Vascular endothelial growth factor receptor-2 expression is induced by 17beta-estradiol in ZR-75 breast cancer cells by estrogen receptor alpha/Sp proteins

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.     

Mechanism of transcriptional regulation of LRP16 gene expression by 17-beta estradiol in MCF-7 human breast cancer cells

LRP16 gene expression is induced by 17-betaestradiol (E2) via estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. A previous study also demonstrated that ectopic expression of LRP16 gene promoted MCF-7 cell proliferation. To explore the mechanism of hormone-induced LRP16 gene expression, the LRP16 gene promoter region (-2600 to -24 bp upstream of the LRP16 gene translation starting site) was analyzed in the present study by using different 5'-truncated constructs, and a luciferase reporter. The 5'-flanking sequence of -676 to -24 bp (pGL3-S5) was found to be E2-responsive. After exchange of the fragment from -213 to -24 bp with the TK gene proximal promoter region in pGL3-S5, E2 still induced reporter gene activity in MCF-7 and HeLa cells. Sequence analysis showed that the pGL3-S6 (-676 to -214) sequence contains two motifs that may contribute to E2-induced transactivation; namely, an estrogen-responsive element (ERE) half-site/Sp1 at -246 to -227 bp and an E-box site at -225 to -219 bp. Further deletion and mutation analysis of these two motifs indicated that both the 1/2 ERE and Sp1 binding sites were required for E2 action, while E-box deletion did not affect the luciferase activity in MCF-7 and HeLa cells. The results of gel mobility shift and chromatin immunoprecipitation assays confirmed that both ERalphaand Sp1 were required for hormone-induced transactivation, which involved both ERalphaand Sp1 directly binding to DNA. Taken together, these findings suggest that ERalphaand Sp1 play a role in activation of the human LRP16 gene promoter.     

Transcriptional activation of cathepsin D gene expression by growth factors

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.     

Identification and characterization of a response element in the EGFR promoter that mediates transcriptional repression by 1,25-dihydroxyvitamin D3 in breast cancer cells

Reduction of epidermal growth factor receptor (EGFR) mRNA and protein by 1,25-dihydroxyvitamin D3 has been documented in MCF7, T47D, and BT549 breast cancer cells. In the present report, functional mapping of the EGFR promoter in BT549 cells has revealed a sequence of DNA between nucleotide positions -536 and -478 that resembles a consensus vitamin D response element (VDRE) and confers a vitamin D response upon both the homologous and a minimal heterologous promoter. In vitro footprinting and gel shift assays demonstrate the presence of an unidentified nuclear factor that is required for strong binding of the vitamin D receptor (VDR) to this putative VDRE. An Sp1 binding site was also identified in close proximity and shown to bind Sp1 from nuclear extract. Mutational analysis and functional studies using a minimal heterologous promoter provide evidence that the VDR in concert with an unknown nuclear partner mediates basal EGFR repression through displacement of Sp1 which is augmented in the presence of a ligand.     

Interaction of Sp1 with the human gamma globin promoter: binding and transactivation of normal and mutant promoters

We have analyzed the binding of Sp1, a ubiquitously expressed transactivator, to the promoter region of the gamma genes. Low-affinity Sp1 sites were found at -50 and -200. A high-affinity site was detected at -140, over the CACCC sequence. To analyze the function of these sites, Drosophila SL-2 cells, which lack Sp1, were cotransfected with an Sp1 expression plasmid and gamma globin promoter-CAT constructs. In these assays, the gamma promoter was significantly stronger in the presence than in the absence of Sp1. Thus, the three Sp1 sites in the gamma promoter allow binding as well as transactivation of the promoter. The majority of this transactivation was due to the strong binding site at -140 because introduction of a point mutation at -144 (CACCC----AACCC) reduced Sp1-dependent promoter strength by 57%. Analysis of the -200 region suggested that in the wild-type promoter, Sp1 binding at this site contributes little to promoter strength. However, a point mutation (-198 T----C) associated with hereditary persistence of fetal hemoglobin (HPFH) dramatically increased the affinity of this site for Sp1 and significantly increased Sp1 dependent promoter strength in SL-2 cells. Three other point mutations associated with HPFH did not significantly affect the interaction of Sp1 with the -200 region.