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Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor

ABSTRACT: Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system.

ORGANISM(S): Homo sapiens

PROVIDER: GSE75603 | GEO | 2016/12/01

SECONDARY ACCESSION(S): PRJNA304681

REPOSITORIES: GEO

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Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-RÂ´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Experiment Overall Design: HUVEC in passage 1 were cultured in serum-free EC medium until cells reached confluency. HUVEC were stimulated by 100 nM LTD4 (n = 4) or 10 nM thrombin (n = 4) or both agonists concomitantly (n = 3). The immediate-early gene programs of these experimental groups were identified at 60 minutes. In addition, one HUVEC preparation was stimulated with 100 nM LTD4 for 6 h and 24 h.

Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-R´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Keywords: Leukotriene Transcriptome, Thrombin Transcriptome, HUVEC, Immediate-Early Gene Expression, Cysteinyl Leukotriene 2 Receptor Gene Signature in HUVEC

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Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor

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