Abstract
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 x 10(5) 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.
Highlights
We reported previously that RKO cells were characterized by their elevated display of uPAR (3 ϫ 105 receptors/cell), whereas the GEO cells were equipped with approximately 104 binding sites/cell [38]
Nuclear run on experiments indicated that the larger amount of urokinase-type plasminogen activator receptor (u-PAR) mRNA in the RKO cells could be ascribed to a higher rate of transcription of this gene (Fig. 1B)
We report that a previously uncharacterized AP-1 motif located 184 nucleotides upstream of the major transcriptional start site is required both for the elevated expression of the gene in a cell line (RKO), which overexpresses the u-PAR (3 ϫ 105/cell), as well as the phorbol 12-myristate 13-acetate (PMA)-inducible expression in a receptor-deficient cell line (GEO)
Summary
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. Several studies have shown that urokinase can bind to a Mr 55,000 – 60,000 heavily glycosylated, disulfide-linked, cell surface receptor and with high affinity (KD, ϳ0.5 nM) [3, 4]. The urokinase-type plasminogen activator receptor (u-PAR) has been implicated in tissue remodeling in a number of physiological and pathological processes, such as re-epithelialization in skin wounds and tumor cell invasion and metastasis [7, 8]. A study by Kook et al [8] demonstrated that the expression of an antisense u-PAR cDNA in HEp3 squamous cell carcinoma cells decreased their invasiveness into a modified chorioallantoic membrane. The 5Ј flanking sequence of the u-PAR gene contains putative binding sites for AP-1, AP-2, Sp1, and NF-B but no potential TATA or CAAT boxes, and there is evidence that suggests the involvement of a proximal Sp1 motif in the basal transcription of the gene [19, 20]
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