Smith J D, Miyata M, Ginsberg M, Grigaux C, Shmookler E, Plump A S
Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, New York 10021-6399, USA.
J Biol Chem. 1996 Nov 29;271(48):30647-55. doi: 10.1074/jbc.271.48.30647.
RAW 264 mouse macrophage cells were stably transfected with human apolipoprotein E (apoE) expression vectors. Clonal derivatives were characterized for expression of the human apoE2, apoE3, and apoE4 isoforms. An apoE4-expressing clonal cell line and a non-expressing clonal control cell line were loaded overnight with either [3H]cholesterol or [3H]choline. The cells were washed and incubated for 24 h in serum-free medium with or without the addition of 8-bromo-cyclic AMP (8-Br-cAMP). Only the apoE-secreting cells and only in the presence of 8-Br-cAMP released large amounts of labeled cholesterol or phosphatidylcholine into the medium. Mass analyses of cellular free and esterified cholesterol confirmed the results of the labeling studies; a decrease in cellular cholesterol content was observed in the 8-Br-cAMP-treated apoE-secreting cells, concurrent with an increase in cholesterol found in the medium. FPLC analysis of the medium demonstrated that 8-Br-cAMP treatment of the apoE-secreting cells led to an increased size fraction and amount of a peak of secreted cholesterol which comigrated with apoE. The 8-Br-cAMP-mediated increase in cholesterol efflux was also observed in non-apoE-secreting cells incubated with exogenous apoE or apoAI, and the effect of apoE was saturable. The apoE2, apoE3, and apoE4 isoforms were equally efficient in promoting 8-Br-cAMP-dependent cholesterol efflux. Reductive methylation of apoE abolished its ability to promote 8-Br-cAMP-dependent cholesterol efflux. Brefeldin A and monensin, inhibitors of protein processing through the Golgi, both blocked the 8-Br-cAMP stimulation of cholesterol efflux to exogenous apoE. 8-Br-cAMP induced specific apoE and apoAI binding, but not apoE degradation, by the RAW cells. We present a model wherein cAMP induces a membrane apolipoprotein receptor that does not lead to endocytosis and degradation, but instead promotes the transfer of lipids to apolipoproteins, which can then be released from the cell.
将人载脂蛋白E（apoE）表达载体稳定转染RAW 264小鼠巨噬细胞。对克隆衍生物进行人apoE2、apoE3和apoE4亚型表达特征分析。将一个表达apoE4的克隆细胞系和一个不表达的克隆对照细胞系用[3H]胆固醇或[3H]胆碱负载过夜。洗涤细胞后，在无血清培养基中培养24小时，添加或不添加8-溴环磷酸腺苷（8-Br-cAMP）。只有分泌apoE的细胞且仅在存在8-Br-cAMP的情况下才会向培养基中释放大量标记的胆固醇或磷脂酰胆碱。对细胞游离胆固醇和酯化胆固醇的质量分析证实了标记研究的结果；在经8-Br-cAMP处理的分泌apoE的细胞中观察到细胞胆固醇含量降低，同时培养基中的胆固醇增加。对培养基的快速蛋白质液相色谱（FPLC）分析表明，8-Br-cAMP处理分泌apoE的细胞会导致分泌胆固醇峰的大小分级增加且量增加，该峰与apoE共迁移。在用外源性apoE或apoAI孵育的非分泌apoE的细胞中也观察到8-Br-cAMP介导的胆固醇流出增加，且apoE的作用是可饱和的。apoE2、apoE3和apoE4亚型在促进8-Br-cAMP依赖性胆固醇流出方面同样有效。apoE的还原甲基化消除了其促进8-Br-cAMP依赖性胆固醇流出的能力。布雷菲德菌素A和莫能菌素是通过高尔基体进行蛋白质加工的抑制剂，二者均阻断了8-Br-cAMP对外源性apoE刺激的胆固醇流出。8-Br-cAMP诱导RAW细胞特异性结合apoE和apoAI，但不诱导apoE降解。我们提出了一个模型，其中环磷酸腺苷（cAMP）诱导一种膜载脂蛋白受体，该受体不会导致内吞作用和降解，而是促进脂质向载脂蛋白的转移，然后载脂蛋白可从细胞中释放。
