title: The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells.
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.
authors.affiliation: Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
authors.forename: Sheng-Qian
authors.initials: SQ
authors.lastname: Wu
authors.forename: Takashi
authors.initials: T
authors.lastname: Minami
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Donovan
authors.forename: William C
authors.initials: WC
authors.lastname: Aird
mesh_terms: D020816:Amino Acid Motifs; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D051766:Early Growth Response Protein 1; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D005813:Genes, Synthetic; D006801:Humans; D017874:Immediate-Early Proteins; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis; D017384:Sequence Deletion; D027602:Serum Response Element / Q000187:drug effects*; D026362:Serum Response Factor / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
chemical_list: D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D026362:Serum Response Factor; D014157:Transcription Factors; D008156:Luciferases; C482128:MAP2K2 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D013917:Thrombin