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

Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (-402/-350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region -152/-135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.

Sp3 inhibits Sp1-mediated activation of the cardiac troponin T promoter and is downregulated during pathological cardiac hypertrophy in vivo.

Combinatorial interactions between cis elements and trans-acting factors are required for regulation of cardiac gene expression during normal cardiac development and pathological cardiac hypertrophy. Sp factors bind GC boxes and are implicated in recruitment and assembly of the basal transcriptional complex. In this study, we show that the cardiac troponin T (cTnT) promoter contains a GC box that is necessary for basal and cAMP-mediated activity of cTnT promoter constructs transfected in embryonic cardiomyocytes. Cardiac nuclear proteins bind the cTnT GC box in a sequence-specific fashion and consist of Sp1, Sp2, and Sp3 protein factors. By chromatin immunoprecipitation, Sp1 binds the cTnT promoter "in vivo." Cotransfected Sp1 trans-activates the cTnT promoter in cardiomyocytes in culture. Sp3 represses Sp1-mediated transcriptional activation of the cTnT gene in embryonic cardiomyocytes. Sp3 repression of Sp1-mediated cTnT promoter activation is dose dependent, inferring a mechanism of competitive binding/inhibition. To evaluate the role of Sp factors in cardiac gene expression in vivo, we have established a clinically relevant animal model of pathological cardiac hypertrophy where the fetal cardiac program is activated. In this animal model, cardiac hypertrophy results from increased left-right shunting, volume loading of the left ventricle, and pressure loading of the right ventricle. Sp1 expression is increased in all four hypertrophied cardiac chambers, whereas Sp3 expression is diminished. This observation is consistent with the in vitro activating function of Sp1 and inhibitory effects of Sp3 on activity of cTnT promoter constructs. Sp factor levels are modulated during the hypertrophic cardiac program in vivo.