ABC Transporters in Inflammation and Lipid Homeostasis

炎症和脂质稳态中的 ABC 转运蛋白

• 批准号: 7647664
• 负责人: Peter A Edwards
• 金额: $ 43.21万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7647664
• 关键词: ABCG2 gene; ATP-Binding Cassette Transporters; Address; Adoptive Transfer; Affect; Apolipoprotein E; Apoptosis; Apoptotic; Arterial Fatty Streak; Arteries; Arts; Atherosclerosis; Attenuated; Biological Assay; Biotinylation; Blood; Bone Marrow Transplantation; Cell Line; Cell surface; Cells; Cholesterol; Cytoplasmic Tail; Data; Development; Disease; Endoplasmic Reticulum; Engineering; Event; Figs - dietary; Fluorescence; Gene Expression; Gene Proteins; Gene Targeting; Genes; Homeostasis; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Knockout Mice; Lead; Lesion; Life; Lipids; Lipoproteins; Lung; Metabolism; Microscopy; Movement; Mus; Mutation; Myocardial Infarction; Pathway interactions; Phospholipids; Physiological; Predisposition; Primary Cell Cultures; Process; Production; Proteins; Publishing; Reporting; Research Personnel; Role; Sterols; Surface; T-Cell Proliferation; T-Lymphocyte; Testing; Vesicle; attenuation; base; cell type; gene induction; in vitro Assay; in vivo; insight; macrophage; mouse model; novel; oxidized lipid; programs; response; sterol homeostasis

During the current grant period we characterized a number of LXR target genes including Abcgl, that is thought to function as a sterol transmembrane transporter. Abcgl was shown to be expressed at high levels, in a number of cell types including macrophages, B and T cells. We used cultured cells and Abcgl-/- mice to demonstrate that ABCG1 functions to flux sterols away from the endoplasmic reticulum to exogenous sterol acceptors, such as HDL. Loss of ABCG1 was associated with massive lipid accumulation, cytokine activation, inflammation and an increase in IgM-secreting B-1 cells. Importantly, bone marrow transplantation (BMT) studies identified a role for ABCG1 in the development of atherosclerosis. In collaboration with Project 6 we also demonstrated an unexpected and novel role for ABCG1 in T cell proliferation. We now propose to use two new mouse models, Abcgl-/-apoE-/- and Abcgl-/-Rag-/- double knockout mice: i) to test the hypothesis that loss of ABCG1 attenuates the development of atherosclerosis as a result of increased apoptosis, and to then identify the apoptotic pathway/mechanism involved, ii) to test the hypothesis that loss of ABCG1 from B and T cells (and possibly B-1 cells) also affects atherosclerosis. The latter studies will utilize adoptive transfer of B and T cells into Abcgl-/-RAG-/- mice followed by BMT into Ldlr-/- recipient mice. In preliminary studies we have identified multiple oxysterols that accumulate in Abcgl- /- macrophages in vivo. We now propose to better characterize the lipids and oxysterols that accumulate and then determine the mechanism by which these specific oxysterols/lipids promote apoptosis both in vivo and in vitro and induce massive cytokine expression, especially in macrophages lacking ABCGL We will also use state-of-the-art spinning disc microscopy and fluorescent-tagged wild type or mutant ABCG1 to follow intracellular movement of the transporter as a means of understanding how ABCG1 functions to flux sterols through intracellular vesicles to the cell surface. Finally, we will use a new cell-based assay to test the hypothesis that ABCG1, but but not ABCA1, functions to promote the efflux of specific sterols away from the endoplasmic reticulum.

在当前赠款期间，我们表征了许多LXR靶基因，包括ABCGL，即 被认为充当固醇跨膜转运蛋白。显示ABCGL以高水平表示 在许多细胞类型中，包括巨噬细胞，B和T细胞。我们使用培养的细胞和ABCGL - / - 小鼠 证明ABCG1可从内质网向外源性固醇发挥作用。 受体，例如HDL。 ABCG1的损失与大量脂质积累，细胞因子有关 激活，炎症和分泌B-1细胞的增加。重要的是，骨髓 移植（BMT）研究确定了ABCG1在动脉粥样硬化发展中的作用。在 与项目6合作，我们还展示了ABCG1在T细胞中的意外和新颖的作用 增殖。我们现在建议使用两种新的鼠标型号ABCGL - / - APOE-/ - 和ABCGL - / - rag - / - double 敲除小鼠：i）检验以下假设：ABCG1的丧失会减弱动脉粥样硬化的发展 凋亡增加的结果，然后确定涉及的凋亡途径/机制，ii）测试 假设B和T细胞（以及可能的B-1细胞）丧失ABCG1也会影响动脉粥样硬化。这 后者的研究将利用B和T细胞的继发性转移到ABCGL - / - 抹布 - / - 小鼠，然后BMT进入 LDLR - / - 接收者小鼠。在初步研究中，我们已经确定了在ABCGL-中积累的多种氧 / - 体内巨噬细胞。现在，我们建议更好地表征积累的脂质和氧 然后确定这些特定的氧化醇/脂质在体内促进凋亡的机制 体外并诱导大量的细胞因子表达，尤其是在缺乏ABCGL的巨噬细胞中，我们还将 使用最先进的旋转盘显微镜和荧光标记的野生型或突变体ABCG1遵循 转运蛋白的细胞内运动是一种理解ABCG1如何发挥通量固醇的手段 通过细胞内囊泡进入细胞表面。最后，我们将使用新的基于单元的基于细胞的分析来测试 假设ABCG1，但没有ABCA1的作用，可以促进特定固醇的外排。 内质网。