Abstract 11425: MT1-MMP Forms a Complex with Gi and Regulates eNOS Expression at Posttranscriptional Mechanism in Oxidized LDL Stimulated-endothelial Cells
Background: Matrix metalloproteinases (MMPs) are involved in pathogenesis of atherosclerosis. We recently have shown that silencing of membrane type1 MMP (MT1-MMP) suppressed the oxidized LDL (ox-LDL) triggered-downregulation of endothelial nitric oxide synthase (eNOS) and the production of reactive oxygen species in endothelial cells. However, it is still unknown about the detailed molecular mechanism of MT1-MMP-related eNOS regulation. In this study, we investigated the correlation between eNOS gene transcription and MT1-MMP as well as involvement of heterotrimec G protein in ox-LDL stimulated-endothelial cells.
Methods: Cultured human aortic endothelial cells were stimulated with ox-LDL. MT1-MMP expression was silenced by siRNA. The activity of MT1-MMP was evaluated by fluorescent assay. The level of eNOS mRNA was determined by RT-PCR and promoter activity of eNOS was evaluated by luciferase assay. GTP-loading of RhoA was assessed by pull-down assays. Pertussis toxin (PTX) was used for inhibition of Gi. Actin stress fibers assembly was demonstrated by falloidin staining. Association of Gi with MT1-MMP was detected by immunoprecipitation.
Results: eNOS protein expression and mRNA levels were downregulated by ox-LDL stimulation, which is inhibited by silencing of MT1-MMP. However, promoter activity of eNOS was not changed by neither ox-LDL stimulation nor MT1-MMP inhibition. These results indicated that eNOS regulation by ox-LDL or MT1-MMP was occurred at posttranscriptional level in endothelial cells. Ox-LDL induced rapid RhoA activation and actin stress fibers reorganization, which is inhibited by PTX and siRNA for MT1-MMP. Ox-LDL triggered-eNOS downregulation was suppressed by pretreatment of PTX. Furthermore, immunoprecipitation revealed that MT1-MMP formed a complex with Gi in endothelial cells.
Conclusions: We demonstrated for the first time that MT1-MMP is involved in eNOS regulation at posttranscriptional mechanism mediated via RhoA dependent-actin stress fibers reorganization in ox-LDL stimulated-endothelial cells. In addition, we provided a new finding that MT1-MMP associated with Gi in endothelial cells. MT1-MMP may be a new therapeutic target for treating endothelial dysfunction.
- © 2010 by American Heart Association, Inc.