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.
Keywords: PFKFB3; TNF-α; endothelial cells; glycolysis; inflammation.