Abstract 524: Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) expression in macrophages by cAMP via PKA/PI3K/CREB
CD39, a transmembrane enzyme, inhibits platelet reactivity and inflammation by catalyzing the phosphohydrolysis of ATP and ADP. Cyclic AMP (cAMP), an essential second messenger, regulates a number of RNA transcriptional events through the Protein Kinase A (PKA)/Phosphoinositide-3 kinase (Pi3K)/cAMP response element binding protein (CREB) pathway. In silico analysis revealed a cAMP Response Element (CRE) in the CD39 promoter, which led to the hypothesis that cAMP might regulate CD39 expression. The murine macrophage cell line RAW was pretreated with PKA inhibitors (Rp-cAMPS 10 uM, or H89 20 uM), or a Pi3K inhibitor (LY294002, LY, 50 uM) for 30 min., plus a membrane permeant cAMP analogue, 8-Bromo-cAMP 250 uM. Immunohistochemistry showed (and Western blot confirmed) that CD39 expression was greatly increased in cells treated with 8-Br-cAMP compared to untreated cells or cells co-incubated with 8-Br-cAMP plus Rp-cAMPS. RT-qPCR showed that CD39 mRNA was induced 138 fold in RAW cells treated with 8-Br-cAMP, compared to untreated controls (p < 0.001). Pretreated with H89 or LY resulted in 95% reduction of 8-Br-cAMP-induced CD39 mRNA expression (p < 0.001 for both). Luciferase activity of the RAW cells transfected with CD39 promoter was increased 10.4 fold in cells treated with 8-Br-cAMP, compared to untreated controls (p < 0.001). Luciferase activity was reduced 65% in RAW cells pretreated with H89 or LY compared to RAW cells treated only with 8-Br-cAMP (p < 0.001). After treatment with 8-Br-cAMP, luciferase activity of the RAW cells transfected with a CRE site mutation plasmid was reduced 83% compared to cells transfected with wild type CD39 promoter (p < 0.001). The higher CREB binding activity to consensus CRE probe was detected by EMSA in nuclear extracts from the RAW cells treated with 8-Br-cAMP, compared to the RAW cells pretreated with H89 or LY, and untreated control. Preincubation of 8-Br-cAMP treated RAW cell nuclear extracts with anti-CREB antibody blocked by > 50% the binding activity of CREB to CRE probe. These data identify cAMP as a potentially important regulator of macrophage CD39 expression through the PKA/Pi3K/CREB pathway. These data reveal the molecular pathways underpinning transcriptional regulation of a key vascular homeostatic mediator.