Identification of a cis-Acting Sequence in the Human Plasminogen Activator Inhibitor Type-1 Gene That Mediates Transforming Growth Factor-β1 Responsiveness in Endothelium in Vivo

Gang Dong,Andrew H Schulick,Mary Beth Deyoung,David A Dichek

doi:10.1074/jbc.271.47.29969

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.

Highlights

plasminogen activator inhibitor type-1 (PAI-1) is expressed by both vascular smooth muscle and endothelial cells (EC) [4]
Endothelial cells were infected with AdPAI800␤-Gal, AdIAP800␤-Gal, AdPAI82␤-Gal, or AdRSV␤-Gal at 4 ϫ 108 pfu/ml followed by addition of transforming growth factor-␤1 (TGF-␤1) protein or vehicle (Fig. 2). ␤-Galactosidase expression was up-regulated by TGF-␤1 in a dose-related manner in EC transduced with AdPAI800␤-Gal and AdIAP800␤-Gal, reaching maximal levels at 10 ng/ml
TGF-␤1 up-regulated ␤-gal expression by a mean of 9.5-fold in EC transduced with AdPAI800␤-Gal and by a mean of 21-fold in EC transduced with AdIAP800␤-Gal

Summary

Introduction

PAI-1 is expressed by both vascular smooth muscle and endothelial cells (EC) [4]. PAI-1 is up-regulated in vivo in association with atherosclerosis [5], endotoxemia [6], thrombosis [7], and vascular injury [8]. Despite the likely importance of PAI-1 regulation by TGF-␤1 in EC in vivo, virtually all of the data on the mechanisms of regulation of PAI-1 gene expression have been produced in transformed hepatocyte and fibroblast lines in vitro [17,18,19,20,21, 25]. While these studies are informative, the extent to which one may extrapolate the results of gene regulation experiments across cell types and from in vitro to in vivo is unclear. We report the use of this animal model to define a functional cis-acting sequence in the human PAI-1 promoter that mediates up-regulation of EC gene expression in response to TGF-␤1 administration in an intact animal

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