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

Abstract

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.