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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/EBP?, C/EBP?, and C/EBP? 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/EBP?, C/EBP? or C/EBP? 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/EBP?, C/EBP? or C/EBP? upregulated the luciferase level controlled by the ET-1 gene promoter. The direct interaction of C/EBP?, C/EBP? or C/EBP? 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.

SUBMITTER: Manea SA

PROVIDER: S-EPMC3871648 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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High glucose-induced increased expression of endothelin-1 in human endothelial cells is mediated by activated CCAAT/enhancer-binding proteins.

Manea Simona-Adriana SA Todirita Andra A Manea Adrian A

PloS one 20131223 12

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 glucos ...[more]

PMID: 24376792

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Project description:BACKGROUND: Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ? (C/EBP?) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBP? in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBP?-null glial cultures. METHODS: Due to fertility and mortality problems associated with the C/EBP?-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBP?-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBP? DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. RESULTS: C/EBP? mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ? (IFN?). Quantitative chromatin immunoprecipitation showed binding of C/EBP? to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN? induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1? and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBP?. In addition, neurotoxicity elicited by LPS+IFN?-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBP? in microglia. CONCLUSIONS: These findings show involvement of C/EBP? in the regulation of pro-inflammatory gene expression in glial activation, and demonstrate for the first time a key role for C/EBP? in the induction of neurotoxic effects by activated microglia.

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High glucose-induced increased expression of endothelin-1 in human endothelial cells is mediated by activated CCAAT/enhancer-binding proteins.

Publications

High glucose-induced increased expression of endothelin-1 in human endothelial cells is mediated by activated CCAAT/enhancer-binding proteins.

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