Department of Biochemistry and Biomedical Sciences, Thrombosis and Atherosclerosis Research Institute, Hamilton Health Sciences, McMaster University, Hamilton, ON, Canada.
Department of Biochemistry and Biomedical Sciences, Thrombosis and Atherosclerosis Research Institute, Hamilton Health Sciences, McMaster University, Hamilton, ON, Canada.
J Lipid Res. 2024 May;65(5):100541. doi: 10.1016/j.jlr.2024.100541. Epub 2024 Apr 5.
HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1 mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1's lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1 mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.
高密度脂蛋白（HDL）携带神经鞘氨醇-1-磷酸（S1P），并刺激包括巨噬细胞和内皮细胞在内的不同细胞中的信号通路，这些信号通路参与动脉粥样硬化斑块的形成。HDL 通过 S1P 发出信号依赖于 HDL 受体清道夫受体 B 型，I 类（SR-B1）和 S1P 受体 1（S1PR1），当这两种受体在 HEK293 细胞系中异源过表达时，它们相互作用。在这项研究中，我们着手测试 SR-B1 和 S1PR1 是否在培养的原代小鼠巨噬细胞和动脉粥样硬化斑块中相互作用。我们使用内源性表达带有 eGFP-（S1pr1 小鼠）标签的 S1PR1 的敲入小鼠，结合邻近连接分析来证明 HDL 刺激原代巨噬细胞中 SR-B1 和 S1PR1 之间的物理相互作用，这依赖于 HDL 相关的 S1P，并且可以被 SR-B1 的脂质转移活性抑制剂或 S1PR1 拮抗剂阻断。我们还证明，一种合成的 S1PR1 选择性激动剂 SEW2871 刺激 SR-B1 和 S1PR1 之间的相互作用，并且这种相互作用也被 SR-B1 的脂质转运活性抑制剂阻断。此外，我们在缺乏载脂蛋白 E 的 S1pr1 小鼠的动脉粥样硬化斑块中检测到大量的 SR-B1/S1PR1 复合物。用 S1PR1 拮抗剂 Ex26 治疗 12 小时可破坏动脉粥样硬化斑块中的 SR-B1-S1PR1 相互作用。这些发现表明，SR-B1 和 S1PR1 在培养的原代巨噬细胞中和在小鼠的动脉粥样硬化斑块中形成配体依赖性复合物，并为 SR-B1 和 S1PR1 如何参与介导 HDL 信号以激活巨噬细胞中的抗动脉粥样硬化反应提供了机制上的见解。
