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

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.

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A zinc complex that suppresses the expression of a reactive sulfur species-producing enzyme, cystathionine γ-lyase, in cultured vascular endothelial cells

Fundamental Toxicological Sciences ◽

10.2131/fts.5.181 ◽

2018 ◽

Vol 5 (6) ◽

pp. 181-184

Author(s):

Akane Takahashi ◽

Musubu Takahashi ◽

Tomoya Fujie ◽

Takato Hara ◽

Eiko Yoshida ◽

...

Keyword(s):

Endothelial Cells ◽

Zinc Complex ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Sulfur Species ◽

Reactive Sulfur Species

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Identification of Fer Tyrosine Kinase Localized on Microtubules as a Platelet Endothelial Cell Adhesion Molecule-1 Phosphorylating Kinase in Vascular Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e03-02-0080 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3553-3564 ◽

Cited By ~ 48

Author(s):

Naoko Kogata ◽

Michitaka Masuda ◽

Yuji Kamioka ◽

Akiko Yamagishi ◽

Akira Endo ◽

...

Keyword(s):

Endothelial Cells ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Adhesion Molecule ◽

Intracellular Signaling ◽

Vascular Endothelial Cells ◽

Expression Cloning ◽

Vascular Endothelial ◽

Cell Contacts ◽

Cell Cell

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

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Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.11.5930 ◽

2000 ◽

Vol 97 (11) ◽

pp. 5930-5935 ◽

Cited By ~ 273

Author(s):

K. S. Russell ◽

M. P. Haynes ◽

D. Sinha ◽

E. Clerisme ◽

J. R. Bender

Keyword(s):

Endothelial Cells ◽

Binding Sites ◽

Intracellular Signaling ◽

Vascular Endothelial Cells ◽

Membrane Binding ◽

Vascular Endothelial ◽

Membrane Binding Sites

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PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314479 ◽

2020 ◽

Vol 40 (7) ◽

pp. 1748-1762

Author(s):

Sumith R. Panicker ◽

Indranil Biswas ◽

Hemant Giri ◽

Xiaofeng Cai ◽

Alireza R. Rezaie

Keyword(s):

Endothelial Cells ◽

Protein Kinase C ◽

Protein Kinase ◽

Intracellular Signaling ◽

Vascular Endothelial Cells ◽

Dominant Negative ◽

Vascular Endothelial ◽

Kinase C ◽

Pkc Protein Kinase C ◽

Anti Inflammatory

Objective: Native and latent conformers of AT (antithrombin) induce anti-inflammatory and proapoptotic signaling activities, respectively, in vascular endothelial cells by unknown mechanisms. Synd-4 (syndecan-4) has been identified as a receptor that is involved in transmitting signaling activities of AT in endothelial cells. Approach and Results: In this study, we used flow cytometry, signaling assays, immunoblotting and confocal immunofluorescence microscopy to investigate the mechanism of the paradoxical signaling activities of high-affinity heparin (native) and low-affinity heparin (latent) conformers of AT in endothelial cells. We discovered that native AT binds to glycosaminoglycans on vascular endothelial cells via its heparin-binding D-helix to induce anti-inflammatory signaling responses by recruiting PKC (protein kinase C)-δ to the plasma membrane and promoting phosphorylation of the Synd-4 cytoplasmic domain at Ser179. By contrast, the binding of latent AT to endothelial cells to a site(s), which is not competed by the native AT, induces a proapoptotic effect by localizing PKC-δ to the perinuclear/nuclear compartment in endothelial cells. Overexpression of a dominant-negative form of PKC-δ resulted in inhibition of anti-inflammatory and proapoptotic signaling activities of both native and latent AT. Conclusions: These results indicate that the native and latent conformers of AT may exert their distinct intracellular signaling effects through differentially modulating the subcellular localization of PKC-δ in endothelial cells.

