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FASEB J. 2013 Nov;27(11):4419-28. doi: 10.1096/fj.13-234625. Epub 2013 Jul 30.
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.
细胞外酶 CD39 通过抑制 ATP 和 ADP 转化为 AMP 来抑制血栓形成和炎症。然而，CD39 的转录和翻译后调节机制尚不清楚。本文中，我们发现 CD39 水平受磷酸二酯酶 3（PDE3）抑制剂的调节。用 PDE3 抑制剂西洛他唑和米力农处理 RAW 巨噬细胞和人脐静脉内皮细胞（HUVEC），然后用 qRT-PCR、免疫沉淀/Western blot、免疫荧光染色、放射性薄层色谱、孔雀绿法和 ELISA 进行分析。HUVEC 表达高水平的 CD39 蛋白（西洛他唑增加 2 倍[P<0.05]，米力农增加 2.5 倍[P<0.01]），而巨噬细胞 CD39 mRNA 和蛋白在 PDE3 抑制后均升高。西洛他唑和米力农处理后 HUVEC 的 ATP 酶活性分别增加 25%（P<0.05 和 P<0.01），ADP 酶活性分别增加 47%和 61%（P<0.001）。用 8-Br-cAMP 处理后可溶性 CD39 也呈剂量依赖性增加，与对照组相比增加了 60%（1 mM，P<0.001）。8-Br-cAMP 处理后的蛋白显示 CD39 的泛素化减少了 43%，与对照组相比。根据西洛他唑、米力农和 8-Br-cAMP 的溶解度，每个实验都包含 DMSO 或 PBS 溶剂对照。这些结果表明，PDE3 抑制在翻译后水平上调节内皮细胞 CD39。
