Abstract
CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser(133)) and ATF2 (Thr(71)) in a PKA-, PI3K-, and ERK-dependent fashion. Chromatin immunoprecipitation assays demonstrated that binding of phosphorylated CREB1 and ATF2 to cAMP-response element-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, PI3K, and ERK inhibition, whereas transfection of Creb1 and Atf2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated Creb1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of Cd39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator.
Highlights
CD39 is an integral membrane protein expressed on the surface of vascular and immune cells
In silico analysis of the Cd39 promoter region revealed, among potential regulatory sites, several Cyclic AMP (cAMP)-response element (CRE)2-like motifs, one of which was in a region proximal enough to the transcriptional start point to be of interest. cAMP, an essential second messenger that acts largely through its downstream effector, protein kinase A (PKA), regulates a diverse array of physiologic processes, ranging from metabolic control to cellular proliferation, by altering basic patterns of gene expression [10]. cAMP is known to be a critical regulator of vascular homeostasis that parallels the function of CD39 in regulating inflammation, coagulation, vasodilation, and barrier function
The binding of phosphorylated ATF2 to the CRE binding site decreased 62% (p Ͻ 0.0001), 72% (p Ͻ 0.0001), and 79% (p Ͻ 0.0001) in cells treated with 8-Br-cAMP and either H89, LY294002, or PD98059, compared with cells that were only treated with 8-Br-cAMP (Fig. 6B). These results indicate that the binding of phosphorylated CREB and phosphorylated ATF2 to the CRE binding site in 8-Br-cAMP-treated RAW cells closely involves the activities of PKA, phosphoinositide 3-kinase (PI3K), and ERK
Summary
CD39 (ectonucleoside triphosphate diphosphohydrolase 1) is an integral membrane protein expressed on the surface of vascular and immune cells. Once catalytic subunit translocation has occurred, it subsequently phosphorylates a serine residue of CREB (cAMP-response element-binding protein) at position 133 (Ser133), an important step for the activation and dimerization of CREB [11]. ATF2, whose known target genes include interferon-␥, tumor necrosis factor-␣, and cyclin A (16 –18), is activated upon its phosphorylation by stress-activated kinases in response to stress and cytokine stimuli [19, 20]. These are just several examples of the complex regulatory control possible for genes whose promoters contain CRE motifs
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