Transactivation of the Urokinase-type Plasminogen Activator Receptor Gene through a Novel Promoter Motif Bound with an Activator Protein-2α-related Factor

Abstract

The urokinase receptor overexpressed in invasive cancers promotes laminin degradation. The current study was undertaken to identify cis elements and trans-acting factors activating urokinase receptor expression through a footprinted (؊148/؊124) region of the promoter containing putative activator protein-2-and Sp1-binding motifs. Mobility shifting experiments using nuclear extract from a high urokinase receptorexpressing cell line (RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct from, activator protein-2␣ bound to this region. Mutations preventing the binding of the activator protein 2␣-related factor diminished urokinase receptor promoter activity. In RKO cells, the expression of a negative regulator of activator protein-2 function diminished urokinase receptor promoter activity, protein, and laminin degradation. Conversely, urokinase receptor promoter activity in low urokinase receptor-expressing GEO cells was increased by activator protein-2␣A expression. Although using GEO nuclear extract, little activator protein-2␣-related factor bound to the footprinted region, phorbol 12-myristate 13-acetate treatment, which induces urokinase receptor expression, increased complex formation. Mutations preventing the activator protein-2␣-related factor and Sp1/Sp3 binding reduced urokinase receptor promoter stimulation by this agent. Thus, the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the urokinase receptor is mediated partly through trans-activation of the promoter via a sequence (؊152/ ؊135) bound with an activator protein-2␣-related factor.

Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors

“…The same was observed for a promoter construct mutated for the binding of the AP-2-like transcription factor, described in a previous work (Allgayer et al, 1999c), and Sp1/Sp3 simultaneously ( Figure 4b, lanes 5 and 9). In addition, a u-PAR promoter construct deleted for region À148/À124 unable to bind the AP-2-like protein, Sp1 and Sp3 was not inhibited by pdcd4 expression in contrast to the wild-type u-PAR promoter ( Figure 4c, lanes 4 and 5).…”

“…We previously reported that u-PAR gene expression in cultured colon cancer is largely due to a transcriptional activation of the gene, and diverse cis-elements of the u-PAR promoter were characterized as being essential for this regulation (Wang et al, 1994;Lengyel et al, 1996;Allgayer et al, 1999c). We, therefore, asked as to whether Pdcd4 is able to regulate u-PAR gene expression at the promoter level.…”

“…We, therefore, asked as to whether Pdcd4 is able to regulate u-PAR gene expression at the promoter level. In GEO cells being characterized by high endogenous Pdcd4 and low endogenous u-PAR (Allgayer et al, 1999b;Allgayer et al, 1999c) a siRNA specifically downregulating endogenous Pdcd4 led to a significant increase of activity of a luciferase reporter driven by À398 bp upstream of the main transcriptional initiation site of the u-PAR gene (Figure 3a). The À398 bp corresponds to the basal u-PAR promoter as described previously (Lengyel et al, 1996;Allgayer et al, 1999c).…”

“…In GEO cells being characterized by high endogenous Pdcd4 and low endogenous u-PAR (Allgayer et al, 1999b;Allgayer et al, 1999c) a siRNA specifically downregulating endogenous Pdcd4 led to a significant increase of activity of a luciferase reporter driven by À398 bp upstream of the main transcriptional initiation site of the u-PAR gene (Figure 3a). The À398 bp corresponds to the basal u-PAR promoter as described previously (Lengyel et al, 1996;Allgayer et al, 1999c). In RKO cells, being characterized by a high endogenous u-PAR promoter activity and u-PAR gene expression (Allgayer et al, 1999a, c), transient transfections with a CAT reporter driven by the À398 bp u-PAR promoter, and increasing amounts of a pdcd4 expression construct, were performed.…”

“…As can be seen (Figure 4a), the inhibitory effect of pdcd4 expression on the extended u-PAR promoter was abolished with a promoter construct deleted for region À402/À350 which contains putative Sp1, NF-1 and GATA-2 sequences (Hapke et al, 2001). Next, we asked as to whether an AP-1 consensus motif (À190/À171, (Lengyel et al, 1996)), and a combined motif that bound an AP-2-like protein, Sp1 and Sp3 (Allgayer et al, 1999c) within the À398 bp basal u-PAR promoter, which we had characterized extensively in previous works, might participate in Pdcd4-mediated u-PAR promoter regulation. Transient transfections and CAT-assays of RKO cells with CAT-reporter-constructs driven by mutations abolishing the binding of AP-1 transcription factors to region À190/À171, Sp1/Sp3 to region À152/ À135, and AP-2/Sp1 and Sp3 to region À152/À135, respectively, were conducted in parallel to transfections with a wild-type-(À398)-u-PAR promoter-CAT reporter, and compared for their ability to abolish Pdcd4-induced promoter suppression ( Figure 4b).…”