Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells

https://doi.org/10.1016/j.bbrc.2009.06.100Get rights and content

Abstract

To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-κB subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-κB. Assay using NF-κB reporter revealed that TrxR1 upregulated transcriptional activity of NF-κB. This study suggests that TrxR1 enhances ROS generation, NF-κB activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.

Introduction

The view that atherosclerosis is indeed a chronic inflammatory disease initiated by monocyte adhesion to activated endothelial cells (EC) is now widely accepted [1]. A bulk of evidences suggests that the inflammatory response in vascular injury involves recruitment and activation of monocytes through activation of monocyte chemoattractant protein-1 (MCP-1) [1]. As a potent CC chemokine for monocytes, MCP-1 plays an essential role in the recruitment of monocytes/macrophages to vascular lesions [2]. Expression of MCP-1 is tightly regulated and has been shown to occur at the transcriptional level by various stimuli such as oxidized low-density lipoprotein and TNF-α, lipopolysaccharide (LPS) [3]. In addition, many other factors were also reported to down modulate MCP-1 gene expression such as estradiol, troglitazone and TGF-β1[3]. It has been reported that reactive oxygen species (ROS) derived from Rac1-activated NADPH oxidase upregulated TNF-α-induced MCP-1 expression, while the antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), significantly inhibited the IL-4-induced MCP-1 mRNA expression in human vascular endothelial cells [4]. Depletion of the endogenous Nox4 by transfection of siRNA for Nox4 into human aortic endothelial cells resulted in a failure to induce ROS generation and subsequent expression of MCP-1 in response to LPS [5]. However, no investigation on the possible role of thioredoxin reductases, which play important role in regulation of cellular redox status, in regulating MCP-1 release and expression has been reported.

Thioredoxin reductases are members of the nucleotide-disulfide oxidoreductase family and ubiquitously found in mammalian tissues. All mammalian TrxRs are homodimeric selenocysteine-containing enzymes and the major selenoproteins expressed in human vascular endothelial cells [6]. They reduce and thereby activate thioredoxins which serves as reducing equivalent and catalyzes many redox reactions [7]. In human coronary atherosclerotic specimens, Trx expression is enhanced throughout the vessel wall and the greatest increases were observed in endothelial cells and infiltrating macrophages within the neointimal plaques [8], [9]. An almost twofold increase in TrxR1 mRNA was recently found in the atherosclerotic plaques relative to surrounding healthy areas of the artery specimen taken from the same patients [10]. The high expression of Trx and TrxR in the atherosclerotic plaques suggests that Trx and TrxR may cooperate to work for antioxidant defense mechanisms in atherosclerosis. However, up to now, there is no evidence to show if TrxR really protects the vascular endothelium from forming atherosclerotic plaque. For this reason, we established two endothelial cell models, the human endothelial-like EAhy926 cells either overexpressing TrxR1 or having their endogenous TrxR1 knocked down, and investigated how TrxR1 modulate the expression of MCP-1 in the endothelial-like cells. It was surprisingly found that TrxR1 upregulated, rather than downregulated the MCP-1 release and expression.

Section snippets

Materials and methods

Plasmids construction. Human TrxR1 cDNA (GenBank Accession No. BG772375) was PCR amplified from original plasmid containing TrxR1 cDNA using the 5′ primer, 5′-CGGAATTCGCCACCATGGACGGCCCTGAAGATCTTC-3′ containing an EcoRI site and a Kozak consensus sequence upstream of the start cordon ATG, and the 3′ primer, 5′-GCGGATCCGCCAAATGAGATGAGGACGTGA-3′ including a BamHI site. The PCR product was subcloned into the mammalian bicistronic expression vector pIRESneo2, which permits the translation of two

Overexpression of TrxR1 upregulates MCP-1 secretion

To know if TrxR1 plays any role in regulation of MCP-1 in vascular endothelium, a stable TrxR1-overexpressing EAhy926 cell line was established by transfecting the Lab-constructed TrxR1-expression vector, pIRESneo2-TrxR1, into cells. The TrxR1 activity in the transfected cells was determined as DTNB reduction rate. As Fig. 1A shows, activity of TrxR1 in the TrxR1-overexpressing cells (EAhy-TrxR1 cells) was 34% higher than that in controls (EAhy-neo cells), which reflects the abundance of TrxR1

Discussion

Since regulatory roles of cytosolic thioredoxin and many other antioxidants are dependent on the activity of cytosolic thioredoxin reductase, TrxR1 together with Trx1 has been recognized as an essential component for cellular redox control and antioxidant defense [21], [22]. Besides that, as increased expression of TrxR was found in human atherosclerotic plaques and foam cells [10], it seems logical that TrxR would protect the vascular endothelium from forming atherosclerotic plaque. However,

Acknowledgments

We sincerely thank Drs. C.J. Edgell, V.N. Gladyshev and L. Hajjar for providing us cell line and plasmids. This work was supported by the National Natural Science Foundation of China (Nos. 30330250 and 30770513).

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      This information afforded us an opportunity to target the removal of selenoproteins and their subsequent reexpression without disrupting the protein internal coding sequence of the mRNA (Yoo et al., 2007b). Other investigators have also used RNAi technology to target the removal of TR1 in various normal or cancer cells (e.g., see Eriksson et al., 2009; Honeggar et al., 2009; Liu and Shen, 2009; Watson et al., 2008) and the reader is referred to these studies to see additional approaches of targeting TR1 knockdown that vary from those described herein. One advantage of using mouse cancer cell lines in RNAi technology studies is that the resulting protein-deficient cells can be injected into mice that are not immunocompromised to further examine the effect of the loss of the targeted protein on, for example, tumorigenicity and metastasis.

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