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Volume 271, Number 47, Issue of November 22, 1996 pp. 29969-29977
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, February 21, 1996, and in revised form, August 1, 1996)

Gang Dong , Andrew H. Schulick , Mary Beth DeYoung Dagger and David A. Dichek Dagger

From the Molecular Hematology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the Dagger  Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100

The mechanism of regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene by transforming growth factor-beta 1 (TGF-beta 1) 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-beta 1. beta -Gal expression was up-regulated 10-20-fold by TGF-beta 1 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-beta 1 up-regulated beta -gal expression at the mRNA level, congruently with TGF-beta 1 up-regulation of expression of the endogenous PAI-1 gene. The constructs were transduced into intact rat carotid endothelium, and TGF-beta 1 was injected systemically. In vivo, TGF-beta 1 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-beta 1 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.


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