Abstract 20370: Fibrinolytic Regulation by CD39: Transcriptional Attenuation of Hypoxic-PAI-1 Induction Through c-Myc/Hif-1α Competition
CD39 is an ectonucleotidase that regulates platelet function by phosphohydrolyzing ATP and ADP. In the setting of ischemic injury, CD39 may also maintain blood fluidity by promoting fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) is known to suppress fibrinolysis under conditions of hypoxia. We hypothesized that CD39 might down-regulate PAI-1 expression and function, thus promoting a fibrinolytic milieu. RAW 264.7 macrophages were transfected with either a CD39 overexpression construct, an overexpression control vector, a CD39 shRNA construct, or a shRNA control vector. The transfected RAW cells were then incubated in either hypoxia or normoxia for 16 hours, and PAI-1 mRNA, protein and function were measured. Data were all compared to the cells transfected with control vectors.
Results: showed that CD39 overexpression caused PAI-1 mRNA, protein, and activity to be decreased by 87%, 98%, and 75% respectively in normoxia, and by 43%, 98%, and 84% respectively in hypoxia. In contrast, when CD39 was silenced, CD39-deficient cells showed a 13-, 12-, and 1.3-fold increase in PAI-1 mRNA, protein, and activity respectively in normoxia, and a 2-, 15-, and 1.2-fold increase respectively in hypoxia. To explore mechanisms by which CD39 overexpression could regulate PAI-1 transcription, CD39-overexpressing RAW cells were transfected with PAI-1 promoter-luciferase report constructs; CD39 overexpression caused luciferase activity to decrease by 55% in hypoxia. ChIP analysis revealed that CD39 overexpression caused a 61% decrease in HIF1α binding to the PAI-1 promoter, whereas c-MYC binding to the PAI-1 promoter increased 4.12-fold. This result also coincided with an increase of c-MYC protein in nuclei of CD39 overexpressing cells. After 5 hours of treatment with the c-MYC inhibitor 10058-F4 (50 μM), PAI-1 mRNA was increased by 117-fold in CD39-overexpressing cells. (p < 0.05 for all reported data). These data are the first to identify CD39 as an important negative regulator of macrophage PAI-1 expression via the upregulation of c-MYC, which competes with HIF-1α binding to the PAI-1 promoter. Thus in addition to its known anti-platelet effects, CD39 may sustain blood fluidity by a previously unexpected perturbation in fibrinolytic regulation.
- © 2010 by American Heart Association, Inc.