title: High glucose-induced increased expression of endothelin-1 in human endothelial cells is mediated by activated CCAAT/enhancer-binding proteins.
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.
authors.affiliation: Institute of Cellular Biology and Pathology 'Nicolae Simionescu' of the Romanian Academy, Bucharest, Romania.
authors.forename: Simona-Adriana
authors.initials: SA
authors.lastname: Manea
authors.affiliation: Institute of Cellular Biology and Pathology 'Nicolae Simionescu' of the Romanian Academy, Bucharest, Romania.
authors.forename: Andra
authors.initials: A
authors.lastname: Todirita
authors.affiliation: Institute of Cellular Biology and Pathology 'Nicolae Simionescu' of the Romanian Academy, Bucharest, Romania.
authors.forename: Adrian
authors.initials: A
authors.lastname: Manea
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D019332:Endothelin-1 / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D019332:Endothelin-1; D005947:Glucose