title: Identification of a 251-bp fragment of the PAI-1 gene promoter that mediates the ethanol-induced suppression of PAI-1 expression.
abstract: BACKGROUND
  Moderate alcohol consumption reduces the risk for coronary heart disease. This cardioprotection may be due to ethanol enhancement of fibrinolysis. Fibrinolysis involves the interaction of plasminogen activators (PAs) and the plasminogen activator inhibitor type-1 (PAI-1). Factor(s) that decrease endothelial cell (EC) PAI-1 expression increase fibrinolysis and may decrease the risk for cardiovascular disease.
  
  
  METHODS
  Five promoter deletion fragments were generated from a 1.1-kb PAI-1 promoter fragment and ligated to a luciferase reporter gene. Cultured human umbilical vein endothelial cells (HUVECs) were transiently transfected with these PAI-1 deletion constructs. A 251-base pair (bp) fragment of the PAI-1 promoter, positions -800 to -549, was cloned upstream of a heterologous promoter/enhancer. ECs luciferase activity was measured in the absence/presence of 20 mM ethanol. Electrophoresis mobility shift assays were performed with nuclear extracts from untreated and ethanol-treated ECs using this 251-bp fragment.
  
  
  RESULTS
  Deletion analysis showed a region between position -800 and -549 mediated ethanol repression of luciferase activity. This 251-bp promoter fragment also repressed the activity of a heterologous promoter/enhancer in the presence of ethanol. Using the labeled 251-bp fragment, nuclear extracts from ethanol-treated ECs contained two inducible bands and one enhanced band. Non-ethanol treated nuclear extracts also contained a band that was not observed in ethanol-treated samples. Competition using 100-fold molar excess of unlabeled probe abolished these four bands.
  
  
  CONCLUSIONS
  Repression of PAI-I gene transcription in cultured HUVECs exposed to ethanol may involve the interaction of several transcription factors with binding sites localized between positions -800 and -549 of the PAI-1 gene promoter.
authors.affiliation: Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham, 35294-2170, USA. hgrenett@cardio.dom.uab.edu
authors.forename: H E
authors.initials: HE
authors.lastname: Grenett
authors.forename: P E
authors.initials: PE
authors.lastname: Wolkowicz
authors.forename: R L
authors.initials: RL
authors.lastname: Benza
authors.forename: J K
authors.initials: JK
authors.lastname: Tresnak
authors.forename: C G
authors.initials: CG
authors.lastname: Wheeler And
authors.forename: F M
authors.initials: FM
authors.lastname: Booyse
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects* / Q000235:genetics; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D002492:Central Nervous System Depressants; D017395:Plasminogen Activator Inhibitor 1; D014157:Transcription Factors; D000431:Ethanol