Volume 271, Number 38, Issue of September 20, 1996 pp. 23176-23184
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Requirement of an Upstream AP-1 Motif for the Constitutive and Phorbol Ester-inducible Expression of the Urokinase-type Plasminogen Activator Receptor Gene

(Received for publication, May 6, 1996, and in revised form, July 9, 1996)

Ernst Lengyel , Heng Wang ^¶ , Evan Stepp ^¶ , Jose Juarez ^¶ , Yao Wang , William Doe , Curt M. Pfarr ^'' and Douglas Boyd ^¶

From the  Department of Obstetrics and Gynecology, Technical University of Munich, 81675 Munich, Germany, ^¶ Department of Tumor Biology, M. D. Anderson Cancer Center, Houston, Texas 77030,  Mucosal Inflammation and Cancer Group, Division of Molecular Medicine, John Curtin School of Medical Research, ANU, Australian Capital Territory 2601, Australia, and ^'' Unite des Virus Oncogenes, Department of Biotechnology, Pasteur Institute, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix proteolysis by accelerating plasmin formation at the cell surface. The present study was undertaken to identify elements in the u-PAR promoter required for the elevated expression of this binding site. Toward this end, we used two cultured colon cancer cell lines; one (RKO) has a transcriptionally activated u-PAR gene, and the other (GEO) overexpresses the receptor only after phorbol ester treatment. A chloramphenicol acetyltransferase (CAT) reporter driven by 398 nucleotides of 5 regulatory sequence of the u-PAR gene was strongly activated in the RKO cells, which displays approximately 3 × 10⁵ receptors/cell. A region of this promoter between 197 and 8 was required for optimal expression, as indicated using a CAT reporter driven by 5 deleted fragments. DNase I footprinting revealed three protected regions (I, 190 to 171; II, 148 to 124; and III, 99 to 70) in this part of the promoter. Mutation of an AP-1 binding site at 184 within region I reduced activation of the promoter by 85%. Deletion of either region II or III also reduced promoter activity by over 60%. An oligonucleotide spanning the AP-1 motif at 184 bound, specifically, nuclear factors from RKO cells, and antibodies specific for Jun-D, c-Jun, or Fra-1 proteins supershifted the complex indicating the presence of these proteins. The amount of these factors was reduced in GEO cells in which the u-PAR gene is only weakly transcriptionally activated. Expression of a vector encoding a wild-type Jun-D cDNA increased u-PAR promoter activity in GEO cells. Conversely, transfection of RKO cells with a transactivation domain-lacking Jun-D expression construct resulted in a dose-dependent decrease in u-PAR promoter activity. Treatment of GEO cells with phorbol ester increased u-PAR mRNA and the activity of a CAT reporter driven by the wild-type but not the AP-1 (-184)-mutated u-PAR promoter, and this was associated with a strong induction in the amount of Jun-D, c-Jun, and c-Fos. Methylation interference studies using a fragment of the u-PAR promoter (spanning 201 to 150) bound with nuclear extracted proteins from RKO cells, and phorbol 12-myristate 13-acetate-treated and -untreated GEO cells showed that the contact points corresponded to the AP-1 binding site at 184. Thus, the elevated expression of u-PAR in RKO cells, which constitutively produces this binding site, as well as in phorbol 12-myristate 13-acetate-stimulated GEO cells requires an AP-1 motif located 184 bp upstream of the transcriptional start site.

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