SP1 SITES MEDIATE ACTIVATION OF THE PLASMINOGEN-ACTIVATOR INHIBITOR-1PROMOTER BY GLUCOSE IN VASCULAR SMOOTH-MUSCLE CELLS

This study was designed to characterize the direct effects of hypergly cemia on plasminogen activator inhibitor-1 (PAI-1) expression in cultu red vascular smooth muscle cells, Glucose induced dose- and time-depen dent increases of PAI-1 mRNA expression in rat aortic smooth muscle (R ASM) cells in vitro, Using a series of luciferase reporter gene constr ucts containing PAI-1 5'-flanking sequence (from -6.4 kilobase to -42 base pairs (bp)) transfected into RASM, we found that glucose (25 mM) consistently induced a 4-fold increase in luciferase activity, with th e response localized to sequence between -85 and -42 bp. Mutagenesis o f two putative Sp1-binding sites located in the region of interest ess entially obliterated the glucose-response. Electrophoretic mobility sh ift assays with radiolabeled oligonucleotides containing the two putat ive Sp1-binding sites from PAI-1 promoter and nuclear extracts from RA SM cells revealed that glucose treatment markedly changed the mobility pattern of the major protein-DNA complexes. Supershift assay showed t hat transcription factor Sp1 was present in the complexes under contro l and hyperglycemic conditions. These results suggest that glucose reg ulates PAI-1 gene expression in RASM cells through an effect on two ad jacent Sp1 sites located between -85 and -42 bp of the PAI-1 5'-flanki ng region and that the release of a transcriptional repressor from the Sp1 complexes may explain the activation of the PAI-1 gene under high glucose conditions in RASM cells.