Abstract 1629: An MT1-MMP-LOX-1 Axis Regulates RhoA and Rac1 Activation through Gi Induced by Oxidized Low-Density Lipoprotein in Endothelial Cells
Background: Lectin-like oxidized-LDL receptor-1 (LOX-1) is a major receptor for oxidized LDL (oxLDL). Small GTP-binding protein RhoA is crucial for the regulation of endothelial nitric oxide synthase (eNOS), whereas Rac1 is required for reactive oxygen species (ROS) generation. It was reported that oxLDL-induced Rac1/NADPH oxidase activity is through LOX-1. Recent studies have shown that membrane type1 matrix metalloproteinase (MT1-MMP) is associated with signal transduction including Gi-mediated pathway. In this study we investigated the correlation between RhoA and Rac1 activation and MT1-MMP through LOX-1 in oxLDL-induced endothelial dysfunction.
Methods: The activities of small GTP-binding proteins were assessed by pull-down assays in cultured human aortic endothelial cells (ECs). For inhibition of LOX-1, JTX92, a neutral antibody to LOX-1, was used and we inhibited Gi by pertussis toxin (PTX). MMPs were inhibited by tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2 and small-interfering RNA (siRNA). Immunofluorescent staining and immunoprecipitation were performed to investigate the association of LOX-1 and MT1-MMP.
Results: OxLDL activated RhoA and Rac1 within 5 and 15 minutes in cultured ECs, respectively, which were inhibited by JTX92 and PTX. TIMP-2 but not TIMP-1 also suppressed oxLDL-triggered RhoA and Rac1 activation. These results suggested that MT-MMPs but not secreted MMPs were essential for LOX-1/Gi-mediated RhoA and Rac1 activation. Thus we focused on MT1-MMP in the oxLDL-triggered signaling pathway. Inhibition of MT1-MMP prevented the oxLDL-induced RhoA and Rac1 activation, as well as the RhoA-dependent eNOS downregulation and Rac1-mediated ROS generation. Immunofluorescent staining demonstrated that LOX-1 and MT1-MMP were co-localized in cultured ECs. Importantly, immunoprecipitation revealed that MT1-MMP was associated with the LOX-1 in the cultured ECs as well as in the aortas of Watanabe heritable hyperlipidemic rabbit.
Conclusion: The present study provides novel evidence that MT1-MMP directly associates with LOX-1 and its MT1-MMP/LOX-1 axis regulates oxLDL-induced endothelial dysfunction mediated via the RhoA and Rac1 activation through Gi.