The Signaling Pathway of Oxysterol-Induced Apoptosis in Macrophages.

<p>Oxidized low-density lipoproteins (OxLDL) mediate many of the pathological events associated with atherosclerosis. Oxysterols, the major cytotoxic component of OxLDL, induce apoptosis in macrophages by a calcium flux mediated activation of cytosolic phospholipase A₂ resulting in the release of arachidonic acid (AA). Inhibition of AA metabolism has been shown to protect macrophages from oxysterol-induced apoptosis. The current study explores the steps in the oxysterol-induced apoptosis signaling pathway in murine macrophages subsequent to the liberation of AA. To elucidate this mechanism, two oxysterols, 7-ketocholesterol and 25-hydroxycholesterol (25-OHC), were used to induce apoptosis in murine macrophage cell lines (P388D1, and Raw 264.7) and mouse peritoneal macrophages (MPMs). Pharmacological inhibition of eicosanoid synthesis or genetic knockout of important eicosanoid biosynthetic genes had no significant effect on the induction of apoptosis by oxysterols in macrophages. The induction of apoptosis in macrophage cell lines and MPMs by oxysterols and OxLDL was suppressed by Sandoz 58-035, an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT). Furthermore, in comparison to wild-type MPMs, ACAT-1 deficient MPMs were found to be resistant to apoptosis induced by oxysterols or OxLDL. Macrophages treated with 7KC accumulated ACAT-derived cholesteryl and 7-ketocholesteryl esters. An inhibitor of cholesterol trafficking, U18666A, specifically prevented the accumulation of cholesteryl esters, but not 7-ketocholesteryl esters nor the induction of apoptosis. An inhibitor of cPLA₂ prevented the accumulation of 7-ketocholesteryl esters. This inhibition was reversed by supplementing oleic acid or AA; however, only AA supplementation restored the induction of apoptosis. These results suggest that oxysterols not only initiate the apoptosis pathway by activating cPLA₂, but also participate in the downstream signaling pathway when esterified by ACAT to form arachidonyl oxysterols. We also demonstrate that macrophages lacking the cannabinoid type-2 (CB₂) receptor have reduced levels of apoptosis when treated with oxysterols or OxLDL in comparison to wild-type macrophages and that a CB₂ specific antagonist blocks oxysterol-induced apoptosis in macrophages suggesting that the CB₂ receptor is involved in this pathway, perhaps by interacting with the arachidonyl oxysterols.</p>

Name thesis:degree_name

PhD (Doctor of Philosophy)

Level thesis:degree_level

Dissertation - restricted

Discipline thesis:degree_discipline

Biomedical Sciences

Year dc:date.issued

2005

Author dc:creator

• Freeman, Natalie Elaine

• Cannabinoid Receptors
• ACAT
• Eicosanoids
• Oxysterols
• Oxidized low density lipoproteins
• Macrophages
• Apoptosis
• Oxysteryl Ester
• Medical Sciences
• Medicine and Health Sciences

Statement dc:rights

• Copyright by the authors.

Repository record dc:identifier

https://dc.etsu.edu/etd/1063

OAI identifier oai:identifier

oai:dc.etsu.edu:etd-2220