CD68通过Vav1调控CD36介导泡沫细胞形成的机制

Foam cells resulted from lipid laden macrophages are critical cellular components atherosclerosis. CD36 has been found to be a key scavenger receptor that engulfs lipids and initiates inflammatory response of macrophagic foam cells. Among the downstream molecules of CD36, Vav1 can be phosphorylated by binding of Ox-LDL and trigger activation of JNK. Genetic ablation of CD36 or Vav1 by CRISPR/Cas9 genome editing tool in RAW264.7 macrophages leads to significantly reduced Ox-LDL uptake and foam cell formation. However, it is poorly understood how the CD36 signaling pathway is regulated even though the its significance is well appreciated. By affinity purification of Vav1-OST fusion protein from Ox-LDL stimulated macrophages harvested in knockin mice, we recently found that Vav1 formed protein complex with CD68, another scavenger receptor recirculating between the lysosome and plasma membrane. We found that Ox-LDL stimulation upregulates CD68 expression, independent on presence of CD36. Based on multiple experimental evidences, we postulate that CD68 plays a critical role in checking the activity of “CD36-Vav1-JNK” signaling axis, by means of translocate Vav1 into lysosome. In the proposed project, we will first analyze the structural basis that conditions interaction between Vav1 and CD68. As CD68 has no determined function in signaling pathway implicated in the foam cell formation, and CD36 plays a dominant role that may mask the effect of CD68, we will study the signaling events triggered by CD68 in the CD36 knockout macrophages. It is also intriguing whether CD68 has more broad function in degradation of other molecules implicated in foam cell formation and inflammatory response. Finally, it is critical to determine the role of CD68 in vivo, by establishing a mouse model on the ApoE and CD68 double knockout background. The proposed project provides a completely novel view how the key signaling pathway downstream CD36 in foam cell formation is regulated by another member of the scavenger receptor family molecule CD68, therefore it is fundamental for understanding occurrence of atherosclerosis and could be beneficial for development to therapeutic interventions for the atherosclerotic diseases.

巨噬细胞源性泡沫细胞是动脉粥样硬化斑块的关键组分。CD36受体刺激及下游Vav1发生磷酸化，是激活JNK形成泡沫细胞的关键。但“CD36-Vav1-JNK”信号轴如何被调控仍然未知。申请人将Vav1-OST基因敲入小鼠巨噬细胞用Ox-LDL处理，质谱分析发现刺激后Vav1与CD68形成高丰度复合物。而CD68主要分布在溶酶体，并在溶酶体与细胞膜之间循环，后者可能对Vav1在溶酶体降解起着关键作用。有趣的是，Ox-LDL促进CD68的表达且不依赖CD36。上述结果提示CD68在调控“CD36-Vav1-JNK”信号轴中起着重要作用。本课题将研究Vav1与CD68相互作用的结构域基础、CD68在蛋白相互作用水平如何调控脂代谢信号，及活体水平CD68对小鼠动脉粥样硬化的影响。本课题将揭示CD68调控泡沫细胞形成的全新机制，对深入理解泡沫细胞的形成机制具有重要意义。课题已有相应研究基础，可行性好。

在动脉粥样硬化中，衍生于血液单核细胞的巨噬细胞促进了非溶解性炎症，随后形成最初的坏死核心，最终引发血栓性血管疾病。然而，人们对炎症细胞，特别是巨噬细胞是如何促进动脉粥样硬化的知之甚少。CD68是一种独特的D类清道夫受体(SRD)家族成员，在单核细胞/巨噬细胞中特异性表达，并在氧化低密度脂蛋白(ox-LDL)刺激下显著上调。尽管如此，髓系特异性CD68是否影响以及如何影响动脉粥样硬化仍有待确定。为了明确CD68在体内对动脉粥样硬化的作用和机制，我们在ApoE背景下构建了全敲和髓系特异性敲除CD68的缺陷小鼠。高脂饮食喂养， CD68全敲和髓系特异性缺失小鼠的动脉粥样硬化程度较对照组均显著减轻，并伴随着粥样斑块儿中免疫/炎性细胞负荷和坏死核心含量的显著减少，但斑块中的平滑肌细胞含量显著增加。体外实验显示，在ox-LDL诱导下，巨噬细胞中的CD68缺乏导致细胞凋亡减弱。此外，CD68的缺失抑制了ROS的产生，而抑制ROS产生则可显著逆转上述凋亡趋势。我们进一步明确了CD68缺陷是通过使丝裂原活化蛋白激酶（MAPK）通路失活而影响凋亡的。我们发现并证实了CD68是一种巨噬细胞特异性的“ROS-MAPK-凋亡”信号轴的调节因子，从而为CD68作为冠状动脉疾病的一种危险因子提供了一种新颖的未知机制。因此，针对CD68的抑制性治疗可能为缓解心血管疾病提供一种有效的手段。