Transcriptional Induction of Cystathionine γ-Lyase, a Reactive Sulfur-Producing Enzyme, by Copper Diethyldithiocarbamate in Cultured Vascular Endothelial Cells.

Tomoya Fujie,Hiroshi Naka,Toshiyuki Kaji,Musubu Takahashi,Yoshito Kumagai,Hideyo Takahashi,Akane Takahashi,Kosho Makino,Chika Yamamoto,Asuka Soyama,Takato Hara

doi:10.3390/ijms21176053

Abstract

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.

Highlights

Blood vessels ubiquitously exist in human organs
We studied the vascular toxicology of heavy metals using a vascular endothelial cell culture system and found that cadmium and lead cause specific functional damage to these cells
Because the response of vascular endothelial cells to exogenous factors is often cell density-dependent [32,33,34,35,36], both dense and sparse cultures of the cells were used in this experiment

Summary

Introduction

Blood vessels ubiquitously exist in human organs. Vascular endothelial cells cover the luminal surfaces of blood vessels in monolayers that serve as barriers between the blood and the parenchymal cells of the organs. Clarification of the intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes will contribute to our understanding of zinc metabolism and heavy metal toxicity in vascular endothelial cells. Such studies will contribute to our understanding of cellular defense mechanisms, because reactive sulfur species are protective against oxidative stress [10]. One week of protein-restricted diet in mice increased CSE expression, with a corresponding decrease in vein graft disease, suggesting ATF4 involvement in this expression [31] Beyond these studies, little is known about the intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes. The present study was undertaken to reveal bio-organometallic sensitive pathways mediating endothelial cell CSE expression

Results

Discussions

Materials

Cell Culture and Treatments

Western Blot Analysis

Statistical Analyses