oxidized low density lipoprotein; 25-OHC, 25-hydroxycholesterol; ETYA, 5,8,11,14-eicosatetraynoic acid; AACOCF3, arachidonyl trifluoromethyl ketone; GFP, green fluorescent protein; myr, myristoylated; BH3, BCL-2 homology-3; STAT, signal transducer and activator of transcription. 1The abbreviations used are: ox-LDL, oxidized low density lipoprotein; 25-OHC, 25-hydroxycholesterol; ETYA, 5,8,11,14-eicosatetraynoic acid; AACOCF3, arachidonyl trifluoromethyl ketone; GFP, green fluorescent protein; myr, myristoylated; BH3, BCL-2 homology-3; STAT, signal transducer and activator of transcription. The constitutive uptake by macrophages of ox-LDL is through specialized scavenger receptors, resulting in these cells becoming lipid-laden foam cells (2.Steinbrecher U.P. Biochim. Biophys. Acta. 1999; 1436: 279-298Crossref PubMed Scopus (188) Google Scholar). The formation of such cells is the hallmark of atherosclerosis. Furthermore, ox-LDL has been shown to be cytotoxic to macrophages (3.Hardwick S.J. Hegyi L. Clare K. Law N.S. Carpenter K.L. Mitchinson M.J. Skepper J.N. J. Pathol. 1996; 179: 294-302Crossref PubMed Scopus (123) Google Scholar) through a process requiring such receptors (4.Wintergerst E.S. Jelk J. Rahner C. Asmis R. Eur. J. Biochem. 2000; 267: 6050-6059Crossref PubMed Scopus (89) Google Scholar, 5.Rusiñol A.E. Yang L. Thewke D. Panini S.R. Kramer M.F. Sinensky M.S. J. Biol. Chem. 2000; 275: 7296-7303Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This cytotoxicity could be very important in the atherogenicity of ox-LDL through the lysis of foam cells and the concomitant deposition of lipids in the coronary vasculature. The cytotoxic effects of ox-LDL have been shown to proceed, at least in part, through apoptotic pathways, in general (reviewed in Refs. 6.Martinet W. Kockx M.M. Curr. Opin. Lipidol. 2001; 12: 535-541Crossref PubMed Scopus (112) Google Scholar and 7.Colles S.M. Maxson J.M. Carlson S.G. Chisholm G.M. Trends Cardiovasc. Med. 2001; 11: 131-138Crossref PubMed Scopus (167) Google Scholar), as well as in macrophages, in particular (3.Hardwick S.J. Hegyi L. Clare K. Law N.S. Carpenter K.L. Mitchinson M.J. Skepper J.N. J. Pathol. 1996; 179: 294-302Crossref PubMed Scopus (123) Google Scholar, 8.Muller K. Dulku S. Hardwick S.J. Skepper J.N. Mitchinson M.J. Atherosclerosis. 2001; 156: 133-144Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 9.Panini S.R. Yang L. Rusiñol A.E. Sinensky M.S. Bonventre J.V. Leslie C.C. J. Lipid Res. 2001; 42: 1678-1686Abstract Full Text Full Text PDF PubMed Google Scholar). The cholesterol oxidation products (oxysterols) found in ox-LDL (10.Schroepfer G.J. Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (823) Google Scholar) have been recognized as a probable basis for its cytotoxicity (11.Chisolm G.M. Ma G. Irwin K.C. Martin L.L. Gunderson K.G. Linberg L.F. Morel D.W. DiCorleto P.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11452-11456Crossref PubMed Scopus (191) Google Scholar, 12.Sevanian A. Hodis H.N. Hwang J. McLeod L.L. Peterson H. J. Lipid Res. 1995; 36: 1971-1986Abstract Full Text PDF PubMed Google Scholar), at least in part, via apoptotic mechanisms (7.Colles S.M. Maxson J.M. Carlson S.G. Chisholm G.M. Trends Cardiovasc. Med. 2001; 11: 131-138Crossref PubMed Scopus (167) Google Scholar, 13.Panini S.R. Sinensky M.S. Curr. Opin. Lipidol. 2001; 12: 529-533Crossref PubMed Scopus (107) Google Scholar, 14.Harada-Shiba M. Kinoshita M. Kamido H. Shimokado K. J. Biol. Chem. 1998; 273: 9681-9687Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). A model compound for such oxysterols is 25-hydroxycholesterol (25-OHC), which has been shown to induce apoptosis in monocyte-macrophage (9.Panini S.R. Yang L. Rusiñol A.E. Sinensky M.S. Bonventre J.V. Leslie C.C. J. Lipid Res. 2001; 42: 1678-1686Abstract Full Text Full Text PDF PubMed Google Scholar, 15.Aupeix K. Weltin D. Mejia J.E. Christ M. Marchal J. Freyssinet J.-M. Bischoff P. Immunobiology. 1995; 194: 415-428Crossref PubMed Scopus (102) Google Scholar, 16.Harada K. Ishibashi S. Miyashita T. Osuga J.-I. Yagyu H. Ohashi K. Yazaki Y. Yamada N. FEBS Lett. 1997; 411: 63-66Crossref PubMed Scopus (49) Google Scholar) and lymphoid (17.Bansal N. Houle A. Melnykovych G. FASEB J. 1991; 5: 211-216Crossref PubMed Scopus (114) Google Scholar, 18.Christ M. Luu B. Mejia J.E. Moosbrugger I. Bischoff P. Immunology. 1993; 78: 455-460PubMed Google Scholar) cell lines in the range of 1-10 μm. Prior studies have been consistent with the activation of the mitochondrial death pathway by oxysterols with its canonical cytochrome c release (19.Yang L. Sinensky M.S. Biochem. Biophys. Res. Commun. 