Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells.

Abstract

Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤ 20 Gy) and time-dependent (6-72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-L-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.

• Authors: Kimimasa Sakata, Takashi Kondo, Natsumi Mizuno, Miki Shoji, Hironobu Yasui, Tohru Yamamori, Osamu Inanami, Hiroki Yokoo, Naoki Yoshimura, Yuichi Hattori
• Journal: Vascular pharmacology (Vascul Pharmacol) Vol. 70 Pg. 55-65 (Jul 2015) ISSN: 1879-3649 [Electronic] United States
• PMID: 25869503 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Antioxidants
• Protein Kinase Inhibitors
• Reactive Oxygen Species
• NOS3 protein, human
• Nitric Oxide Synthase Type III
• Proto-Oncogene Proteins c-akt
• PRKCB protein, human
• Protein Kinase C beta
• Extracellular Signal-Regulated MAP Kinases

Topics

• Antioxidants (pharmacology)
• Cells, Cultured
• Dose-Response Relationship, Radiation
• Enzyme Activation
• Extracellular Signal-Regulated MAP Kinases (metabolism)
• Human Umbilical Vein Endothelial Cells (drug effects, enzymology, radiation effects)
• Humans
• Nitric Oxide Synthase Type III (metabolism)
• Phosphorylation
• Protein Kinase C beta (antagonists & inhibitors, genetics, metabolism)
• Protein Kinase Inhibitors (pharmacology)
• Proto-Oncogene Proteins c-akt (metabolism)
• RNA Interference
• Reactive Oxygen Species (metabolism)
• Signal Transduction (drug effects)
• Time Factors
• Transfection
• Up-Regulation