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PMID: 20178980 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

cAMP/CREB-mediated transcriptional regulation of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) expression.

The Journal of biological chemistry ·Vol. 285 ·No. 19 ·2010-05-07 ·Pages 14791-805

Liao H, Hyman MC, Baek AE, Fukase K, Pinsky DJ

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.

MeSH Terms
Activating Transcription Factor 2/genetics,metabolism Animals Antigens, CD/genetics,metabolism Apyrase/genetics,metabolism Blotting, Western Cells, Cultured Chromatin Immunoprecipitation Cyclic AMP/metabolism Cyclic AMP Response Element-Binding Protein/antagonists & inhibitors,genetics,metabolism Cyclic AMP-Dependent Protein Kinases/metabolism Flow Cytometry Fluorescent Antibody Technique Gene Expression Regulation Macrophages, Peritoneal/cytology,metabolism Male Mice Mice, Inbred C57BL Mutagenesis, Site-Directed Phosphorylation Promoter Regions, Genetic/genetics RNA, Messenger/genetics,metabolism Rats Reverse Transcriptase Polymerase Chain Reaction Signal Transduction
Chemicals
Activating Transcription Factor 2 Antigens, CD Atf2 protein, mouse Creb1 protein, mouse Cyclic AMP Response Element-Binding Protein RNA, Messenger Cyclic AMP Cyclic AMP-Dependent Protein Kinases Apyrase CD39 antigen
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Liao Hui
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Hyman Matthew C
Baek Amy E
Fukase Keigo
Pinsky David J
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
1083-351X
DOI
10.1074/jbc.M110.116905
Published
2010-05-07
Epub
2010-00-23
Pages
14791-805
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2863166
Subset
IM
Grants
NHLBI NIH HHS · R01HL085149 · United States
NHLBI NIH HHS · R01HL055397 · United States
NHLBI NIH HHS · R01 HL085149 · United States
NHLBI NIH HHS · R01 HL086676 · United States
NHLBI NIH HHS · P01HL089407 · United States
NHLBI NIH HHS · R01 HL055397 · United States
NHLBI NIH HHS · R01HL086676 · United States
NHLBI NIH HHS · P01 HL089407 · United States
External Links
PubMed source DOI: 10.1074/jbc.M110.116905 PMC Full Text View journal details
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