High Glucose-Induced Increased Expression of Endothelin-1 in Human Endothelial Cells Is Mediated by Activated CCAAT/Enhancer-Binding Proteins

Simona-Adriana Manea,Adrian Manea,Andra Todirita

doi:10.1371/journal.pone.0084170

Abstract

High glucose-induced endothelial dysfunction is partially mediated by the down-stream pathophysiological effects triggered by increased expression of endothelin-1 (ET-1). The molecular control mechanisms of ET-1 synthesis are yet to be discovered. Members of the CCAAT/enhancer-binding proteins (C/EBP) family are important regulators of key metabolic processes, cellular differentiation and proinflammatory genes. In this study, we aimed at elucidating the role of C/EBP in mediating the high glucose effect on ET-1 expression in human endothelial cells (EC). Human umbilical vein cells (EAhy926) and primary cultures of human aortic EC were exposed to high levels of glucose (16.5–25 mM). Real-time PCR, Western blot, enzyme-linked immunosorbent assay, ET-1 promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate ET-1 regulation. High glucose activated C/EBPα, C/EBPβ, and C/EBPδ in a dose-dependent manner. It also promoted significant increases in ET-1 gene and peptide expression. Chemical inhibition of JNK, p38MAPK and ERK1/2 diminished significantly the high glucose-induced nuclear translocation of C/EBP and ET-1 expression. Silencing of C/EBPα, C/EBPβ or C/EBPδ greatly reduced the high glucose-induced upregulation of ET-1 mRNA, pre-pro-ET-1, and ET-1 secretion. The expression of various C/EBP isoforms was selectively downregulated by siRNA-mediated gene silencing. In silico analysis indicated the existence of typical C/EBP elements within human ET-1 gene promoter. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ upregulated the luciferase level controlled by the ET-1 gene promoter. The direct interaction of C/EBPα, C/EBPβ or C/EBPδ proteins with the ET-1 promoter in high glucose-exposed EC was confirmed by chromatin immunoprecipitation assay. High glucose-induced ET-1 expression is mediated through multiple mechanisms. We present evidence that members of the C/EBP proinflammatory transcription factors are important regulators of ET-1 in high glucose-exposed human endothelial cells. High glucose-induced activation of C/EBP-related signaling pathways may induce excessive ET-1 synthesis, thus promoting vasoconstriction and dysfunction of the vascular wall cells in diabetes.

Highlights

Transactivation assays showed that high glucose induced a significant up-regulation of the luciferase level directed the promoter of ET-1 gene, a process that was associated with the activation of CCAAT/enhancer-binding proteins (C/EBP) transcriptional responses (Figure 2A)
Several lines of evidence indicate that the vascular inflammation- and growthpromoting transcription factors NF-kB and AP-1 are important regulators of ET-1 expression. [15,27] In accordance to this, we have demonstrated previously that the pro-inflammatory signaling pathway, Jak/STAT, mediates the up-regulation of ET-1 biosynthesis in human endothelial cells (EC) exposed to high glucose concentration. [16]
It has been demonstrated that inhibition of C/EBP transcriptional activity by decoy oligodeoxynucleotide reduces the restenosis after angioplasty in hypercholesterolemic rabbits, a process that may be partially mediated by the reduction in ET-1 expression

Summary

Introduction

Hyperglycemia, the primary clinical manifestation of diabetes, contributes to diabetic complications [1] by inducing vascular inflammation, oxidative stress, impaired vascular relaxation, changing vascular cell metabolism, altering the vascular matrix molecules, and circulating proteins/lipoproteins. [2,3,4] the precise mechanisms by which hyperglycemia induce pathological outcomes and the molecular nature of its downstream effectors is still a debatable issue.Convincing evidence exists that the endothelin system plays an important role in the pathophysiology of diabetes-associated cardiovascular diseases. [5] The endothelin system comprises biological active peptides called endothelins, endothelin converting enzymes, and specific cellular receptors. [6,7,8] Endothelins regulate important physiological processes including vascular tonus [9], cellular growth and proliferation. [10] in pathological conditions such as diabetes mellitus, dysregulation of the endothelin system, characterized by enhanced expression, activity or responsiveness of different constituents contributes to dysfunction of the vascular cells. [11,12]Hyperglycemia-induced vascular deleterious effects are partially mediated by the endothelin-1 (ET-1). [10] in pathological conditions such as diabetes mellitus, dysregulation of the endothelin system, characterized by enhanced expression, activity or responsiveness of different constituents contributes to dysfunction of the vascular cells. Evidence is accumulating that the basic-leucine zipper transcription factor family, CCAAT/enhancer-binding proteins (C/ EBP), plays a major role in cellular differentiation and function. C/EBPs form homo- or heterodimers and interact with the cytidine-cytidine-adenosine-adenosine-thymidine box motif in the enhancers and promoters of target genes, and regulate important biological activities such as metabolism, cellular proliferation, growth, and differentiation. Based on the fact that C/EBPs transduce the effects of numerous pro-inflammatory and growth-related stimuli, we examined the role of C/EBP in mediating high glucose-induced ET-1 level in cultured EC. We provide evidence that C/EBPa, C/ EBPb and C/EBPd are activated by high glucose and that MAPK signaling, and C/EBPa, -b, and –d isoforms are coordinately involved in the regulation of ET-1 expression in high glucoseexposed endothelial cells

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