FREE CHOLESTEROL LOADING INDUCED APOPTOSIS AND PHOSPHOLIPID COMPOSITION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Studies involving pharmacologic or molecular biologic manipulation of Group VIA Phospholipase A2 (iPLA2¿) activity in cultured cells suggest that iPLA2¿ participates in ER-stress induced apoptosis, and mouse peritoneal macrophages undergo apoptosis in response to ER stress when loaded with free cholesterol. We recently generated iPLA2¿-null mice and reported that their macrophages exhibit defective transcriptional responses to viral infection. Here we demonstrate that iPLA2¿-null mouse peritoneal macrophages are less sensitive than wild-type macrophages to apoptosis induced by free cholesterol loading, although wild-type and iPLA2¿-null macrophages exhibit similar increases in expression of the ER stress-inducible transcriptional regulator CHOP. Arachidonic acid is released and lysophosphatidylcholine (LPC) accumulates in wild-type macrophages upon cholesterol loading, consistent with PLA2 activation, and these responses are attenuated in iPLA2¿-null macrophages. Despite suggestions that iPLA2¿ is a housekeeping enzyme that regulates LPC levels and arachidonate incorporation into phosphatidylcholine (PC), we find that iPLA2¿-null macrophages incorporate [3H]arachidonic acid into PC as readily as wild-type macrophages. Electrospray ionization mass spectrometric analyses indicate that arachidonate-containing PC species are equally abundant in iPLA2¿-null and wild-type macrophages, and no differences between the two genotypes were observed in the composition of sphingomyelin, ceramide, or glycerophospho-ethanolamine, -glycerol, -serine, or -inositol lipids. While we find no evidence for a housekeeping role, these and previous findings indicate that iPLA2¿-null mouse macrophages exhibit phenotypic abnormalities in signaling processes and that iPLA2¿ participates in ER stress-induced apoptosis.