FREE CHOLESTEROL LOADING INDUCED APOPTOSIS AND PHOSPHOLIPID COMPOSITION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 涉及培养细胞中VIA族磷脂酶A2（iPLA 2）活性的药理学或分子生物学操作的研究表明，iPLA 2参与ER应激诱导的细胞凋亡，并且小鼠腹腔巨噬细胞在负载游离胆固醇时响应于ER应激而发生细胞凋亡。我们最近产生了iPLA 2-null小鼠，并报告说它们的巨噬细胞对病毒感染表现出有缺陷的转录反应。在这里，我们证明了iPLA 2-null小鼠腹腔巨噬细胞比野生型巨噬细胞对游离胆固醇负荷诱导的细胞凋亡不敏感，尽管野生型和iPLA 2-null巨噬细胞在ER应激诱导的转录调节因子CHOP的表达方面表现出相似的增加。在胆固醇负荷后，花生四烯酸被释放，溶血磷脂酰胆碱（LPC）在野生型巨噬细胞中积累，与PLA 2活化一致，这些反应在iPLA 2缺失的巨噬细胞中减弱。尽管有人认为iPLA 2 <$是一种管家酶，可以调节LPC水平和花生四烯酸掺入磷脂酰胆碱（PC），但我们发现iPLA 2 <$-null巨噬细胞与野生型巨噬细胞一样容易将[3 H]花生四烯酸掺入PC。电喷雾电离质谱分析表明，含花生四烯酸盐的PC物种同样丰富的iPLA 2-null和野生型巨噬细胞，两种基因型之间没有差异，鞘磷脂，神经酰胺，或甘油磷酸-乙醇胺，-甘油，-丝氨酸，或-肌醇脂质的组成中观察到。虽然我们没有发现管家作用的证据，但这些和以前的研究结果表明，iPLA 2 <$-null小鼠巨噬细胞在信号传导过程中表现出表型异常，iPLA 2 <$参与ER应激诱导的细胞凋亡。

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.