CCN1 acutely increases nitric oxide production via integrin αvβ3–Akt–S6K–phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis

• Published in: Free radical biology & medicine Vol. 89; pp. 229 - 240
• Main Authors: Hwang, Soojin, Lee, Hyeon-Ju, Kim, Gyungah, Won, Kyung-Jong, Park, Yoon Shin, Jo, Inho
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2015
• Subjects: Akt; Animals; Aorta - cytology; Aorta - drug effects; Aorta - metabolism; Blotting, Western; Cattle; CCN1; Cells, Cultured; Cysteine-Rich Protein 61 - pharmacology; Endothelial nitric oxide synthase; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Free radicals; Human Umbilical Vein Endothelial Cells - cytology; Human Umbilical Vein Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Integrin; Integrin alphaVbeta3 - metabolism; Male; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Phosphorylation - drug effects; Proto-Oncogene Proteins c-akt - metabolism; Rats; Rats, Sprague-Dawley; Retina - cytology; Retina - drug effects; Retina - metabolism; Ribosomal Protein S6 Kinases - metabolism; S6K; Serine - chemistry; Serine - metabolism; Vasodilation; Vasodilator Agents - pharmacology; Endothelial nitric oxide synthase; Phosphorylation; Integrin; Free radicals; Nitric oxide; S6K; Akt; CCN1; Vasodilation
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.08.005

Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser1177), but not that of eNOS-Thr495 or eNOS-Ser114. The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser1177 phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser473 and S6K-Thr389. However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser1177 phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser1177 phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3–Akt–S6K–eNOS-Ser1177 phosphorylation, suggesting an important role for CCN1 in vasodilation. •CCN1 acutely increases p-eNOS-Ser1177 and NO production in HUVECs.•Integrin αvβ3, Akt, and S6 kinase mediate CCN1-stimulated p-eNOS-Ser1177 and NO production.•VEGF is unlikely to be involved in CCN1-stimulated p-eNOS-Ser1177 and NO production.•CCN1 increases acetylcholine-induced aortic relaxation in an isolated rat aorta ex vivo model.•CCN1-stimulated p-eNOS-Ser1177 and NO production are true for other types of EC, HRMECs and BAECs.