Okoro Emmanuel U, Zhang Hongfeng, Guo Zhongmao, Yang Fang, Smith Carlie, Yang Hong
Department of Physiology, Meharry Medical College, Nashville, Tennessee, United States of America.
Department of Physiology, Meharry Medical College, Nashville, Tennessee, United States of America; Department of Pathology, Central Hospital of Wuhan, Wuhan City, People's Republic of China.
PLoS One. 2015 Aug 28;10(8):e0136895. doi: 10.1371/journal.pone.0136895. eCollection 2015.
Activation of apolipoprotein E receptor-2 (apoER2) and very low density lipoprotein receptor (VLDLR) inhibits foam cell formation. Reelin is a ligand of these receptors. Here we generated two reelin subregions containing the receptor binding domain with or without its C-terminal region (R5-6C and R5-6, respectively) and studied the impact of these peptides on macrophage cholesterol metabolism. We found that both R5-6C and R5-6 can be secreted by cells. Purified R5-6 protein can bind apoER2 and VLDLR. Overexpression of apoER2 in macrophages increased the amount of R5-6 bound to the cell surface. Treatment of macrophages with 0.2 μg/ml R5-6 elevated ATP binding cassette A1 (ABCA1) protein level by ~72% and apoAI-mediated cholesterol efflux by ~39%. In addition, the medium harvested from cells overexpressing R5-6 or R5-6C (R5-6- and R5-6C-conditioned media, respectively) also up-regulated ABCA1 protein expression, which was associated with accelerated cholesterol efflux and enhanced phosphorylation of phosphatidylinositol 3 kinase (PI3K) and specificity protein-1 (Sp1) in macrophages. The increased ABCA1 expression and cholesterol efflux by R5-6- and R5-6C-conditioned media were diminished by Sp1 or PI3K inhibitors mithramycin A and LY294002. Further, the cholesterol accumulation induced by apoB-containing, apoE-free lipoproteins was significantly less in macrophages incubated with R5-6- or R5-6C-conditioned medium than in those incubated with control conditioned medium. Knockdown of apoER2 or VLDLR attenuated the inhibitory role of R5-6-conditioned medium against lipoprotein-induced cholesterol accumulation. These results suggest that the reelin subregion R5-6 can serve as a tool for studying the role of apoER2 and VLDLR in atherogenesis.
载脂蛋白E受体2（apoER2）和极低密度脂蛋白受体（VLDLR）的激活可抑制泡沫细胞形成。Reelin是这些受体的配体。在此，我们生成了两个包含受体结合域且有或没有其C端区域的Reelin亚区域（分别为R5-6C和R5-6），并研究了这些肽对巨噬细胞胆固醇代谢的影响。我们发现R5-6C和R5-6均可被细胞分泌。纯化的R5-6蛋白可结合apoER2和VLDLR。巨噬细胞中apoER2的过表达增加了与细胞表面结合的R5-6的量。用0.2μg/ml R5-6处理巨噬细胞可使ATP结合盒转运体A1（ABCA1）蛋白水平提高约72%，并使载脂蛋白AI介导的胆固醇外流提高约39%。此外，从过表达R5-6或R5-6C的细胞中收获的培养基（分别为R5-6条件培养基和R5-6C条件培养基）也上调了ABCA1蛋白表达，这与巨噬细胞中胆固醇外流加速以及磷脂酰肌醇3激酶（PI3K）和特异性蛋白-1（Sp1）的磷酸化增强有关。Sp1或PI3K抑制剂光神霉素A和LY294002可减弱R5-6条件培养基和R5-6C条件培养基对ABCA1表达增加和胆固醇外流的作用。此外，与对照条件培养基孵育的巨噬细胞相比，用R5-6条件培养基或R5-6C条件培养基孵育的巨噬细胞中，含载脂蛋白B、不含载脂蛋白E的脂蛋白诱导的胆固醇积累明显减少。敲低apoER2或VLDLR可减弱R5-6条件培养基对脂蛋白诱导的胆固醇积累的抑制作用。这些结果表明，Reelin亚区域R5-6可作为研究apoER2和VLDLR在动脉粥样硬化发生中作用的工具。
