Identification of a cis-acting sequence in the human plasminogen activator inhibitor type-1 gene that mediates transforming growth factor-beta1 responsiveness in endothelium in vivo.
The mechanism of regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene by transforming growth factor-beta1 (TGF-beta1) was studied in vitro and in vivo in endothelial cells. We constructed adenovirus vectors containing PAI-1 5'-flanking sequences driving expression of a beta-galactosidase (beta-gal) reporter gene. Cultured bovine endothelial cells were transduced with the vectors and treated with TGF-beta1. beta-Gal expression was up-regulated 10-20-fold by TGF-beta1 when vectors contained 799-base pair (bp) of 5'-flanking sequence, but only minimally (2-3-fold) from a vector containing only 82-bp of 5' PAI-1 flanking sequence. TGF-beta1 up-regulated beta-gal expression at the mRNA level, congruently with TGF-beta1 up-regulation of expression of the endogenous PAI-1 gene. The constructs were transduced into intact rat carotid endothelium, and TGF-beta1 was injected systemically. In vivo, TGF-beta1 up-regulated endothelium-specific expression of beta-gal 3-fold (p < 0.03) from a vector containing the 799-bp sequence, but did not alter expression from a vector containing the 82-bp sequence. The sequence between -799 and -82 mediates up-regulation of reporter gene expression by TGF-beta1 in endothelial cells in vitro and in vivo. This general method permits the elucidation of mechanisms of gene regulation by physiologic stimuli delivered to the endothelium of intact animals.
Dong
G
G
Molecular Hematology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. david_dichek@quickmail.ucsf.edu
Schulick
A H
AH
DeYoung
M B
MB
Dichek
D A
DA
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002339:Carotid Arteries / Q000378:metabolism; D002417:Cattle; D004730:Endothelium, Vascular / Q000378:metabolism*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008954:Models, Biological; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D016212:Transforming Growth Factor beta