Dubey Raghvendra K, Fingerle Jürgen, Gillespie Delbert G, Mi Zaichuan, Rosselli Marinella, Imthurn Bruno, Jackson Edwin K
From the Department of Reproductive Endocrinology, University Hospital Zurich, Switzerland (R.K.D., M.R., B.I.); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Switzerland (R.K.D.); Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine (D.G.G., Z.M., E.K.J.); and Preclinical Pharma Research 68/209, F. Hoffmann-La-Roche, Basel, Switzerland (J.F.).
Hypertension. 2015 Dec;66(6):1207-19. doi: 10.1161/HYPERTENSIONAHA.115.05912. Epub 2015 Sep 28.
The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression.
本研究的目的是确定腺苷是否以及如何影响人冠状动脉平滑肌细胞（HCASMCs）的增殖。在HCASMCs中，2-氯腺苷（稳定的腺苷类似物）而非N(6)-环戊基腺苷、CGS21680或N(6)-(3-碘苄基)-腺苷-5'-N-甲基脲酰胺可抑制HCASMC增殖（A2B受体特征）。2-氯腺苷增加cAMP，降低ERK和Akt的磷酸化（激活）（已知分别增加细胞周期蛋白D表达和活性的蛋白激酶），并降低细胞周期蛋白D1的水平（促进G1期细胞周期进程的细胞周期蛋白）。此外，2-氯腺苷抑制S期激酶相关蛋白2（Skp2；促进p27(Kip1)的蛋白水解）的表达并上调p27(Kip1)的水平（损害细胞周期蛋白D功能的细胞周期调节因子）。2-氯腺苷还抑制细胞周期蛋白D下游的信号传导，包括视网膜母细胞瘤蛋白的过度磷酸化和细胞周期蛋白A（S期细胞周期蛋白）的表达。敲低A2B受体可阻止2-氯腺苷对ERK1/2、Akt、Skp2、p27(Kip1)、细胞周期蛋白D1、细胞周期蛋白A和增殖的影响。同样，抑制腺苷酸环化酶和蛋白激酶A可消除2-氯腺苷对Skp2的抑制作用以及对p27(Kip1)的刺激作用，并使HCASMCs免受2-氯腺苷介导的抑制。敲低p27(Kip1)也可逆转2-氯腺苷对HCASMC增殖的抑制作用。在体内，动脉周围（大鼠颈动脉）给予2-氯腺苷（20 μmol/L，持续7天）可下调Skp2的血管表达，上调p27(Kip1)的血管表达，并使新生内膜增生减少71%（P<0.05；新生内膜厚度：对照组，37424±18371像素；处理组，10352±2824像素）。总之，腺苷/A2B受体/cAMP/蛋白激酶A轴通过阻断多条在细胞周期蛋白D处汇聚的信号通路（ERK1/2、Akt和Skp2）来抑制HCASMC增殖，细胞周期蛋白D是控制细胞周期进程的关键G1期细胞周期蛋白。
