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.     

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.     

Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor alpha through its interaction with Sp1

In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.     

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.     

Activation of adenosine deaminase in MCF-7 cells through IGF-estrogen receptor alpha crosstalk

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.     

MEK1,2 response element mediates angiotensin II-stimulated plasminogen activator inhibitor-1 promoter activation

The MEK1,2 (MAPK/ERK kinase 1 and 2) pathway mediates the up-regulation of plasminogen activator inhibitor-1 (PAI-1) expression in vascular smooth muscle cells by a variety of hormones, including angiotensin II. Transfection of constitutively active MEKK-1, an upstream activator of the mitogen-activated protein (MAP) kinase pathways, was used to isolate an enhancer element located between -89 and -50 bp in PAI-1 promoter that was activated by MEKK-1 and selectively blocked by the MEK1,2 inhibitor PD98059. Mutational analysis revealed that the MEKK-1 response element (MRE) contained 2 cis-acting Sp1- and AP-1-like sequences, located between -75 to -70 and -63 to -52 bp, respectively. Overexpression of Sp1 enhanced MEKK-1-induced MRE promoter activity and a dominant-negative c-Fos blocked this Sp1 response. The combination of Sp1 and c-Jun or c-Fos was required to activate this MRE. Angiotensin II (Ang II) stimulation increased c-Fos, c-Jun, and Sp1 binding to the MRE by 100-, 4.9-, and 1.9-fold, respectively, and these responses were inhibited by PD98059 and AT1 receptor antagonist candesartan. Intravenous Ang II infusion in rats increased aortic c-Fos binding to the MRE. This MRE sequence mediated a 4-fold increase of MEK1,2-dependent PAI-1/luciferase mRNA expression by angiotensin II stimulation. This report identifies the MEK1,2 response element that mediates angiotensin II-stimulated PAI-1 promoter activation and shows that activation of this element requires Sp1 and AP-1 co-activation.     

The effect of mechanical stretch on cyclooxygenase type 2 expression and activator protein-1 and nuclear factor-kappaB activity in human amnion cells

Stretch of the uterus plays a role in parturition. Uterine stretch also leads to stretch of the fetal membranes, including the amnion, an important source of prostaglandin E2 (PGE2). We tested the hypothesis that stretch of the amnion leads to increased cyclooxygenase (COX)-2 expression and PGE2 synthesis and investigated the mechanisms involved. We obtained amnion from women undergoing term elective cesarean section and isolated amnion epithelial cells. These cells were subjected to 11% static stretch. Stretch increased COX-2 expression and PGE2 production. EMSA studies showed that stretch increased both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) DNA binding at 1 and 6 h. In contrast, IL-1beta increased both AP-1 and NF-kappaB DNA binding at 1 h only. Chromatin immunoprecipitation studies confirmed that stretch increased binding of NF-kappaB to the COX-2 promoter in vivo. Stretch had no effect on inhibitory-kappaBalpha (IkappaBalpha) levels at the early time points but caused a decrease at 4 h. IL-1beta stimulation decreased IkappaBalpha levels after 30 min. MG132, a proteasome inhibitor, inhibited only the second stretch-induced increase in NF-kappaB binding. This suggests that stretch initially activates NF-kappaB via a nonclassical pathway, which does not involve the inhibitory-kappa kinase-induced degradation of IkappaBalpha. The second peak of NF-kappaB activation may be mediated by the classical mechanism. Stretch of the amnion may contribute to increased expression of COX-2- and other AP-1- and NF-kappaB-regulated genes with the onset of labor in the human.     

Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells

The cad gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. Cad gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nM 17beta-estradiol (E2) resulted in a 3- to 5-fold increase in cad mRNA levels in both cell lines. The mechanism of hormone-induced cad gene expression was further investigated using constructs containing the growth-responsive -90 to +115 (pCAD1) region of the cad gene promoter. E2 induced reporter gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the cad gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor alpha interact with the GC-rich region of the cad gene promoter. Moreover, in transactivation assays with pCAD1, hormone-induced transactivation was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1, which silences Sp1 expression in the cells. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the cad gene is also regulated by a nonclassical ERalpha/Sp1-mediated pathway.