title: Citron Rho-interacting kinase mediates arsenite-induced decrease in endothelial nitric oxide synthase activity by increasing phosphorylation at threonine 497: Mechanism underlying arsenite-induced vascular dysfunction.
abstract: We reported that arsenite causes an acute decrease in nitric oxide (NO) production by increasing phosphorylation of endothelial NO synthase at threonine 497 (eNOS-Thr(497)); however, the detailed mechanism has not yet been clarified. Here, we investigated the kinase involving in arsenite-stimulated eNOS-Thr(497) phosphorylation. Although treatment with H-89, a known protein kinase A (PKA) inhibitor, inhibited arsenite-stimulated eNOS-Thr(497) phosphorylation, no inhibition was found in cells treated with other PKA inhibitors, including Rp-8-Br-cAMPS or PKI. Based on previous reports, we also tested whether RhoA mediates arsenite-stimulated eNOS-Thr(497) phosphorylation and found that arsenite causes an acute increase in RhoA activity. Ectopic expression of dominant negative (DN)-RhoA significantly reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. An in vitro phosphorylation assay also revealed that the well-known Rho effectors, Rho-associated protein kinase 1/2 (ROCK1/2), directly phosphorylate eNOS-Thr(497). Y27632, a selective ROCK inhibitor, reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. However, overexpression of a small interfering RNA (siRNA) against ROCK1/2 or DN-ROCK did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation, thereby providing no conclusive evidence of a role for ROCK1/2. Knockdown of PKC-related protein kinase 1/2, another Rho effector, also did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, we found that transfection with an siRNA against citron Rho-interacting kinase (CRIK), the other downstream effector of Rho, significantly reversed the arsenite-induced eNOS-Thr(497) phosphorylation that was accompanied by restoration of eNOS enzymatic activity repressed by arsenite. Moreover, CRIK directly phosphorylated eNOS-Thr(497)in vitro. Finally, we also found that arsenite increased eNOS-Thr(497) phosphorylation and decreased acetylcholine-induced vessel relaxation in rat aortas. In conclusion, we demonstrate that arsenite acutely inhibits eNOS enzymatic activity and vessel relaxation in part by increasing the RhoA/CRIK/eNOS-Thr(497) phosphorylation signaling axis, which provides a molecular mechanism underlying arsenite-induced impaired vascular diseases.
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Yangcheon-gu, Seoul 158-710, South Korea; Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan, Chonbuk, South Korea.
authors.forename: Jungwon
authors.initials: J
authors.lastname: Seo
authors.affiliation: Department of Pharmacology, School of Medicine, Eulji University, Jung-gu, Daejeon 301-746, South Korea.
authors.forename: Du-Hyong
authors.initials: DH
authors.lastname: Cho
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Yangcheon-gu, Seoul 158-710, South Korea.
authors.forename: Hyeon-Ju
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Yangcheon-gu, Seoul 158-710, South Korea.
authors.forename: Min-Sun
authors.initials: MS
authors.lastname: Sung
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Yangcheon-gu, Seoul 158-710, South Korea.
authors.forename: Jee Young
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Department of Medical Science, School of Medicine, Konkuk University, Chungju 380-701, South Korea.
authors.forename: Kyung-Jong
authors.initials: KJ
authors.lastname: Won
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Yangcheon-gu, Seoul 158-710, South Korea.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Yangcheon-gu, Seoul 158-710, South Korea. Electronic address: inhojo@ewha.ac.kr.
authors.forename: Inho
authors.initials: I
authors.lastname: Jo
mesh_terms: D000818:Animals; D018053:Arsenites / Q000633:toxicity*; D002417:Cattle; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D042783:Endothelial Cells / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014652:Vascular Diseases / Q000139:chemically induced*; D014664:Vasodilation / Q000187:drug effects; D054460:rho-Associated Kinases / Q000502:physiology
chemical_list: D018053:Arsenites; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines; D013449:Sulfonamides; D013912:Threonine; D052250:Nitric Oxide Synthase Type III; C115635:citron-kinase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C015001:arsenite
keywords: Arsenite; Citron Rho-interacting kinase; Endothelial nitric oxide synthase; Ex vivo vessel relaxation; Nitric oxide; Phosphorylation; Rho; Vascular disease