Abstract
Thrombin signaling in endothelial cells provides an important link between coagulation and inflammation. We report here that thrombin induces endogenous Egr-1 mRNA and Egr-1 promoter activity in primary human endothelial cells by approximately 6-fold and 3-fold, respectively. In transient transfection assays, deletion of the 3' cluster of serum response elements (SREs), but not the 5' cluster of SREs, resulted in a loss of thrombin response. When coupled to a heterologous core promoter, a region spanning the 3' SRE cluster contained information for thrombin response, whereas a region spanning the 5' SRE cluster had no such effect. A point mutation of the most proximal SRE (SRE-1), but not of the proximal Ets motif or upstream SREs, abrogated the response to thrombin. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated cells displayed increased binding of total and phosphorylated serum response factor (SRF) to SRE-1. Thrombin-mediated induction of Egr-1 was blocked by inhibitors of MEK1/2, but not by inhibitors of protein kinase C, phosphatidylinositol 3-kinase, or p38 mitogen-activated protein kinase (MAPK). Taken together, these data suggest that thrombin induces Egr-1 expression in endothelial cells by a MAPK-dependent mechanism that involves an interaction between SRF and SRE-1.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Motifs
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Cells, Cultured / cytology
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Cells, Cultured / drug effects
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Culture Media, Serum-Free / pharmacology
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DNA-Binding Proteins / biosynthesis
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DNA-Binding Proteins / genetics*
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Early Growth Response Protein 1
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Electrophoretic Mobility Shift Assay
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Endothelium, Vascular / cytology
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Endothelium, Vascular / drug effects*
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Enzyme Inhibitors / pharmacology
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Genes, Reporter
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Genes, Synthetic
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Humans
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Immediate-Early Proteins*
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Luciferases / biosynthesis
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Luciferases / genetics
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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MAP Kinase Signaling System / drug effects
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Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
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Point Mutation
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Protein Binding / drug effects
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Transport / drug effects
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Protein-Tyrosine Kinases / antagonists & inhibitors
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RNA, Messenger / biosynthesis
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Sequence Deletion
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Serum Response Element / drug effects*
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Serum Response Factor / pharmacology*
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Thrombin / pharmacology*
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Transcription Factors / biosynthesis
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Transcription Factors / genetics*
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Transcriptional Activation / drug effects
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Transfection
Substances
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Culture Media, Serum-Free
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DNA-Binding Proteins
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EGR1 protein, human
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Early Growth Response Protein 1
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Enzyme Inhibitors
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Immediate-Early Proteins
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RNA, Messenger
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Serum Response Factor
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Transcription Factors
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Luciferases
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MAP2K2 protein, human
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Protein-Tyrosine Kinases
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Protein Serine-Threonine Kinases
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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MAP2K1 protein, human
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Mitogen-Activated Protein Kinase Kinases
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Thrombin