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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

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
Authors
Seo, JungwonCho, Du-HyongLee, Hyeon-JuSung, Min-SunLee, Jee YoungWon, Kyung-JongPark, Jung-HyunJo, Inho
Ewha Authors
조인호서정원박정현이현주이지영
SCOPUS Author ID
조인호scopus; 이현주scopus; 이지영scopusscopus
Issue Date
2016
Journal Title
FREE RADICAL BIOLOGY AND MEDICINE
ISSN
0891-5849JCR Link

1873-4596JCR Link
Citation
FREE RADICAL BIOLOGY AND MEDICINE vol. 90, pp. 133 - 144
Keywords
ArseniteVascular diseaseNitric oxideEndothelial nitric oxide synthasePhosphorylationRhoCitron Rho-interacting kinaseEx vivo vessel relaxation
Publisher
ELSEVIER SCIENCE INC
Indexed
SCI; SCIE; SCOPUS WOS scopus
Document Type
Article
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. (C) 2015 Elsevier Inc. All rights reserved.
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DOI
10.1016/j.freeradbiomed.2015.11.020
Appears in Collections:
의과대학 > 의학과 > Journal papers
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