Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells.
This study was designed to characterize the direct effects of hyperglycemia on plasminogen activator inhibitor-1 (PAI-1) expression in cultured vascular smooth muscle cells. Glucose induced dose- and time-dependent increases of PAI-1 mRNA expression in rat aortic smooth muscle (RASM) cells in vitro. Using a series of luciferase reporter gene constructs 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 the response localized to sequence between -85 and -42 bp. Mutagenesis of two putative Sp1-binding sites located in the region of interest essentially obliterated the glucose-response. Electrophoretic mobility shift assays with radiolabeled oligonucleotides containing the two putative Sp1-binding sites from PAI-1 promoter and nuclear extracts from RASM cells revealed that glucose treatment markedly changed the mobility pattern of the major protein-DNA complexes. Supershift assay showed that transcription factor Sp1 was present in the complexes under control and hyperglycemic conditions. These results suggest that glucose regulates PAI-1 gene expression in RASM cells through an effect on two adjacent Sp1 sites located between -85 and -42 bp of the PAI-1 5'-flanking 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.
Chen
Y Q
YQ
Cardiovascular Division, Departments of Medicine and Pharmacology, Vanderbilt University Medical Center, Nashville Veterans Affairs Medical Center, Nashville, Tennessee 37232, USA.
Su
M
M
Walia
R R
RR
Hao
Q
Q
Covington
J W
JW
Vaughan
D E
DE
D000818:Animals; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016296:Mutagenesis; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D016329:Sp1 Transcription Factor; D005947:Glucose