Regulation of tPA in endothelial cells exposed to cyclic strain: role of CRE, AP-2, and SSRE binding sites.
We have previously reported that exposure of cultured bovine aortic endothelial cells (EC) to 10% average strain resulted in an increase in tissue plasminogen activator (tPA) mRNA, immunoreactive tPA protein, and tPA activity in the medium. The present study was designed to examine the regulation of tPA gene expression in EC by cyclic strain. We performed a functional analysis of the tPA promoter by transfecting bovine aortic EC with a 1.4-kilobase (kb) construct of the human tPA promoter coupled to chloramphenicol acetyltransferase. We found that subjecting the EC to 10% average strain (and not 6% average strain) resulted in a 2.6-fold increase in activity of the 1.4-kb tPA promoter by 4 h. Analysis of deletion mutants of the promoter transfected into EC demonstrated a 60% drop-off in activity between position -145 and -105. Deoxyribonuclease I protection analysis of the segment downstream of position -196 suggested involvement of activator protein-2 (AP-2) and adenosine 3',5'-cyclic monophosphate-responsive element (CRE)-like binding sites, which was confirmed by electrophoretic mobility shift assays. Site-directed mutants of either the AP-2 or CRE-like regions resulted in a 65% decrease in activity compared with the wild type. Double mutations abolished basal transcription and any strain-induced activity. A shear stress responsive element (SSRE) binding site is present at -945, but site-directed mutants did not show any drop in activity compared with wild type by cyclic strain. These studies demonstrate that cyclic strain regulates tPA gene transcription in bovine aortic EC and that this transcriptional activation is dependent on factors that are similar to those activated with phorbol ester.
Sumpio
B E
BE
Department of Surgery (Vascular), Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Chang
R
R
Xu
W J
WJ
Wang
X J
XJ
Du
W
W
D000818:Animals; D001013:Aorta, Thoracic; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013314:Stress, Mechanical; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D010959:Tissue Plasminogen Activator