title: CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.
abstract: 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-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). 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-Ser(1177) 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-Ser(473) and S6K-Thr(389). 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-Ser(1177) 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-Ser(1177) 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-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.
authors.affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea.
authors.forename: Soojin
authors.initials: S
authors.lastname: Hwang
authors.affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea.
authors.forename: Hyeon-Ju
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea.
authors.forename: Gyungah
authors.initials: G
authors.lastname: Kim
authors.affiliation: Department of Medical Science, School of Medicine, Konkuk University, Chungju 380-701, Republic of Korea.
authors.forename: Kyung-Jong
authors.initials: KJ
authors.lastname: Won
authors.affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea.
authors.forename: Yoon Shin
authors.initials: YS
authors.lastname: Park
authors.affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea. Electronic address: inhojo@ewha.ac.kr.
authors.forename: Inho
authors.initials: I
authors.lastname: Jo
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D055515:Cysteine-Rich Protein 61 / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D039302:Integrin alphaVbeta3; D014665:Vasodilator Agents; D009569:Nitric Oxide; D012694:Serine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases
keywords: Akt; CCN1; Endothelial nitric oxide synthase; Free radicals; Integrin; Nitric oxide; Phosphorylation; S6K; Vasodilation