JME
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Journal of Molecular Endocrinology (2007) 39 289-304    DOI: 10.1677/JME-07-0043
© 2007 Society for Endocrinology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, S.
Right arrow Articles by Safe, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, S.
Right arrow Articles by Safe, S.

Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells

 Shaheen Khan1, Fei Wu2, Shengxi Liu3, Qian Wu2 and Stephen Safe1,3

Departments of 1 Veterinary Physiology and Pharmacology and 2 Biochemistry and Biophysics,, Texas A & M University, College Station, Texas 77843-4466, USA 3 The Institute of Biosciences and Technology,, Texas A & M University Health Science Center, 2121 West Holcombe Boulevard, Houston, Texas 77030, USA

(Correspondence should be addressed to S Safe; Email: ssafe{at}cvm.tamu.edu)

The authors have nothing to disclose.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Deletion analysis of several 17ß-estradiol (E2)-responsive genes have identified GC-rich sites that are associated with hormone-induced transactivation in MCF-7 breast cancer cells. However, the role of individual specificity proteins (Sps) in mediating hormone-induced gene expression has not been unequivocally determined. In transient transfection studies using E2-responsive GC-rich promoters from the E2F1, carbamoylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), and retinoic acid receptor {alpha} (RAR{alpha}) genes, RNA interference using small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) decreased both basal and E2-induced transactivation. The contributions of individual Sp proteins to basal and E2-induced activity were promoter dependent. iSp1, iSp3, and iSp4 also significantly inhibited hormonal induction of E2F1, CAD, and RAR{alpha} mRNA levels; however, the enhanced inhibitory effects of the latter two small inhibitory RNAs suggest that Sp3 and Sp4 play a major role in estrogen receptor {alpha}/Sp-mediated gene expression in MCF-7 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The estrogen receptor (ER) is a member of the nuclear receptor superfamily of transcription factors and is required for mediating 17ß-estradiol (E2)-induced responses in multiple tissues and organs (Chow et al. 1992, Turner et al. 1994, Farhat et al. 1996, Clark 1998). Two major ER subtypes (ER{alpha} and ERß) and multiple splice variants have been identified and these various forms exhibit both overlapping and different activities (Hall et al. 2001, Olefsky 2001, Nilsson & Gustafsson 2002, Matthews & Gustafsson 2003). ER{alpha} and ERß exhibit the typical domain structures of nuclear receptors, and similarities in their DNA binding (C) and ligand binding (EF) domains are correlated with comparable mechanisms of DNA binding and similar but not identical interactions with various estrogenic ligands (Hall et al. 2001, Olefsky 2001, Nilsson & Gustafsson 2002, Matthews & Gustafsson 2003). The N-terminal regions (AB) of human ER{alpha} and ERß exhibit only 17% amino acid homology and this variability may account for some of the differences between the ER subtypes such as those observed in the murine uterus where ERß tends to inhibit ER{alpha}-dependent uterotrophic responses (Krege et al. 1998, Weihua et al. 2000).

The classical ER mechanism of action involves ligand-induced formation of an ER homodimer which interacts with estrogen responsive elements (EREs; Klein-Hitpass et al. 1986, Cowley et al. 1997, Hyder et al. 1999) in target gene promoters and recruits cofactors necessary for transactivation. There is an increasing evidence that the classical genomic ER–ERE complex formation is only one of several genomic and non-genomic pathways of estrogen action (Blobel & Orkin 1996, Delfino & Walker 1999, Kelly & Wagner 1999, Kushner et al. 2000, Watson et al. 2002, Razandi et al. 2004, Safe & Kim 2004, Levin 2005). Genomic ER associates with other transcription factors such as the activating protein-1 (AP-1) complex, nuclear factor kappa B (NF{kappa}B), and specificity proteins (Sps) to modulate ligand-dependent gene expression. For example, studies in this laboratory and others have demonstrated that deletion and mutational analysis of promoters derived from several E2-responsive genes contain GC-rich motifs that bind Sp proteins (Safe & Kim 2004). Some of the genes that fall into this category are important for cell proliferation, angiogenesis, and nucleotide metabolism and include E2F1, retinoic acid receptor {alpha} (RAR{alpha}), carbamoylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), bcl-2, DNA polymerase {alpha}, vascular endothelial growth factor (VEGF), VEGFR receptor 2 (VEGFR2), progesterone receptor, creatine kinase B, thymidylate synthase, insulin-like growth factor binding protein 4, epidermal growth factor receptor, receptor for advanced glycation end products, low density lipoprotein receptor, vitamin D receptor, pS2, LRP16, metastasis associated protein 3, and SK3 (Porter et al. 1996, 1997, Duan et al. 1998, Sun et al. 1998, 2005, Dong et al. 1999, Qin et al. 1999, Wang et al. 1999, 2002, Xie et al. 1999, 2000, Byrne et al. 2000, Salvatori et al. 2000, Saville et al. 2000, Stoner et al. 2000, 2004, Tanaka et al. 2000, Castro-Rivera et al. 2001, Li et al. 2001, Samudio et al. 2001, Bruning et al. 2003, Jacobson et al. 2003, Khan et al. 2003, Ngwenya & Safe 2003, Schultz et al. 2003, 2005, Fujita et al. 2004, Bardin et al. 2005, Zhao et al. 2005, Higgins et al. 2006b). In addition, RNA interference studies with a small inhibitory RNA (siRNA) for Sp1 (iSp1) show that after transfection with iSp1, there was a significant decrease in hormone-induced G0/G1 to S-phase progression, suggesting that ER{alpha}/Sp1 plays an important role in this process (Abdelrahim et al. 2002).

