凝集素样氧化低密度脂蛋白受体1（LOX-1）在巨噬细胞中介导的功能及机制研究

On stimulation, LOX-1-mediated signal pathway can activate endothelial cells. However, the precise signal mechanism remains to be further clarified and especially, the signal protein(s), which binds the cytoplasmic tail of LOX-1, is unknown. In contrast, there are few reports about the function of LOX-1 on macrophages and dendritic cells as well as LOX-1-mediated signal pathway. Recently, we, for the first time, demonstrate that LOX-1 function as the receptor for heat shock protein 60 (Hsp60), a danger signal for the immune system. LOX-1 recognizes the C-terminus of Hsp60 to take up Hsp60 into cells. Although previous reports indicate that Toll-like receptor 2 and 4 (TLR2/4) function as the signal receptors for Hsp60, the endocytosis of Hsp60 is required for the Hsp60-mediated signal transduction. Also, we observed that Hsp60-C could activate ERK1/2 via LOX-1 and however, Hsp60-C did not stimulate TLR2/4 to activate NF-κB, which implies that TLR2/4 do not response to Hsp60-C. In this study, we will stimulate LOX-1 on macrophages by Hsp60-C to investigate the release of cytokines and the activation of macrophages. Furthermore, we will identify the signal protein(s) directly binding to the cytoplasmic tail of LOX-1 by yeast two-hybrid assay and by immunoprecipitation together with 2-DE/MS. Moreover, we will determine LOX-1-mediated signal pathway in macrophages and the role of LOX-1 together with its mechanism in the regulation of the function of TLR2/4. Therefore, this study will lay a foundation to clarify the function of LOX-1 and its mechanism in the immune system.

刺激内皮细胞表面LOX-1可使其活化，但详细的信号机制还未阐明，特别是LOX-1结合的信号蛋白未被鉴定。相比，LOX-1在巨噬细胞和树突细胞中的功能及机制鲜有报道。我们首次阐明了LOX-1作为免疫危险信号Hsp60的受体，LOX-1识别Hsp60的C末端（Hsp60-C）介导Hsp60的结合和内化。虽然TLR2/4被报道为Hsp60的信号受体，但Hsp60的内化对Hsp60活化信号是必须的。我们观察到Hsp60-C能刺激LOX-1活化ERK1/2，但不刺激TLR2/4介导的NF-κB活化。利用这一特性，本项目拟通过Hsp60-C刺激巨噬细胞，阐明LOX-1介导的细胞因子分泌和细胞活化功能。并通过酵母双杂交和免疫沉淀-质谱分析鉴定LOX-1直接结合的信号蛋白，阐明LOX-1介导的信号通路及其在调节TLR2/4信号和功能中的作用和机制，为揭示LOX-1在免疫系统中发挥的功能及其机制奠定基础。

凝集素样氧化低密度脂蛋白受体1（LOX-1）广泛表达于内皮细胞、巨噬细胞和树突细胞等细胞表面。虽然LOX-1在心血管系统和免疫系统中被广泛研究，然而LOX-1活化的机制和功能仍有待进一步研究。本研究利用Hsp60的C末端（Hsp60-C）研究LOX-1在巨噬细胞中介导的信号和功能。我们的研究表明，LOX-1单独能够介导Hsp60-C的结合和内化，LOX-1抗体能够抑制Hsp60-C与LOX-1的结合。进一步的研究表明，LOX-1能够与APPL1相互作用，并与APPL1共定位于细胞内。Hsp60-C能够明显促进下游的p38、NFκB的磷酸化，同时促进细胞因子IL-6、MCP-1、MIP-1α和BAFF等分泌。TLR2/4缺失不损害Hsp60-C激活p38、NFκB的磷酸化，然而LPS能够促进LOX-1的表达，促进LOX-1介导的细胞因子分泌。同时，通过Hsp60-C与 OVA形成融合抗原，特异靶向LOX-1，能够明显促进OVA抗体的产生。该研究的开展，为进一步揭示Hsp60作为自身抗原在自身免疫性疾病中的发病机制研究奠定了基础，同时为进一步研究LOX-1在免疫系统中的功能机制以及以LOX-1为靶向的免疫治疗研究奠定了基础。