Regulation of ecto-apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3)

Amy E. Baek, Yogendra Kanthi, Nadia R. Sutton, Hui Liao, David J. Pinsky

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)4419-4428
Number of pages10
JournalFASEB Journal
Volume27
Issue number11
DOIs
StatePublished - Nov 2013

Keywords

  • CAMP
  • Endothelium
  • Vascular homeostasis

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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