ABC Transporters in Inflammation and Lipid Homeostasis

在目前的授权期内，我们鉴定了一些LXR靶基因，包括Abcgl，即 被认为具有类固醇跨膜转运蛋白的功能。结果显示，Abcg1在高水平表达， 在许多细胞类型中，包括巨噬细胞、B细胞和T细胞。我们使用培养的细胞和abcgl-/-小鼠 证明Abcg1具有将内质网中的甾醇输送到外源甾醇的功能 受体，如高密度脂蛋白。Abcg1的丢失与大量的脂质堆积、细胞因子有关 活化、炎症和分泌IgM的B-1细胞增加。重要的是，骨髓 移植(BMT)研究证实了Abcg1在动脉粥样硬化发展中的作用。在……里面 通过与Project 6的合作，我们还展示了Abcg1在T细胞中的一个意想不到的新角色 扩散。我们现在建议使用两种新的小鼠模型，Abcgl-/-apoE-/-和Abcgl-/-Rag-/-Double 基因敲除小鼠：i)验证Abcg1基因缺失减缓AS发展的假说。 细胞凋亡增加的结果，然后确定所涉及的凋亡途径/机制，ii)测试 假设B和T细胞(可能还有B-1细胞)中Abcg1的缺失也会影响动脉粥样硬化。这个 后一项研究将利用B和T细胞过继转移到Abcgl-/-RAG-/-小鼠体内，然后进行骨髓移植 LDLR-/-受体小鼠。在初步研究中，我们已经确定了在Abcgl-1中积累的多种氧固醇。 /-体内的巨噬细胞。我们现在建议更好地描述积累和 然后确定这些特定的氧化类固醇/脂类在体内和 在体外诱导大量细胞因子的表达，特别是在缺乏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型Abcg1进行跟踪 细胞内转运体的运动作为了解Abcg1如何作用于类固醇的一种手段 通过细胞内的小泡到达细胞表面。最后，我们将使用一种新的基于细胞的分析来测试 假设Abcg1，而不是ABCA1，起促进特定甾醇外流的作用 内质网。

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.