MCF-7 cells express multiple Sp proteins including Sp1, Sp3, and Sp4, and the role of these individual proteins in mediating E2-induced transactivation has not been determined. Using the E2F1 gene as a model, we now show that hormonal activation of a GC-rich E2F1 promoter construct is inhibited in cells cotransfected siRNAs for Sp1, Sp3 (iSp3), and Sp4 (iSp4), and using this approach, it was shown that these Sp proteins are also important for hormone-induced expression of E2F1 mRNA and protein. Similar results were observed for two other E2-responsive genes, CAD and RAR{alpha}, demonstrating that all three Sp proteins and particularly Sp3 and Sp4 are important for ER{alpha}/Sp-mediated gene expression in MCF-7 cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells, chemicals, biochemicals, and plasmids

MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in DME/F12 (Sigma Chemical Co.) media supplemented with 5% fetal bovine serum (FBS; JRH Biosciences, Lenaxa, KS, USA; or Atlanta Biologicals Inc., Norcross, GA, USA), 1.5 g/l sodium bicarbonate, and 10 ml/l antibiotic–antimycotic solution (Sigma). Cells were maintained in incubators at 37 °C under humidified 5% CO2: 95% air. Dimethyl sulfoxide (Me2SO), PBS, and E2 were purchased from Sigma. Reporter lysis buffer and luciferase assay reagent were purchased from Promega Corp. and/or Boehringer Mannheim. ß-Galactosidase activity was measured using Tropix Galacto-Light Plus assay system (Tropix, Bedford, MA, USA). Instant Imager and Lumicount micro-well plate reader were purchased from Packard Instrument Co. (Downers Grove, IL, USA). ß-Actin antibody was obtained from Sigma and all other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human ER{alpha} expression plasmid was originally provided by Dr Ming-Jer Tsai (Baylor College of Medicine, Houston, TX, USA) and was recloned into pcDNA3 in this laboratory. Promoter luciferase construct pRAR{alpha}2 (–79/–49) was made by inserting the oligonucleotide spanning –79 to –49 of the human RAR{alpha}1 promoter into the pGL2 basic luciferase reporter plasmid (Promega Corp). CAD promoter luciferase construct pCAD2 (–90/+25) was kindly provided by Dr Peggy Farnham (University of Wisconsin, Madison, WI, USA). E2F1 promoter luciferase construct pE2F1h was made in this laboratory as described (Wang et al. 1999). Plasmid pSp13 containing three consensus Sp1 binding sites linked to a luciferase reporter gene was cloned into pXP2 plasmid in this laboratory.

Transient transfection assays of small inhibitory RNA

Validated, non-targeting small inhibitory RNA (siRNA; Silencer Negative Control siRNA; iNS1) was purchased from Ambion (Austin, TX, USA) and siRNAs for Sp1, Sp3, and Sp4 were purchased from Dharmacon Research (Lafayette, CO, USA). MCF-7 cells were seeded in DME/F12 medium supplemented with 2.5% charcoal-stripped serum overnight in 12-well plates. After 16–20 h, siRNA duplexes were transfected using Lipofectamine 2000 Reagent (Invitrogen Life Technologies). After 24 h, reporter plasmids (200 ng) were cotransfected with ER{alpha} expression plasmid (150 ng) and 50 ng pCDNA3.1–His-LacZ using GeneJuice (Novagen, EMD Biosciences Inc., Madison, WI, USA) according to the manufacturer's protocol. Cells were then treated for 36 h with Me2SO or E2 and harvested in 100 µl of cell lysis buffer (Promega Corp). Luciferase activities in the various treatment groups were performed on 20 µl of cell extract using the luciferase assay system (Promega Corp.) in a luminometer (Packard Instrument Co., Meriden, CT, USA) and results were normalized to ß-galactosidase enzyme activity, which was carried out on 20 µl of cell extract. Sequences of siRNA oligonucleotides for Sp1, Sp3, and Sp4 are summarized below.

