title: Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin.
abstract: Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S-nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen-glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear-localized Nrf2, as demonstrated through co-immunoprecipitation and immunocytochemical assay. Paralleling the ischemia-induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD-induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S-nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S-nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO-1 expression, paralleled with the restored heme oxygenase-1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia-evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.
authors.affiliation: Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, China.
authors.forename: Rong-rong
authors.initials: RR
authors.lastname: Tao
authors.forename: Ji-Yun
authors.initials: JY
authors.lastname: Huang
authors.forename: Xue-jing
authors.initials: XJ
authors.lastname: Shao
authors.forename: Wei-feng
authors.initials: WF
authors.lastname: Ye
authors.forename: Yun
authors.initials: Y
authors.lastname: Tian
authors.forename: Mei-hua
authors.initials: MH
authors.lastname: Liao
authors.forename: Kohji
authors.initials: K
authors.lastname: Fukunaga
authors.forename: Yi-jia
authors.initials: YJ
authors.lastname: Lou
authors.forename: Feng
authors.initials: F
authors.lastname: Han
authors.forename: Ying-mei
authors.initials: YM
authors.lastname: Lu
mesh_terms: D000704:Analysis of Variance; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006651:Histocytochemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007511:Ischemia / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008550:Melatonin / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects* / Q000502:physiology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000975:Antioxidants; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D009566:Nitrates; C002744:3-nitrotyrosine; D014443:Tyrosine; D005947:Glucose; D008550:Melatonin; D010100:Oxygen