Induction of metallothionein isoforms by copper diethyldithiocarbamate in cultured vascular endothelial cells

Abstract

-Metallothionein (MT) plays a central role in cellular defense against heavy metals and oxidative stress. Since the induction of MT requires the activation of metal response element (MRE)-binding transcription factor-1 (MTF-1) by binding of zinc ions, inorganic zinc is regarded as a typical MT inducer. However, in a previous report, we showed that inorganic zinc could not induce MT in vascular endothelial cells. While it is suggested that endothelial MT presents mechanisms different from those of other cell types, these remain unclear. In this study, we investigated whether the induction of endothelial MT expression involves the Nrf2-ARE pathway using copper(II) bis(diethyldithiocarbamate), termed Cu10, using a culture system of bovine aortic endothelial cells. Cu10 induced MT-1/2 protein expression and increased the expression of mRNAs for MT-1A, MT-1E, and MT-2, MT isoforms expressed in the cells. Cu10 activated not only the MTF-1-MRE, but also the Nrf2-ARE pathway. MTF-1 knockdown resulted in the repression of Cu10-induced MT-1 and -2 expression. Cu10-induced MT-1 expression was down-regulated by Nrf2 knockdown. However, MT-2 expression was not affected by Nrf2 knockdown. These results suggest that the expression of endothelial MT is up-regulated by the Nrf2-ARE pathway as well as by the MTF-1-MRE pathway. Moreover, MT-1 regulation mechanisms differ from that of MT-2. Specifically, the present data support the hypothesis that MT-1 participates in the biological defense system, while MT-2 mainly regulates intracellular zinc metabolism.

Copper diethyldithiocarbamate as an inhibitor of tissue plasminogen activator synthesis in cultured human coronary endothelial cells

“…Such studies should investigate the physical characteristics of Cu10 that are required for the specific biological activities, including endothelial metallothionein induction (Fujie et al, 2016b) and the t-PA synthesis shown in the present study, as well as activation of nuclear factor, erythroid 2-like 2 (Fujie et al, 2016d).…”

“…In that study, it was found that the transcriptional induction of MT-1, an isoform of metallothionein, requires the activation of both metal response elements and antioxidant response element pathways by binding metal regulatory transcription factor-1 and nuclear factor, erythroid 2-like 2, respectively (Fujie et al, 2016b;Fujie et al, 2016c). However, activation of only metal response element is required for transcriptional induction of MT-2, which is another isoform of metallothionein.…”

“…We hypothesized that copper diethyldithiocarbamate (Cu10), which is a copper complex that exhibits interesting biological activities including endothelial metallothionein induction (Fujie et al, 2016b) and activation of nuclear factor, erythroid 2-like 2 (Fujie et al, 2016d), may provide a useful tool for analyzing new mechanisms that regulate endothelial fibrinolytic activity. Herein, we report on the findings of our experiments, in which we show that Cu10 suppresses the fibrinolytic activity of human coronary endothelial cells in culture.…”

Induction of Syndecan-4 by Organic–Inorganic Hybrid Molecules with a 1,10-Phenanthroline Structure in Cultured Vascular Endothelial Cells

“…This suggested that organic–inorganic hybrid molecules might be a good tool to analyze biological systems in certain cases. We subsequently confirmed this by successfully analyzing intracellular signaling pathways that mediate endothelial metallothionein induction using copper/zinc complexes [20,21]. …”

Synergistic cytotoxicity caused by forming a complex of copper and 2,9-dimethyl-1,10-phenanthroline in cultured vascular endothelial cells

“…Additionally, we have reported on the use of zinc diethyldithiocarbamate (Fujie et al, 2016b), copper diethyldithiocarbamate (Fujie et al, 2016c), and tris(pentafluorophenyl)stibane (Fujie et al, 2016d) as a molecular probe for use in the analysis of metallothionein induction in vascular endothelial cells. We have also recently found that expression of syndecan-4 (a small heparan sulfate proteoglycan), can be induced through activation of the hypoxia-inducible factor-1α/β pathway via inhibition of prolyl hydroxylase-domain-containing protein 2 in vascular endothelial cells, which is accomplished by using hybrid molecules with a 1,10-phenanthroline structure (Hara et al, 2017).…”