β₃A-Integrin Downregulates the Urokinase-Type Plasminogen Activator Receptor (u-PAR) through aPEA3/ets Transcriptional Silencing Element in the u-PAR Promoter

Abstract

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that ␤ 3 -integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition ␤ 3 -integrin regulates u-PAR expression. Overexpression of ␤ 3 -integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (؊1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced ␤ 3 -integrin expression. A region between ؊398 and ؊197 bp of the u-PAR promoter was critical for ␤ 3 -integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at ؊248 bp substantially impaired the ability of ␤ 3 -integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when ␤ 3 -integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the ␤ 3 A-integrin isoform but not by other ␤ 3 -integrin isoforms and required the cytoplasmic membrane NITY 759 motif. Moreover, overexpression of the short but not the long isoform of the ␤ 3 -integrin adapter protein ␤ 3 -endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of ␤ 3 -integrin and u-PAR at the cell surface, ␤ 3 signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

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

“…To corroborate these results with an extended promoter construct including additional upstream regulatory motifs, the experiment was repeated using a luciferase reporter driven by À1498 bp upstream of the main transcriptional start site of the u-PAR gene. This construct contains additional putative enhancer/ silencer motifs (Hapke et al, 2001). As can be seen, an inhibition of activity could also be observed for the Figure 1 Reciprocal expression of Pdcd4 and u-PAR in 14 cancer cell lines, and inverse correlation of Pdcd4 with u-PAR protein/ mRNA in resected colorectal cancer.…”

“…As a first step to narrow down putative sequences within the extended u-PAR promoter (À1498), luciferase constructs driven by diverse deletions of defined promoter regions (Hapke et al, 2001) were compared to the wild-type À1498 promoter for Pdcd4-induced promoter suppression, in transient transfections of RKO cells. 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).…”

“…As a first step to narrow down putative sequences within the extended u-PAR promoter (À1498), luciferase constructs driven by diverse deletions of defined promoter regions (Hapke et al, 2001) were compared to the wild-type À1498 promoter for Pdcd4-induced promoter suppression, in transient transfections of RKO cells. 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.…”

“…Since our results regarding promoter region À152/ À135 indicated an important role of Sp family transcription factors in Pdcd4-mediated u-PAR gene expression, we performed additional EMSA analyses for region À402/À350, which contains a putative Sp-1, GATA-2 and NF-1 motifs (Hapke et al, 2001) and which was indicated as an additional mediator in previous reporter experiments (see Figure 4a). EMSA analysis for the GATA-2 and NF-1 motif within this region did not show any differences in transcription factor binding between pdcd4-and mock-transfected cells (data not shown).…”

“…Further promoter regions containing putative transcription factor binding sites, e.g. À99/À70 (Sp-1 motifs driving basal expression), or À402/À350 (putative Sp-1, GATA-2 and NF-1 motifs) have been described (Hapke et al, 2001). Thus, expression of the invasion-related gene u-PAR is transcriptionally regulated by diverse promoter elements.…”