Genotype-specific transcriptional regulation of PAI-1 gene by insulin, hypertriglyceridemic VLDL, and Lp(a) in transfected, cultured human endothelial cells.
Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease. Variations in plasma PAI-1 levels have been attributed to variations in the PAI-1 gene, and associations between PAI-1 levels and PAI-1 genotypes suggest that PAI-1 expression may be regulated in a genotype-specific manner by insulin, hypertriglyceridemic (HTG) very low density lipoprotein (VLDL), or lipoprotein(a) [Lp(a)]. Polymerase chain reaction-amplified 1106-bp fragments of the promoter of the 1/1 and 2/2 PAI-1 genotypes were sequenced and showed 5 regions of small nucleotide differences in the 1/1 versus 2/2 PAI-1 promoters that consistently occurred with high frequency. These fragments were ligated into the luciferase reporter gene, and 1/1 and 2/2 PAI-1 genotype human umbilical vein endothelial cell (HUVEC) cultures were transiently transfected with their respective p1PAI110/luc and p2PAI110/luc constructs and vice versa. Insulin induced an approximately 12- to 16-fold increase in luciferase activity in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p1PAI110/luc construct. HTG-VLDL and Lp(a) induced luciferase activity by approximately 14- to 16- and approximately 8- to 11-fold, respectively, in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p2PAI110/luc construct. The positive control interleukin-1 showed an approximately 7- to 12-fold response in the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with either of the constructs. These cross-over results demonstrate that regulation of either the 1/1 or 2/2 PAI-1 genotype by its respective inducer is due to the promoter itself and not to some factor(s) expressed differently in the 1/1 or 2/2 PAI-1 genotype HUVEC cultures.
Grenett
H E
HE
Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, USA.
Benza
R L
RL
Fless
G M
GM
Li
X N
XN
Davis
G C
GC
Booyse
F M
FM
D002478:Cells, Cultured; D015247:Deoxyribonuclease HindIII / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005838:Genotype; D006801:Humans; D015228:Hypertriglyceridemia / Q000097:blood / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology*; D017270:Lipoprotein(a) / Q000494:pharmacology*; D008079:Lipoproteins, VLDL / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014470:Umbilical Cord / Q000166:cytology / Q000187:drug effects
D007328:Insulin; D017270:Lipoprotein(a); D008079:Lipoproteins, VLDL; D017395:Plasminogen Activator Inhibitor 1; D015247:Deoxyribonuclease HindIII