Abstract 19436: cAMP Regulation of Vascular Endothelial Ectoenzyme ENTPD1

Abstract

Ectonucleoside triphosphate diphosphohydrolase 1 (or CD39), an enzyme expressed on the plasmalemma of leukocytes and endothelial cells, serves as a potent antithrombotic agent by sequentially phosphohydrolyzing ATP and ADP, to generate AMP. CD39 is thus able to locally deplete levels of extracellular ATP and ADP, which would otherwise promote inflammation and platelet aggregation by binding P2X and P2Y classes of purinergic receptors found on leukocytes and platelets. The only known pathway for the transcriptional regulation of CD39 was described in recent work by this lab, which demonstrated that increased cAMP levels led to CD39 upregulation in murine macrophages through binding to the cAMP response elements (CRE) in the CD39 promoter with a consequent increase in CD39 transcription. Here, in contrast, we show that human endothelial cells (from umbilical vein; HUVEC) express less CD39 in response to elevated cAMP levels. Furthermore, we demonstrate that blockade of a major cAMP effector protein, protein kinase A (PKA), restores CD39 expression. Treatment of HUVEC cells with 250 μM 8-Br-cAMP, a cell permeable analogue of cAMP, led to a 2-fold decrease in CD39 protein from whole cell lysates in Western blots (18 hrs, p<0.001, n=5), and this pattern persisted in time course assays from 8 hours to 24 hours. This result was confirmed with immunoprecipitation for CD39 in HUVEC supernatants, where 8 hours of 8-Br-cAMP treatment at 250 μM led to a 2.2-fold decrease in CD39 protein. ELISA measurements of HUVEC treated with 20 μM of the PKA inhibitor H89 led to a 2.8-fold increase compared to control of CD39 protein from HUVEC supernatants (p=0.0021, n=6). This was confirmed by Western blot, where co-treatment with 250 μM 8-Br-cAMP and 20 μM H89 for 8 hours led to a 50% increase measured by Western blot in CD39 protein compared to control in whole cell lysates (p<0.05, n=3). Together, these data demonstrate that endothelial expression of CD39, a potent antithrombotic and anti-inflammatory enzyme, is modulated by the cAMP/PKA pathway, and contributes to basic understanding of the regulatory pathways governing CD39.