Huang Y, Ghosh M J, Lopes-Virella M F
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
J Lipid Res. 1997 Jan;38(1):110-20.
We have previously shown that uptake of low density lipoprotein-containing immune complexes (LDL-IC) by human monocyte-derived macrophages led to the transformation of these cells into foam cells and induced a paradoxical increase in receptor-mediated binding of 125I-labeled LDL due to an increase in the number of LDL receptors (LDL-R). The same metabolic changes are also observed in PMA-treated THP-1 cells after incubation for 2 h with 150 microg/ml of immune complexes containing either native, oxidized (ox), or malondialdehyde (mda) LDL. After stimulation, PMA-treated THP-1 cells showed not only a 40-fold increase in 125I-labeled LDL binding but also a 40-fold increase in the immunoreactive LDL-R protein, confirming that the increase in LDL binding is due to an increase LDL-R number. In this study we have investigated, in PMA-treated THP-1 cells, the regulatory mechanism(s) responsible for the increased receptor-mediated binding of LDL induced by LDL-IC. By Northern blot and nuclear run-on analysis we have shown transcriptional activation of the LDL-R gene with a 7-fold increase in the LDL-R mRNA level in LDL-IC stimulated cells. Due to the marked difference between the increase in LDL-R mRNA and LDL-R protein, we estimated LDL-R mRNA stability using a inhibitor chase method and have shown that LDL-IC did not alter the LDL-R mRNA stability in THP-1 cells. We have also demonstrated, using cycloheximide as a inhibitor of protein synthesis, that the marked increase in LDL-R protein observed in LDL-IC-stimulated THP-1 cells resulted from de novo synthesis of LDL-R protein. To determine whether the increase in transcriptional activity of the LDL-R gene was secondary to changes in the cholesterol regulatory pool we performed experiments in which the cell cholesterol content was modified by the addition of either 25-hydroxycholesterol and mevalonate or inhibitors of ACAT activity (SA-58035 and progesterone). These experiments showed that the enhanced LDL-R expression was not affected by the addition of any of the above compounds. In conclusion, LDL-IC induced both transcriptional and post-transcriptional activation of the LDL-R gene in PMA-treated THP-1 cells and this induction was independent of the free cholesterol content of these cells.
我们之前已经表明，人单核细胞衍生的巨噬细胞摄取含低密度脂蛋白的免疫复合物（LDL-IC）会导致这些细胞转变为泡沫细胞，并由于低密度脂蛋白受体（LDL-R）数量增加而使受体介导的125I标记的低密度脂蛋白结合出现反常增加。在用150微克/毫升含天然、氧化（ox）或丙二醛（mda）低密度脂蛋白的免疫复合物孵育2小时后，在经佛波酯（PMA）处理的THP-1细胞中也观察到了相同的代谢变化。刺激后，经PMA处理的THP-1细胞不仅125I标记的低密度脂蛋白结合增加了40倍，而且免疫反应性LDL-R蛋白也增加了40倍，证实低密度脂蛋白结合增加是由于LDL-R数量增加所致。在本研究中，我们在经PMA处理的THP-1细胞中研究了负责LDL-IC诱导的受体介导的低密度脂蛋白结合增加的调节机制。通过Northern印迹和核转录分析，我们发现LDL-R基因转录激活，在LDL-IC刺激的细胞中LDL-R mRNA水平增加了7倍。由于LDL-R mRNA增加与LDL-R蛋白增加之间存在显著差异，我们使用抑制剂追踪法估计了LDL-R mRNA的稳定性，并表明LDL-IC不会改变THP-1细胞中LDL-R mRNA的稳定性。我们还使用环己酰亚胺作为蛋白质合成抑制剂证明，在LDL-IC刺激的THP-1细胞中观察到的LDL-R蛋白显著增加是由于LDL-R蛋白的从头合成。为了确定LDL-R基因转录活性的增加是否继发于胆固醇调节库的变化，我们进行了实验，通过添加25-羟基胆固醇和甲羟戊酸或ACAT活性抑制剂（SA-58035和孕酮）来改变细胞胆固醇含量。这些实验表明，添加上述任何一种化合物均不影响LDL-R表达的增强。总之，LDL-IC在经PMA处理的THP-1细胞中诱导了LDL-R基因的转录和转录后激活，并且这种诱导与这些细胞的游离胆固醇含量无关。
