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

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. 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. 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. Introduction Hyperglycemia, the primary clinical manifestation of diabetes, contributes to diabetic complications [1] by inducing vascular inflammation, oxidative stress, impaired Desacetylnimbin vascular relaxation, changing vascular cell metabolism, altering the vascular matrix molecules, and circulating proteins/lipoproteins. [2]C[4] Nevertheless, the precise mechanisms by which hyperglycemia induce pathological outcomes and the molecular nature of its down-stream 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]C[8] Endothelins regulate important physiological processes including vascular tonus [9], cellular growth and proliferation. [10] However, 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). Increased synthesis of ET-1, the main effector of the endothelin system, induces vasoconstriction, dysfunction of endothelial cells (EC), phenotypic alteration of smooth muscle cells, vascular remodeling, inflammation and oxidative stress. [13] Multiple mitogenic signaling pathways [(e.g., mitogen-activated protein kinases (MAPK), Janus kinase (Jak)] and pro-inflammatory transcription factors such as nuclear factor kB (NF-kB), activator protein 1 (AP-1), and members of the signal transducer and activator of transcription (STAT) family have been implicated in the regulation of ET-1 Desacetylnimbin expression. [14]C[16] However, the precise molecular pathways responsible for increased ET-1 level in diabetes are not totally deciphered. 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. [17] The C/EBP family consists of six members (C/EBP-, -, -, -, -, -) each with a distinct cell and tissue distribution. Upon activation, 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. [18] Various members of the C/EBP family, namely C/EBP, -, and C have been proved to regulate the expression of many cytokines, chemokines, growth factors, acute phase proteins, and immunoglobulins. [17], [19] Still, the precise function of C/EBPs in the cardiovascular system is still a matter of debate. Based on the fact that C/EBPs transduce the effects of numerous pro-inflammatory and growth-related stimuli, Desacetylnimbin we examined the role Desacetylnimbin of C/EBP in mediating high glucose-induced ET-1 level in cultured EC. We provide evidence that C/EBP, C/EBP and C/EBP are activated by high glucose and that MAPK signaling, and C/EBP, -, and C isoforms are coordinately involved in the regulation of ET-1 expression in high glucose-exposed endothelial cells. Materials and Methods Materials General chemicals and reagents, antibodies, siRNA, and molecular biology kits were derived from Sigma-Aldrich (Germany), Santa Cruz Biotechnology (USA), Invitrogen (Austria), Qiagen (Germany), R&D Systems (Austria). The enzyme-linked immunosorbent assay (ELISA)-based endothelin-1 detection kit was obtained VBCH from Biomedica (Austria). The pGL2 basic reporter vector carrying the human ET-1 gene promoter was.