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: Rong-rong Tao, Ji-Yun Huang, Xue-jing Shao, Wei-feng Ye, Yun Tian, Mei-hua Liao, Kohji Fukunaga, Yi-jia Lou, Feng Han, Ying-mei Lu
• Journal: Journal of pineal research (J Pineal Res) Vol. 54 Issue 3 Pg. 271-81 (Apr 2013) ISSN: 1600-079X [Electronic] England
• PMID: 22946793 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2012 John Wiley & Sons A/S.

Chemical References

• Antioxidants
• Intracellular Signaling Peptides and Proteins
• KEAP1 protein, human
• Kelch-Like ECH-Associated Protein 1
• NF-E2-Related Factor 2
• NFE2L2 protein, human
• Nitrates
• 3-nitrotyrosine
• Tyrosine
• Glucose
• Melatonin
• Oxygen

Topics

• Analysis of Variance
• Antioxidants (metabolism, pharmacology)
• Cell Line
• Endothelial Cells (drug effects, metabolism)
• Glucose (metabolism)
• Histocytochemistry
• Humans
• Intracellular Signaling Peptides and Proteins (metabolism)
• Ischemia (metabolism)
• Kelch-Like ECH-Associated Protein 1
• Melatonin (pharmacology)
• Microscopy, Fluorescence
• NF-E2-Related Factor 2 (metabolism)
• Nitrates (metabolism)
• Oxygen (metabolism)
• Stress, Physiological (drug effects, physiology)
• Tyrosine (analogs & derivatives, metabolism)