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

Abstract

Long tracts of CCG trinucleotide or CCGNN pentanucleotide repeats in DNA have previously been shown to resist assembly into nucleosomes. This may provide a molecular explanation for the nature of certain rare, folate-sensitive fragile sites in human chromosomes that contain expanded CCG triplet tracts. Further, it is known that methylation of CpG dinucleotides at or near these fragile sites enhances the fragile phenotype. Here DNAs containing 76 tandem CCG triplets or 48 CCGNN pentanucleotide repeats were methylated with SssI methylase at three different levels of methylation. Using competitive nucleosome reconstitution/gel shift assays, the ability of these DNAs and a mixed sequence DNA from the pUC19 plasmid were compared in their ability to assemble into nucleosomes. DNA methylation had no significant effect on nucleosome formation over the pUC 19 fragment. However, the highly methylated DNAs containing 76 CCG triplets or 48 CCGNN pentanucleotide repeats were 2.0 +/- 0. 2-fold and 2.1 +/- 0.3-fold less efficient in nucleosome assembly than the respective unmethylated forms, and 4.4 +/- 0.4-fold and 12. 6 +/- 1.6-fold less efficient than a pUC19 fragment of similar length.

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

“…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.…”

“…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).…”

“…Our present work suggests that the suppression of migration and invasion by Pdcd4 is, at least in part, brought about by a downregulation of the urokinase receptor (u-PAR), which, together with its binding enzyme u-PA and its specific inhibitor PAI-1, has been well established as a proteolytic system promoting invasion/metastasis and a poor clinical prognosis in diverse cancers (Duffy, 1987;Blasi, 1993;Heiss et al, 1996;Muehlenweg et al, 2001). Although it is well established that u-PAR is regulated by many growth factors, oncogenes (Boyd and Brattain, 1989;Lengyel et al, 1996;Allgayer et al, 1999a-c) and signaling cascades promoting proliferation/progression (AguirreGhiso et al, 2001;Aguirre-Ghiso et al, 2003;Ahmed et al, 2003), no tumor suppressor gene has been identified so far regulating u-PAR gene expression (Fuchs and Allgayer, 2003). According to previous studies, the regulation of u-PAR gene expression has largely been observed at the transcriptional level (Wang et al, 1994;Wagner et al, 1995;Lengyel et al, 1996), and correspondingly, we showed that Pdcd4 reduces u-PAR promoter activity, and that siPdcd4 induces u-PAR mRNA, in our cell lines studied, suggesting transcriptional regulation.…”

“…In our recent work, we had shown that a motif spanning region À152/À135 of the u-PAR promoter bound by an AP-2-like transcription factor, Sp3 and Sp1, was required for a high u-PAR gene expression in cultured colon cancer cells, the AP-2-like protein mediating a high constitutive and PMA-inducible expression, Sp1, a high u-PAR gene expression caused by the c-src-oncogene (Gallick and Jessup, 1992;Allgayer et al, 1999a, c). Another AP-1 consensus motif (À190/À171) regulates constitutive and phorbol ester-induced u-PAR gene expression in colon cancer, as well as u-PAR gene expression brought about by mutation-activated K-ras (Lengyel et al, 1996;Allgayer et al, 1999b). A PEA3/etssilencer-motif further upstream (at À248 bp) mediates a downregulation of u-PAR gene expression by b 3 -integrin.…”