On the regulation of the plasminogen activator inhibitor-1 gene expression.

Plasminogen activators and their inhibitors play a pivotal role in maintaining the hemostatic balance in blood and also in other proteolytic processes such as cell migration and tumor invasion. The main physiological inhibitor of the plasminogen activators is plasminogen activator inhibitor-1 (PAI-1). 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 (> or = 10 nM) to monolayer cell cultures induced a 2- to 5.5-fold increase of PAI-1 antigen production within 8-24 h. In HUVEC, PMA (160 nM) induced both the 2.4 kb and 3.4 kb 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. When HUVEC, HT1080 and HeLa, transfected with an 826 bp or a 336 bp PAI-1 gene promoter fragment, were stimulated with 160 nM phorbol 12-myristate 13-acetate (PMA), the CAT activity was induced 4-, 3.5- and 10-fold respectively for both constructs, revealing that PMA responsive sequences are present in the proximal 336 bp of the PAI-1 promoter. Substitution mutations in the regions encompassing nucleotides -78 to -69 (TGGGTGGGGC) or -61 to -54 (TGAGTTCA), but not in the regions -155 to -149 (TGCCTCA) or -84 to -76 (AGTGAGTGG) reduced this induction. Gel electrophoresis of double stranded -65 to -50 oligonucleotides of the PAI-1 promoter region and nuclear extracts from HeLa cells produced a gel shift pattern, similar to that obtained with a AP-1 consensus oligomer, and excess unlabeled AP-1 oligomer reverted binding suggesting that this region of the PAI-1 promoter is an AP-1-like binding site. Gel electrophoresis of double stranded -82 to -65 oligonucleotide with HeLa nuclear extracts revealed a gel shift pattern of three bands. Sp1 consensus oligomer competed with the binding of two of these bands and AP-2 consensus sequence oligomer with the binding to the third band. The -82 to -65 oligomer also bound to purified AP-2 and Sp1 proteins. Southwestern blotting of HeLa nuclear extracts revealed that the labeled oligomer covering region -82 to -65 bound the proteins with molecular weights of 52 kD and 72 kD.(ABSTRACT TRUNCATED AT 400 WORDS)