Department of Critical Care Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
Inflammation. 2019 Feb;42(1):146-155. doi: 10.1007/s10753-018-0880-x.
Endothelial cells play an important role in health and a variety of diseases. Recent evidences show that endothelial cells rely on glycolysis rather than on oxidative phosphorylation to generate energy to support cellular functions such as angiogenesis. However, the effect of endothelial glycolysis on vascular inflammation remains little known. Here, we investigate the role of key glycolytic enzyme PFKFB3 in tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory responses. siRNAs were used to knockdown the expression of PFKFB3. In some experiments, PFKFB3 inhibitors were also used. TNF-α at 20 ng/ml was added to confluent endothelial cells for different time period of stimulation. PFKFB3 expression was examined by RT-PCR and western blotting. Cytokine antibody panel membranes were employed to detect different cytokines/chemokines in culture supernatant of endothelial cells. The determination of monocyte adhesion to endothelial cells after TNF-α treatment was conducted using THP-1 cells. The monocyte attraction was performed using Transwell filters. For further mechanisms, NF-κB-p65 localization was examined by immunofluorescence. Expression of total IκB, phospho-IκB, phospho-NF-κB-p65, and Ikkβ was detected by western blotting. DNA-binding activity of NF-κB was assessed using electrophoretic mobility shift assay. We found that TNF-α increased endothelial PFKFB3 expression. Knockdown of PFKFB3 almost blocked all TNF-α-induced release of the proinflammatory cytokines/chemokines (MCP-1, IL-8, CXCL1, GMCSF, RANTES, TNF-α) and ICAM-1. PFKFB3 knockdown also significantly inhibited TNF-α-induced monocyte adhesion and transmigration. Furthermore, inhibition of PFKFB3 inhibited TNF-α-induced Ikkβ phosphorylation, IκBα phosphorylation and degradation, NF-κB-p65 phosphorylation, nuclear translocation, and DNA-binding activity. Thus, our results demonstrate that glycolytic enzyme PFKFB3 plays a critical role in TNF-α-induced endothelial inflammation.
内皮细胞在健康和多种疾病中发挥着重要作用。最近的证据表明，内皮细胞依赖糖酵解而不是氧化磷酸化来产生能量，以支持细胞功能，如血管生成。然而，内皮糖酵解对血管炎症的影响知之甚少。在这里，我们研究了关键糖酵解酶 PFKFB3 在肿瘤坏死因子-α（TNF-α）诱导的内皮前炎症反应中的作用。使用 siRNA 敲低 PFKFB3 的表达。在一些实验中，还使用了 PFKFB3 抑制剂。将 20ng/ml TNF-α加入到汇合的内皮细胞中，进行不同时间的刺激。通过 RT-PCR 和 Western blot 检测 PFKFB3 的表达。使用细胞因子抗体膜检测内皮细胞培养上清液中的不同细胞因子/趋化因子。使用 THP-1 细胞检测 TNF-α处理后单核细胞与内皮细胞的黏附。使用 Transwell 过滤器进行单核细胞趋化作用。为了进一步研究机制，通过免疫荧光法检测 NF-κB-p65 的定位。通过 Western blot 检测总 IκB、磷酸化 IκB、磷酸化 NF-κB-p65 和 Ikkβ的表达。通过电泳迁移率变动分析评估 NF-κB 的 DNA 结合活性。我们发现 TNF-α增加了内皮细胞 PFKFB3 的表达。PFKFB3 的敲低几乎阻断了所有 TNF-α诱导的促炎细胞因子/趋化因子（MCP-1、IL-8、CXCL1、GMCSF、RANTES、TNF-α）和 ICAM-1 的释放。PFKFB3 敲低也显著抑制了 TNF-α诱导的单核细胞黏附和迁移。此外，PFKFB3 的抑制抑制了 TNF-α诱导的 Ikkβ磷酸化、IκBα磷酸化和降解、NF-κB-p65 磷酸化、核转位和 DNA 结合活性。因此，我们的结果表明，糖酵解酶 PFKFB3 在 TNF-α 诱导的内皮炎症中起着关键作用。
