TRANSFECTION OF HUMAN ENDOTHELIAL-CELLS WITH PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 PROMOTER REPORTER GENE FRAGMENTS REVEALS PHORBOL ESTER INDUCTION OF GENE-EXPRESSION

The endothelium participates in haemostasis through the synthesis and secretion of factors involved in coagulation and fibrinolysis. The present study evaluates the feasibility of transfecting human umbilical vein endothelial cells (HUVEC) with promoter constructs to study the regulation of genes involved in the haemostatic process. Transfection by the calcium phosphate coprecipitation method of 1.5X10(6) HUVEC with 15 mug of a plasmid containing a PAI-1 gene promoter fragment (spanning nucleotides -808 to +18) fused to the chloramphenicol acetyl transferase (CAT) reporter gene produced a basal CAT activity that was 16+/-4.3-fold (mean+/-SEM, n=7) over background. When HUVEC, transfected with an 826bp or a 336bp PAI-1 gene promoter fragment, were stimulated with l60nM phorbol 12-myristate 13-acetate (PMA), the CAT activity was induced 4-fold, revealing that PMA responsive sequences are present in the proximal 336bp of the PAI-1 promoter. The effect of PMA on the endogenous PAI-1 production and on the expression of transfected promoter/reporter gene constructs was studied in HUVEC and in the HT1080 and HeLa cell lines. Addition of PMA (greater-than-or-equal-to 10nM) to monolayer cell cultures induced a 2- to 5.5-fold increase of PAI-1 antigen production within 8724h. In HUVEC, PMA (160nM) induced both the 2.4kb and 3.4kb mRNA species of PAI-1: 3.1+/-1.1 and 1.7+/-0.8-fold (n=6), respectively, within 8 h. Run-on experiments confirmed that this increase is at least partially mediated by increased gene transcription. Thus, HUVEC appear to be suitable for the investigation of the cell-specific regulation of PAI-1 gene expression, allowing the quantitation of the expression products not only of the endogenous PAI-1 gene but also of promoter/reporter gene constructs.