Kou Ruqin, Michel Thomas
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
J Biol Chem. 2007 Nov 9;282(45):32719-29. doi: 10.1074/jbc.M706815200. Epub 2007 Sep 12.
beta-Adrenergic receptors (betaAR) play an important role in vasodilation, but the mechanisms whereby adrenergic pathways regulate the endothelial isoform of nitric-oxide synthase (eNOS) are incompletely understood. We found that epinephrine significantly increases eNOS activity in cultured bovine aortic endothelial cells (BAEC). Epinephrine-dependent eNOS activation was accompanied by an increase in phosphorylation of eNOS at Ser(1179) and with decreased eNOS phosphorylation at the inhibitory phosphoresidues Ser(116) and Thr(497). Epinephrine promoted activation of the small G protein Rac1 and also led to the activation of protein kinase A. All of these responses to epinephrine in BAEC were blocked by the beta(3)AR blocker SR59230A. We transfected and validated duplex small interfering RNA (siRNA) constructs to selectively "knock down" specific signaling proteins in BAEC. siRNA-mediated knockdown of Rac1 completely blocked all beta(3)AR signaling to eNOS and also abrogated epinephrine-dependent cAMP-dependent protein kinase (PKA) and Akt activation. However, siRNA-mediated knockdown of PKA did not affect Rac1 activation by epinephrine but did attenuate Akt activation by epinephrine. These findings indicate that Rac1 is an upstream regulator of beta(3)AR signaling to PKA and to eNOS and identify a novel beta(3)AR --> Rac1 --> PKA --> Akt pathway in endothelium. We exploited the p21-activated kinase pulldown assay to identify proteins associated with activated Rac1 and found that epinephrine stimulated the association of eNOS with Rac1; epinephrine-stimulated eNOS-Rac1 interactions were blocked by the beta(3)AR antagonist SR59230A. Co-transfection of eNOS cDNA with constitutively active Rac1 enhanced beta(3)AR-promoted eNOS-Rac1 association; co-transfection of eNOS with dominant negative Rac1 completely blocked the eNOS-Rac1 association. We also found that epinephrine-induced Rac1 --> PKA --> Akt pathway mediates beta(3)AR-mediated endothelial cell migration. Taken together, our data establish that the small G protein Rac1 is a key regulator of beta(3)AR signaling in cultured aortic endothelial cells with potentially important implications for the pathways involved in adrenergic modulation of eNOS pathways in the vascular wall.
β-肾上腺素能受体（βAR）在血管舒张中起重要作用，但肾上腺素能途径调节一氧化氮合酶（eNOS）内皮型的机制尚未完全明确。我们发现，肾上腺素可显著增加培养的牛主动脉内皮细胞（BAEC）中eNOS的活性。肾上腺素依赖性eNOS激活伴随着eNOS在Ser（1179）位点磷酸化增加，而在抑制性磷酸化位点Ser（116）和Thr（497）处的eNOS磷酸化减少。肾上腺素促进小G蛋白Rac1的激活，还导致蛋白激酶A的激活。BAEC中对肾上腺素的所有这些反应均被β（3）AR阻滞剂SR59230A阻断。我们转染并验证了双链小干扰RNA（siRNA）构建体，以选择性地“敲低”BAEC中的特定信号蛋白。siRNA介导的Rac1敲低完全阻断了所有β（3）AR向eNOS的信号传导，还消除了肾上腺素依赖性cAMP依赖性蛋白激酶（PKA）和Akt的激活。然而，siRNA介导的PKA敲低并不影响肾上腺素对Rac1的激活，但确实减弱了肾上腺素对Akt的激活。这些发现表明，Rac1是β（3）AR向PKA和eNOS信号传导的上游调节因子，并确定了内皮中一条新的β（3）AR→Rac1→PKA→Akt途径。我们利用p21激活激酶下拉试验来鉴定与活化Rac1相关的蛋白，发现肾上腺素刺激eNOS与Rac1的结合；肾上腺素刺激的eNOS-Rac1相互作用被β（3）AR拮抗剂SR59230A阻断。将eNOS cDNA与组成型活性Rac1共转染可增强β（3）AR促进的eNOS-Rac1结合；将eNOS与显性负性Rac1共转染则完全阻断eNOS-Rac1结合。我们还发现，肾上腺素诱导的Rac1→PKA→Akt途径介导β（3）AR介导的内皮细胞迁移。综上所述，我们的数据表明，小G蛋白Rac1是培养的主动脉内皮细胞中β（3）AR信号传导的关键调节因子，对血管壁中肾上腺素能调节eNOS途径所涉及的通路具有潜在重要意义。
