College of Bioengineering, Henan University of Technology, Lianhua Street, Zhengzhou 450001, China.
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China.
Biochim Biophys Acta Mol Cell Res. 2017 Apr;1864(4):613-624. doi: 10.1016/j.bbamcr.2017.01.003. Epub 2017 Jan 10.
The importance of macroautophagy (hereafter referred to as autophagy) in vascular endothelial cell (VEC) biology and dysfunction is increasingly recognized, but the molecular mechanisms of autophagy in VECs in the presence of serum are still poorly understood. Previously, we identified pterostilbene as a potent autophagy inducer of VECs in the presence of serum. In this study, we used pterostilbene as a tool to induce VEC autophagy and identified the differentially expressed genes using high-throughput DAN microarray. The small GTPase Ras-related protein in brain 13 (Rab13) was found to be the most significantly up-regulated gene in pterostilbene-treated human umbilical VECs (HUVECs). Knockdown of Rab13 blocked pterostilbene-induced mTOR inhibition and autophagy, whereas overexpression of the GTP-containing active form of Rab13 induced mTOR inhibition and autophagy in HUVECs. Using a combination of immunofluorescence and co-immunoprecipitation (co-IP) assays, we demonstrated that pterostilbene or up-regulation of the active form of Rab13 promoted the interaction between Rab13 and growth factor receptor-bound protein 2 (Grb2). Knockdown of Grb2 suppressed pterostilbene or up-regulation of the active form of Rab13-induced autophagy. Further mechanistic studies revealed that Rab13 activated the downstream AMP-activated protein kinase (AMPK) and blocked mammalian target of rapamycin (mTOR) signaling by its functional interaction with Grb2 to regulate autophagy in HUVECs. Our study firmly establishes Rab13 as a novel regulator of autophagy in VECs under nutrient-enriched conditions.
自噬（以下简称自噬）在血管内皮细胞（VEC）生物学和功能障碍中的重要性日益受到重视，但在有血清存在的情况下，VEC 中自噬的分子机制仍知之甚少。先前，我们发现紫檀芪是一种在有血清存在的情况下能强烈诱导 VEC 自噬的物质。在这项研究中，我们使用紫檀芪作为诱导 VEC 自噬的工具，并使用高通量 DNA 微阵列鉴定差异表达基因。在紫檀芪处理的人脐静脉内皮细胞（HUVEC）中，发现脑 Ras 相关蛋白 13（Rab13）是上调最显著的基因。Rab13 的敲低阻断了紫檀芪诱导的 mTOR 抑制和自噬，而 Rab13 的 GTP 结合活性形式的过表达则诱导了 HUVEC 中的 mTOR 抑制和自噬。通过免疫荧光和共免疫沉淀（co-IP）实验的组合，我们证明了紫檀芪或 Rab13 的活性形式的上调促进了 Rab13 与生长因子受体结合蛋白 2（Grb2）之间的相互作用。Grb2 的敲低抑制了紫檀芪或 Rab13 活性形式诱导的自噬。进一步的机制研究表明，Rab13 通过与 Grb2 的功能相互作用激活下游 AMP 激活的蛋白激酶（AMPK）并阻断哺乳动物雷帕霉素靶蛋白（mTOR）信号通路，从而调节 HUVEC 中的自噬。我们的研究明确确立了 Rab13 是营养丰富条件下 VEC 自噬的一种新的调节剂。
