Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts.
Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts.
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DOI：
10.9734/ijbcrr/2015/14058
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Medh JD
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文献类型：
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作者：
Akopian D;Kawashima RL;Medh JD
ATP结合盒蛋白A1（ABCA 1）是一种胆固醇转运蛋白，有助于主动转运/清除多余的细胞胆固醇。当细胞积累胆固醇时，ABCA 1表达上调。本研究的目的是确定细胞外磷脂水平与ABCA 1表达和功能之间的任何相关性。将人包皮成纤维细胞与单独的胆固醇或胆固醇和磷脂酰胆碱孵育。分离总RNA并进行终点RT-PCR以比较ABCA 1转录物水平。对细胞裂解物进行Western印迹分析以比较ABCA 1蛋白水平。将细胞装载放射性标记的胆固醇，并在存在和不存在胆固醇受体apoE的情况下测量细胞胆固醇流出。ApoE依赖性外排计算为ABCA 1介导的外排的测量。在这里，我们发现，与单独加载胆固醇的细胞相比，加载胆固醇的人皮肤成纤维细胞与L-α-磷脂酰胆碱（PC）孵育分别使ABCA 1 mRNA和蛋白水平降低93%和57%。类似地，与单独用胆固醇处理的细胞相比，PC处理导致ABCG 1 mRNA水平降低25%，但SR-BI转录物水平没有变化。随后将磷脂处理的细胞与胆固醇受体如apoE孵育24小时，与未用PC处理的细胞中的胆固醇流出相比，ABCA 1介导的胆固醇流出减少65%。在脂质处理本身期间，与单独用胆固醇处理的细胞相比，PC处理的细胞的胆固醇损失大2.7倍。细胞脂质提取物中胆固醇的测量揭示，与仅装载有胆固醇的细胞相比，在磷脂酰胆碱存在下孵育的细胞显著耗尽胆固醇，仅具有20%的胆固醇。因此，磷脂酰胆碱促进细胞胆固醇的去除，从而否定ABCA 1信息、蛋白质和功能的胆固醇依赖性诱导。
ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor. ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-α-phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone. Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone. Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.