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Journal of Lipid Research, Vol. 46, 1933-1943, September 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

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

Natalie E. Freeman^*, Antonio E. Rusinol^*, MacRae Linton, David L. Hachey, Sergio Fazio, Michael S. Sinensky^* and Douglas Thewke^1,*

^* Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0581
Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300
Mass Spectrometry Research Center, Vanderbilt University Medical Center, Nashville, TN 37232-8575

Published, JLR Papers in Press, July 1, 2005. DOI 10.1194/jlr.M500101-JLR200

¹ To whom correspondence should be addressed. e-mail: thewke{at}etsu.edu

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.

Abbreviations: AA, arachidonic acid; AACOCF₃, arachidonyl trifluoromethyl ketone; ACAT, acyl-coenzyme A:cholesterol acyltransferase; cPLA₂, cytosolic phospholipase A₂; ETYA, 5,8,11,14-eicosatetraynoic acid; MPM, mouse peritoneal macrophage; OxLDL, oxidized low density lipoprotein; TUNEL, terminal transferase-mediated dUTP nick end labeling; 7KC, 7-ketocholesterol; 7KC ester, 7-ketocholesteryl ester; 25-OHC, 25-hydroxycholesterol

Supplementary key words 7-ketocholesterol • oxysterol esterification • low density lipoproteins

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