title: Regulation of ecto-apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3).
abstract: The ectoenzyme CD39 suppresses thrombosis and inflammation by suppressing ATP and ADP to AMP. However, mechanisms of CD39 transcriptional and post-translational regulation are not well known. Here we show that CD39 levels are modulated by inhibition of phosphodiesterase 3 (PDE3). RAW macrophages and human umbilical vein endothelial cells (HUVECs) were treated with the PDE3 inhibitors cilostazol and milrinone, then analyzed using qRT-PCR, immunoprecipitation/Western blot, immunofluorescent staining, radio-thin-layer chromatography, a malachite green assay, and ELISA. HUVECs expressed elevated CD39 protein (2-fold [P<0.05] for cilostazol and 2.5-fold [P<0.01] for milrinone), while macrophage CD39 mRNA and protein were both elevated after PDE3 inhibition. HUVEC ATPase activity increased by 25% with cilostazol and milrinone treatment (P<0.05 and P<0.01, respectively), as did ADPase activity (47% and 61%, P<0.001). There was also a dose-dependent elevation of soluble CD39 after treatment with 8-Br-cAMP, with maximal elevation of 60% more CD39 present compared to controls (1 mM, P<0.001). Protein harvested after 8-Br-cAMP treatment showed that ubiquitination of CD39 was decreased by 43% compared to controls. A DMSO or PBS vehicle control was included for each experiment based on solubility of cilostazol, milrinone, and 8-Br-cAMP. These results indicate that PDE3 inhibition regulates endothelial CD39 at a post-translational level.
authors.affiliation: 17240 Medical Science Research Bldg. III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA. abaek@umich.edu.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Baek
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Kanthi
authors.forename: Nadia R
authors.initials: NR
authors.lastname: Sutton
authors.forename: Hui
authors.initials: H
authors.lastname: Liao
authors.forename: David J
authors.initials: DJ
authors.lastname: Pinsky
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics*; D001081:Apyrase / Q000235:genetics*; D000077407:Cilostazol; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D020105:Milrinone / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D054875:Ubiquitination
chemical_list: D015703:Antigens, CD; D004791:Enzyme Inhibitors; D013777:Tetrazoles; D015124:8-Bromo Cyclic Adenosine Monophosphate; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen; D020105:Milrinone; D000077407:Cilostazol
keywords: cAMP; endothelium; vascular homeostasis
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