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Vol. 273, Issue 5, C1441-C1448, November 1997
Department of Surgery (Vascular), Yale University School of Medicine, New Haven, Connecticut 06510
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.
endothelium; hemodynamics; tissue plasminogen activator gene expression
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