Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution
Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution
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DOI：
10.1021/bi991666c
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发表时间：
2000-01-11
期刊：
影响因子：
2.9
通讯作者：
Rothblat, GH
中科院分区：
文献类型：
--
作者：
Kellner-Weibel, G;de la Llera-Moya, M;Rothblat, GH
先前的研究表明，清道夫受体BI（SR-BI）刺激HDL和SR-BI表达细胞之间游离胆固醇（FC）的双向流动。FC通量增强的主要成分似乎独立于HDL与SR-BI的结合而发生，并且可能是由于SR-BI表达导致的膜脂质结构域的变化（I）。在本研究中，SR-BI对细胞胆固醇代谢的影响通过检查SR-BI介导的细胞胆固醇质量的变化、HDL衍生的FC的酯化和膜脂质池的变化来确定。SR-BI表达细胞在含有HDL的培养基中生长导致细胞胆固醇质量增加，其中大部分以酯形式积累。通过SR-BI表达增强HDL衍生的FC的酯化作用，其程度远大于SR-BI介导的FC摄取增加，表明SR-BI介导的对细胞中胆固醇利用的影响。通过比较SR-BI阳性和阴性细胞中的FC酯化率来测试这一观察结果，此时等量的细胞外FC通过环糊精或载脂蛋白AT/磷脂盘吸收，两者都不含胆固醇酯。在这些条件下，SR-BI没有优先刺激胆固醇酯化。这些结果表明，在SR-BI表达细胞中HDL衍生的FC的酯化作用增强是由于细胞FC池的扩大，而不是SR-BI对胆固醇利用的特异性作用。使用两种方法来测试SR-BI表达对膜脂质组织的影响。在第一，细胞FC外源性胆固醇氧化酶的敏感性进行了测试的条件下，有一个优先氧化的小窝胆固醇。与载体转染的细胞或表达相关B类清道夫受体CD 36的细胞相比，发现SR-BI表达大大增加了氧化酶可利用的细胞胆固醇的分数。这些结果表明，SR-BI表达改变了膜游离胆固醇向小窝部分的分布或改变了该膜部分对外源性胆固醇氧化酶的可及性。在第二种方法中，在FC从质膜解吸是流出速率限制的条件下监测细胞FC向高浓度环糊精的流出。表达SR-BI的细胞显示FC在两个动力学池之间的分布发生变化，快速池中FC较多，慢速池中FC较少。这些数据支持一种模型，其中SR-BI表达导致胆固醇重新分布到膜结构域，该膜结构域用于促进FC在细胞和脂蛋白之间的通量。
Previous studies have shown that scavenger receptor BI (SR-BI) stimulates the bidirectional flux of free cholesterol (FC) between HDL and SR-BI-expressing cells. A major component of the enhanced FC flux appears to occur independently of HDL binding to SR-BI and may be due to changes in membrane lipid domains resulting from SR-BI expression (I). In the present study, the impact of SR-BI on cellular cholesterol metabolism was determined by examining SR-BI-mediated changes in cellular cholesterol mass, the esterification of HDL-derived FC, and changes in membrane lipid pools. Growth of SR-BI-expressing cells in medium containing HDL led to increased cellular cholesterol mass, most of which accumulated as ester. The esterification of HDL-derived FC was enhanced by SR-BI-expression to a far greater extent than the SR-BI mediated increase in FC uptake, suggesting an SR-BI-mediated effect on cholesterol utilization in the cell. This observation was tested by comparing FC esterification rates in SR-BI positive and negative cells when equivalent amounts of extracellular FC were taken up via cyclodextrins or apolipoprotein AT/phospholipid disks, neither of which contained cholesteryl ester. Under these conditions, SR-BI did not preferentially stimulate cholesterol esterification. These results indicate that the enhanced esterification of HDL-derived FC in SR-BI-expressing cells is due to the expanded pool of cellular FC and not to a specific effect of SR-BI on cholesterol utilization. Two approaches were used to test the effects of SR-BI expression on membrane lipid organization. In the first, the sensitivity of cellular FC to exogenous cholesterol oxidase was tested under conditions in which there is a preferential oxidation of caveolar cholesterol. SR-BI-expression was found to greatly increase the fraction of cellular cholesterol available to the oxidase as compared to either vector-transfected cells or cells expressing the related class B scavenger receptor CD36, These results suggest that SR-BI expression alters the distribution of membrane-free cholesterol to a caveolar fraction or alters the accessibility of this membrane fraction to exogenous cholesterol oxidase. In the second approach, the efflux of cellular FC to high concentrations of cyclodextrins was monitored under conditions where desorption of FC from the plasma membrane is rate Limiting for efflux. SR-BI-expressing cells showed a shift in the distribution of FC between two kinetic pools with more FC in the fast pool and less in the slow pool. These data support a model in which SR-BI expression leads to a redistribution of cholesterol to membrane domains that serve to facilitate the flux of FC between cells and lipoproteins.