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Copper diethyldithiocarbamate as an activator of Nrf2 in cultured vascular endothelial cells.

ABSTRACT: The interest in organic-inorganic hybrid molecules as molecular probes for biological systems has been growing rapidly. Such hybrid molecules exhibit unique biological activities. Herein, copper(II) bis(diethyldithiocarbamate) (Cu10) was found to activate the transcription factor NF-E2-related factor 2 (Nrf2), which is responsible for regulating antioxidant and phase II xenobiotic enzymes, in vascular endothelial cells. The copper complex rapidly accumulated within cells and induced nuclear translocation of Nrf2, leading to upregulation of the expression of downstream proteins without cytotoxic effects. However, while copper bis(2-hydroxyethyl)dithiocarbamate activated Nrf2, copper ion, diethyldithiocarbamate ligand with or without zinc or iron failed to exhibit this activity. Intracellular accumulation of Cu10 was higher than that of Cu(II) and Cu(I). While the accumulation of copper(II) bis(dimethyldithiocarbamate) was reduced by small interfering RNA (siRNA)-mediated knockdown of the copper transporter CTR1, the knockdown did not affect Cu10 accumulation, indicating that Cu10 rapidly enters vascular endothelial cells via CTR1-independent mechanisms. In addition, copper and iron complexes with other ligands tested could not activate Nrf2, suggesting that the intramolecular interaction between copper and dithiocarbamate ligand is important for the activation of the transcription factor. Cu10 induced the expression of heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and γ-glutamylcysteine synthetase, downstream proteins of Nrf2. It was suggested that Cu10-induced activation of Nrf2 was due to proteasome inhibition as well as binding to Kelch-like ECH-associated protein 1. Since the effects of Cu10 on vascular endothelial cells are unique and diverse, the copper complex may be a good molecular probe to analyze the functions of the cells.

SUBMITTER: Fujie T

PROVIDER: S-EPMC4801994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As toxic substances can enter the circulating blood and cross endothelial monolayers to reach parenchymal cells in organs, vascular endothelial cells are an important target compartment for such substances. Reactive sulfur species protect cells against oxidative stress and toxic substances, including heavy metals. Reactive sulfur species are produced by enzymes, such as cystathionine γ-lyase (CSE), cystathionine β-synthase, 3-mercaptopyruvate sulfurtransferase, and cysteinyl-tRNA synthetase. However, little is known about the regulatory mechanisms underlying the expression of these enzymes in vascular endothelial cells. Bio-organometallics is a research field that analyzes biological systems using organic-inorganic hybrid molecules (organometallic compounds and metal coordinating compounds) as molecular probes. In the present study, we analyzed intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes in cultured bovine aortic endothelial cells, using copper diethyldithiocarbamate (Cu10). Cu10 selectively upregulated CSE gene expression in vascular endothelial cells independent of cell density. This transcriptional induction of endothelial CSE required both the diethyldithiocarbamate scaffold and the coordinated copper ion. Additionally, the present study revealed that ERK1/2, p38 MAPK, and hypoxia-inducible factor (HIF)-1α/HIF-1β pathways mediate transcriptional induction of endothelial CSE by Cu10. The transcription factors NF-κB, Sp1, and ATF4 were suggested to act in constitutive CSE expression, although the possibility that they are involved in the CSE induction by Cu10 cannot be excluded. The present study used a copper complex as a molecular probe to reveal that the transcription of CSE is regulated by multiple pathways in vascular endothelial cells, including ERK1/2, p38 MAPK, and HIF-1α/HIF-1β. Bio-organometallics appears to be an effective strategy for analyzing the functions of intracellular signaling pathways in vascular endothelial cells.

Project description:Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.

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Copper diethyldithiocarbamate as an activator of Nrf2 in cultured vascular endothelial cells.

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