No association of the plasminogen activator inhibitor-1 promoter 4G/5G polymorphism with inhibitor level during basal transcription in vitro.

Abstract

Plasminogen activator inhibitor type 1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease, myocardial infarction, and cerebrovascular events. Previous studies on variations in plasma PAI-1 levels and associations between PAI-1 levels and PAI-1 genotypes have suggested that PAI-1 expression maybe regulated in a genotype-specific manner by insulin, hypertriglyceridemic very low-density lipoprotein, and lipoprotein. We investigated whether basal transcription of the PAI-1 gene also is regulated in a genotype-specific manner. Allele-specific polymerase chain reaction-amplified fragments containing a 4G/5G polymorphism of the PAI-1 gene promoter were ligated into the chloramphenicol acetyltransferase (CAT) reporter gene. The constructs of p4G-CAT or pSG-CAT and pSV-beta-galactosidase as an internal control were transiently cotransfected into human HepG2 hepatoma cells. Electrophoresis mobility shift assays (EMSA) employed a fragment from positions -687 to -664 (4G allele) or from -688 to -664 (5G allele) labeled with adenosine triphosphate tagged with phosphorous 32 in the gamma position and used nuclear extracts of HepG2 cells. Analysis of CAT produced by constructs containing the PAI-1 4G or 5G allele showed similar 3-fold increases in CAT activity in the PAI-1 4G/4G and PAI-1 5G/5G constructs, compared with the CAT activity in the pCAT3-Basic construct. Analyses using the probes containing the 4G or 5G allele site in the EMSA assay revealed no difference in the binding of nuclear protein. Our in vitro assay of basal transcription suggests no difference in the transcriptional activities of the alleles of the PAI-1 4G/5G polymorphism.