Abstract 19209: Ectonucleoside Triphosphate Diphosphohydrolase-1 (cd39) Attenuates Expression f of P-Selectin Glycoprotein Ligand-1 in Hypoxic Raw Cells via Hif-1α
CD39 is a transmembrane enzyme that phosphohydrolyzes ATP and ADP to AMP. CD39 inhibits platelet reactivity and inflammation and thus limits platelet accumulation and leukocyte transmigration to sites of inflammation or injury. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein on the surface of leukocytes which binds with high affinity to P-selectin, to promote tethering and rolling adhesion of leukocytes to activated endothelial cells and platelets. As this leads to increased leukocyte migration to sites of inflammation or injury, we hypothesized that CD39 may regulate PSGL-1 expression on leukocytes under hypoxic conditions. Murine RAW macrophages transfected with a CD39 overexpression construct (CD39Tx) or a pcDNA3.1 vector (VTx) as control were incubated in hypoxia (0.5% O2) or nomoxia. Subsequent flow cytometry showed 35% more cells expressed PSGL-1 after 6 hours of exposure to hypoxia (H) on VTx cells compared to normoxic (N) VTx control (p < 0.0001). The number of cells expressing PSGL-1 was reduced by 14% in H CD39Tx cells compared with H VTx cells (p = 0.0061). Western blotting showed PSGL-1 antigen expression increased 4.9, 7.0 and 3.9 fold after 2, 6, and 16 hours of hypoxia in VTx cells, respectively, compared to N VTx controls; and PSGL-1 antigen was also attenuated 19%, 30%, and 28% respectively in H CD39Tx cells compared with H VTx cells. Quantitative real-time PCR results showed that PSGL-1 mRNA expression increased by 2.1 fold in 6 hours H VTX cells compared with N VTx cells (p < 0.0001) and decreased 40% in H CD39Tx cells compared with H VTx control (p < 0.0001). The PSGL-1 promoter region contains 3 HIF-1α response elements (HRE). The activity of PSGL-1 promoter increased by 1.9-fold in 6 hours H VTx cells compared with N VTx controls (p < 0.0001) and decreased by 61% in H CD39Tx cells compared with H VTx controls (p < 0.0001). HIF-1α protein expression was also reduced in nuclear protein samples of H CD39Tx cells compared to H VTx cells. These data identify CD39 as a critical regulator of macrophage PSGL-1 expression, acting through the HIF-1alpha pathway to squelch inflammatory cell trafficking under hypoxic/ischemic conditions.
- © 2012 by American Heart Association, Inc.