2000; 278: 557-563Crossref PubMed Scopus (43) Google Scholar, 20.Lizard G. Gueldry S. Sordet O. Monier S. Athias A. Miguet C. Bessede G. Lemaire S. Solary E. Gambert P. FASEB J. 1998; 12: 1651-1663Crossref PubMed Scopus (190) Google Scholar). Cytochrome c release from mitochondria is regulated, in turn, through the activation of pro-apoptotic BCL family members, along with possible inactivation of anti-apoptotic BCL family members (21.Kaufmann S.H. Hengartner M.O. Trends Cell Biol. 2001; 11: 526-534Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). In this study, we describe the role of the AKT-regulated BAX/BAD pathway in oxysterol-induced apoptosis of murine macrophage cell lines. Materials—RAW 264.7 cells were purchased from American Type Culture Collection (Manassas, VA). P388D1 cells (MAB variant) were provided by Dr. Edward Dennis (University of California, San Diego, CA) (22.Shinohara H. Balboa M.A. Johnson C.A Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). RPMI 1640 medium and Dulbecco's modified Eagle's medium were from Invitrogen. NovaCell I fetal bovine serum was from Nova-Tech (Grand Island, NE). All other cell culture reagents were obtained from Invitrogen. 5,8,11,14-Eicosatetraynoic acid (ETYA), Ac-DEVD-aldehyde, Ac-DEVD-7-amino-4-trifluoromethyl coumarin, and arachidonyl trifluoromethyl ketone (AACOCF3) were purchased from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). Oxysterols were purchased from STERALOIDS, Inc. (Wilton, NH). All antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA), with the exception of anti-AKT1/2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Myc antibody (Upstate Biotechnology, Inc., Charlottesville, VA), and anti-HSP70 antibody (Stressgen Biotech Corp., Victoria, British Columbia, Canada). Peroxidase-conjugated secondary antibodies were from Pierce. pEGFP-C3 was from Clontech. Proteasome inhibitor I was from Calbiochem. MitoTracker™ Red was from Molecular Probes, Inc. (Eugene, OR). Cell Culture—All cell lines were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. 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 μg/ml streptomycin, and 50 μm 2-mercaptoethanol. P388D1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin. Caspase-3 Assay—P388D1 cells were seeded at 2 × 106/well in 12-well culture plates, and RAW 264.7 cells at 2.5 × 106/well in 6-well culture plates. The medium was supplemented with in or of or was to the medium 2 to the of the and cells were by and at × for The cells were with in lysis buffer A mm (pH mm 10 mm sodium and 10 mm sodium for 10 and at × for at °C. The or was for protein the and for as of were for 2.5 at 37 in buffer mm Hepes (pH 10% and 2 mm μm in the and of a inhibitor at 100 to the of the was a with a with of and of the was by the fluorescent obtained in the of the inhibitor from the fluorescent obtained in the of the inhibitor and to the protein of the treatment was in and the are as the was from Signaling Technology, with and and pEGFP-C3 the of the The was by and RAW 264.7 cells of in RAW 264.7 RAW 264.7 cells were Cells were and with for with cells were observed a In mitochondria were with MitoTracker™ Red the were obtained by of a with a or and was by cells with a that small interfering that for the were and Inc., and The was by the of and P388D1 cells × were with a the and or to the were by in medium 1 for by in were by for of the The were to in medium 10 μg/ml for The cells were and maintained in medium μg/ml were obtained from The of BAX was by cell from the cells, and of cells were with a the of the (Upstate Biotechnology, to the were by in medium 1 for by 10 in μg/ml The were and for of the protein by with antibody Signaling Technology, and anti-Myc were maintained in medium μg/ml P388D1 cells were seeded at 2 × 106/well in 6-well culture plates. the cells were with and with or with to the of the was to for to the of and of Cell and cells were to a density of 2 × in medium supplemented with or of of cells were and with lysis buffer mm (pH mm 1 mm 1 mm 2.5 mm sodium 1 mm 1 mm and 1 μg/ml and for was from the by for 10 at × and the protein in the was by were by and to The were with protein Probes, to of the and of proteins to the The were antibodies for the protein of and the secondary antibodies The proteins of were by as cells were in medium for 2 and with for Cells were and for in medium 1 mm and or 10 μg/ml Cells were as and to with were to the and the of AKT in the was by and AKT of AKT was a AKT kinase Signaling Technology, the antibody to AKT was used to AKT from cell The was with protein in the of and kinase AKT to of was by antibody and by in P388D1 Cells through a have previously induction of apoptosis in a cell line and a monocyte-macrophage cell line as release and (9.Panini S.R. Yang L. Rusiñol A.E. Sinensky M.S. Bonventre J.V. Leslie C.C. J. Lipid Res. 2001; 42: 1678-1686Abstract Full Text Full Text PDF PubMed Google Scholar). of we in of cells and the well of the murine macrophage P388D1 cells in studies of (22.Shinohara H. Balboa M.A. Johnson C.A Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), we P388D1 cells apoptosis in to oxysterols in a a K. Ishibashi S. Miyashita T. Osuga J.-I. Yagyu H. Ohashi K. Yazaki Y. Yamada N. FEBS Lett. 1997; 411: 63-66Crossref PubMed Scopus (49) Google Scholar) that apoptosis in P388D1 cells and as was observed for the other cell that the induction of apoptosis was by the inhibitor and by ETYA, inhibitor of AKT in P388D1 (protein kinase has been well as anti-apoptotic kinase J. Curr. Opin. Cell Biol. 1998; PubMed Scopus Google Scholar) that in cell G. L. Curr. Opin. 1998; PubMed Scopus Google Scholar). 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