Koseki Masahiro, Hirano Ken-Ichi, Masuda Daisaku, Ikegami Chiaki, Tanaka Masaki, Ota Akemi, Sandoval Jose C, Nakagawa-Toyama Yumiko, Sato Satoshi B, Kobayashi Toshihide, Shimada Yukiko, Ohno-Iwashita Yoshiko, Matsuura Fumihiko, Shimomura Iichiro, Yamashita Shizuya
Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
J Lipid Res. 2007 Feb;48(2):299-306. doi: 10.1194/jlr.M600428-JLR200. Epub 2006 Nov 1.
Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of theta-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kappaB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-alpha (TNF-alpha) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kappaB and TNF-alpha were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-alpha secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-alpha secretion.
细胞表面的脂筏被认为作为各种细胞功能的平台非常重要。本研究的目的是了解脂质流出缺陷是否会影响细胞表面的脂筏及其相关的细胞功能。我们研究了来自ATP结合盒转运蛋白A1缺陷（Abca1基因敲除）小鼠的脂质流出缺陷的巨噬细胞。通过以下两种新型探针评估脂筏：一种生物素化且经蛋白酶（枯草杆菌蛋白酶卡尔伯格）切割的θ毒素衍生物和一种聚乙二醇衍生胆固醇的荧光素酯。两种探针均表明，Abca1基因敲除巨噬细胞中的脂筏增加。此外，在用脂多糖（LPS）刺激后，检测了核因子κB的活性、mRNA和细胞内分布以及肿瘤坏死因子-α（TNF-α）的分泌。与野生型巨噬细胞相比，Abca1基因敲除巨噬细胞中LPS诱导的核因子κB激活和TNF-α反应更迅速且加速。用环糊精和制霉菌素修饰脂筏可纠正异常反应，表明脂筏增加与TNF-α分泌异常之间存在关联。我们在此报告，脂质流出缺陷的Abca1基因敲除巨噬细胞在细胞表面表现出脂筏增加且TNF-α分泌加速。