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Mechanism of temporal gradients in shear-induced ERK1/2 activation and proliferation in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.1.h22 ◽

2001 ◽

Vol 281 (1) ◽

pp. H22-H29 ◽

Cited By ~ 62

Author(s):

Xuping Bao ◽

Chuanyi Lu ◽

John A. Frangos

Keyword(s):

Endothelial Cells ◽

Intracellular Signaling ◽

Proliferative Response ◽

Vascular Endothelial Cells ◽

Inhibitory Effect ◽

Steady Shear ◽

Endothelial Cell Proliferation ◽

Vascular Endothelial ◽

Extracellular Signal ◽

Intracellular Signaling Cascade

The aim of the current study was to investigate the intracellular signaling cascade that leads to temporal gradients in shear (TGS)-induced endothelial cell proliferation, with a focus on the involvement of extracellular signal-regulated kinases 1 and 2 (ERK1/2). With the use of well-defined pulsatile, impulse, step, and ramp laminar flow profiles, we found that TGS (impulse flow and pulsatile flow) induced an enhanced and sustained (>30 min) phosphorylation of ERK1/2 relative to step flow (which contains a step increase in shear followed by steady shear), whereas steady shear (ramp flow) alone downregulated activated ERK1/2. Nitric oxide (NO) was found to mediate both the stimulatory effect of TGS and the inhibitory effect of steady shear on endothelial ERK1/2 phosphorylation. Reactive oxygen species (ROS) were also demonstrated to be associated with TGS-induced ERK1/2 phosphorylation. Both Gq/11 and Gi3 were necessary for the activation of ERK1/2 by TGS. Finally, the TGS-induced endothelial proliferative response was abolished by ERK1/2 inhibition. Our study demonstrated the essential role of G proteins, NO, and ROS in TGS-dependent ERK1/2 activation and proliferative response in vascular endothelial cells.

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

The Journal of Toxicological Sciences ◽

10.2131/jts.41.225 ◽

2016 ◽

Vol 41 (2) ◽

pp. 225-232 ◽

Cited By ~ 19

Author(s):

Tomoya Fujie ◽

Yukino Segawa ◽

Eiko Yoshida ◽

Tomoki Kimura ◽

Yasuyuki Fujiwara ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Metallothionein Isoforms ◽

Copper Diethyldithiocarbamate

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Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.1.h266 ◽

2001 ◽

Vol 281 (1) ◽

pp. H266-H274 ◽

Cited By ~ 10

Author(s):

Yoshiyuki Rikitake ◽

Seinosuke Kawashima ◽

Tomosaburo Takahashi ◽

Tomomi Ueyama ◽

Satoshi Ishido ◽

...

Keyword(s):

Endothelial Cells ◽

Tyrosine Phosphorylation ◽

Intracellular Signaling ◽

Phorbol Esters ◽

Vascular Endothelial Cells ◽

Biological Effects ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Vascular Endothelial ◽

Acetoxymethyl Ester

Lysophosphatidylcholine (LPC), a component of oxidized low-density lipoprotein, exerts various biological effects on vascular endothelial cells. However, the intracellular signaling of LPC is poorly understood. In this study, we investigated the involvement of proline-rich tyrosine kinase (PYK2) in LPC signaling in cultured bovine aortic endothelial cells by immunoprecipitation and Western blotting assays. Treatment of cells with LPC promoted a rapid increase in tyrosine phosphorylation of PYK2. LPC-stimulated PYK2 phosphorylation was inhibited by calcium chelators, 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-acetoxymethyl ester, EGTA, protein kinase C (PKC) inhibitor, GF-109203X, or PKC depletion by phorbol esters. PYK2 phosphorylation was inhibited by treatment with cytochalasin D but with neither botulinum C3 transferase nor overexpression of a dominant negative mutant of Rho A. LPC stimulated the association of Shc with PYK2, Shc tyrosine phosphorylation, and Grb2 binding to Shc and induced Ras activation. These results provide evidence that 1) LPC tyrosine phosphorylates PYK2 by calcium- and PKC-dependent mechanisms, 2) the intact cytoskeleton is required for LPC-stimulated PYK2 phosphorylation, and 3) LPC-activated Ras via the PYK2/Shc/Grb2 signaling.

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Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-β1: Induction of Cystathionine γ-Lyase and Cystathionine β-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms222111762 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11762

Author(s):

Musubu Takahashi ◽

Tomoya Fujie ◽

Tsuyoshi Nakano ◽

Takato Hara ◽

Yasuhiro Shinkai ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Molecular Mass ◽

Vascular Endothelial Cells ◽

Vascular Endothelial Cell ◽

High Molecular Mass ◽

Vascular Endothelial ◽

Sulfur Species ◽

Tgf Β1

Transforming growth factor-β1 (TGF-β1) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β1. Bovine aortic endothelial cells in a culture system were treated with TGF-β1 to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β1, induction of RSS-producing enzymes by TGF-β1, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β1 increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β1 regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β1, may modulate the regulation activity in vascular endothelial cells.

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