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
- 주제(키워드) Arsenite , Citron Rho-interactingkinase , Endothelial nitricoxidesynthase , Ex vivo vessel relaxation , Nitric oxide , Phosphorylation , Rho , Vascular disease
- 등재 SCIE, SCOPUS
- 발행기관 Elsevier Inc.
- 발행년도 2016
- 총서유형 Journal
- URI http://www.dcollection.net/handler/ewha/000000120581
- 본문언어 영어
- Published As http://dx.doi.org/10.1016/j.freeradbiomed.2015.11.020
- 저작권 이화여자대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
We reported that arsenite causes an acute decrease in nitric oxide (NO) production by increasing phosphorylation of endothelial NO synthase at threonine 497 (eNOS-Thr497); however, the detailed mechanism has not yet been clarified. Here, we investigated the kinase involving in arsenite-stimulated eNOS-Thr497 phosphorylation. Although treatment with H-89, a known protein kinase A (PKA) inhibitor, inhibited arsenite-stimulated eNOS-Thr497 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-Thr497 phosphorylation and found that arsenite causes an acute increase in RhoA activity. Ectopic expression of dominant negative (DN)-RhoA significantly reversed arsenite-stimulated eNOS-Thr497 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-Thr497. Y27632, a selective ROCK inhibitor, reversed arsenite-stimulated eNOS-Thr497 phosphorylation. However, overexpression of a small interfering RNA (siRNA) against ROCK1/2 or DN-ROCK did not reverse arsenite-stimulated eNOS-Thr497 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-Thr497 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-Thr497 phosphorylation that was accompanied by restoration of eNOS enzymatic activity repressed by arsenite. Moreover, CRIK directly phosphorylated eNOS-Thr497 in vitro. Finally, we also found that arsenite increased eNOS-Thr497 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-Thr497 phosphorylation signaling axis, which provides a molecular mechanism underlying arsenite-induced impaired vascular diseases. © 2015 Elsevier Inc. All rights reserved.
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