The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells.
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.
Wu
Sheng-Qian
SQ
Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Minami
Takashi
T
Donovan
Diana J
DJ
Aird
William C
WC
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
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