  1. 5'-AUC ACU CCA UGG AUG AAA UGA dTdT-3'
  2. 5'-GCG GCA GGU GGA GCC UUC ACU dTdT-3'
  3. 5'-GCA GUG ACA CAU UAG UGA GC dT dT-3'

Preparation of whole cell extracts and western blot analysis

MCF-7 cells were seeded into six-well plates in DME/F12 medium supplemented with 2.5% charcoal-stripped serum. The next day, cells were transfected with siRNAs using Lipofectamine 2000 reagent according to the manufacturer's protocol. After 55–60 h, cells were dosed with Me2SO and E2 for 18 h and then harvested with ice-cold lysis buffer (50 mM HEPES (pH 7.5), 500 mM NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, and 1 mM EGTA) and supplemented with protease inhibitor cocktail (Sigma). Equal amounts of protein from each treatment group were boiled in 1x sample buffer (50 mM Tris–HCl, 2% SDS, 0.1% bromphenol blue, and 175 mM ß-mercaptoethenol) for 5 min and separated on 7.5 or 10% gel, and then transferred to polyvinylidene difluoride membrane (Bio-Rad) overnight at 30 V. Membranes were blocked in Blotto (5% milk, Tris-buffered saline (10 mM Tris–HCl (pH 8.0) and 150 mM NaCl), and 0.05% Tween 20) for 30 min and probed with primary antibodies for 2–4 h. Membranes were washed for 30 min in 1x TBS-Tween, probed with peroxidase-conjugated secondary antibody for 1–2 h, and then washed in 1x TBS-Tween for 30 min. Ten millilitres of HRP substrate (Dupont-NEN, Boston, MA, USA) were added and incubated for 1 min and visualized by autoradiography. Protein band intensities were scanned on a JX-330 scanner (SHARP Corp., Mahwah, NJ, USA) using Adobe Photoshop 3.0 (Adobe Systems Inc., San Jose, CA, USA).

Immunohistochemistry

For ER{alpha}-Sp1 colocalization experiments, MCF-7 cells were seeded onto two-well glass chamber slides (Nalge Nunc International, Naperville, IL, USA) at 100 000 cells per well in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 5% charcoal stripped FBS, incubated in a 37 °C incubator with 5% CO2 for 24 h, and treated with Me2SO vehicle or 10 nM E2 for 1 h. Slides were then washed with PBS, fixed with –20 °C methanol, air dried, and washed with PBS +0.3% Tween 20 (PBS/Tween). Slides were blocked for 1 h with 5% donkey serum in antibody dilution buffer (1% BSA in PBS/Tween), washed with PBS/Tween briefly, incubated with anti-Sp1 PEP-2 (goat) antibody (Santa Cruz) at 1:100 dilution (goat serum for controls) in antibody dilution buffer at 4 °C for 12 h. Slides were washed with PBS/Tween (3x10 min), incubated with donkey anti-goat immunoglobulin G (IgG) fluorescein isothiocyanate (FITC) (Santa Cruz) at 1:200 dilution in antibody dilution buffer for 1 h and washed with PBS/Tween (3x10 min). Slides were subsequently blocked with 5% donkey serum in antibody dilution buffer for 1 h, washed with PBS/Tween briefly, incubated with anti-ER{alpha} H-184 antibody (Santa Cruz) at 1:100 dilution in antibody dilution buffer at 4 °C for 12 h (rabbit serum for controls), washed with PBS/Tween (3x10 min), incubated with donkey anti-rabbit IgG Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) at 1:500 dilution in antibody dilution buffer for 1 h, and washed with PBS/Tween (3x10 min). Slides were finally washed in deionized water, and cover glass mounted using Prolong Gold antifade reagent with DAPI (Molecular Probes). Immunofluorescence images of Sp1 and ER{alpha} were examined using a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY, USA) fitted with an Axiocam high-resolution digital camera. Digital images were captured using Axiovision 4.1 software (Carl Zeiss).

For ER{alpha}-Sp4 colocalization experiments, COS-7 cells were seeded as described above, transfected with 250 ng of ER{alpha} and 250 ng of Sp4 expression plasmid using GeneJuice transfection reagent (Novagen), incubated at 37 °C with 5% CO2 for 24 h, and then treated with Me2SO vehicle or 10 nM E2 for 1 h, and immunostaining experiments were carried out as described above using ER{alpha} D-12 (Santa Cruz) paired with donkey anti-mouse Alexa Fluor 488 (Molecular Probes) and Sp4 V-20 (Santa Cruz) paired with donkey anti-rabbit IgG Alexa Fluor 594 (Molecular Probes) antibodies.

Real-time PCR

Cells were seeded into six-well plates in DME/F12 medium supplemented with 2.5% charcoal-stripped serum overnight. The next day cells were transfected with siRNAs using Lipofectamine 2000 Reagent and, after 55–60 h, cells were treated with Me2SO or E2 for 6 or 12 h. RNA was extracted using Qiagen RNeasy minikit (Qiagen) following the manufacturer's protocol and was reverse transcribed for cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol. The cDNA reaction mixture was then used to carry out PCR using SYBR Green PCR Master Mix from PE Applied Biosystems (Warrington, UK) on an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The relative quantitation of samples was carried out using comparative CT method and TATA binding protein (TBP) was used for normalization. Quantitect primer assays (Qiagen) for Sp1, Sp3, Sp4, E2F1, and CAD were used to perform PCR, whereas primers for RAR{alpha} and TBP were purchased from Integrated DNA technologies (Coralville, IA, USA) and are as follows:

  1. 5'-TCC ATG CCG CCT CTC ATC-3'
  2. 5'-CGG CTG TCC GCT CAG AGT-3'
  3. 5'-TGC ACA GGA GCC AAG AGT GAA-3'
  4. 5'-CAC ATC ACA GCT CCC CAC CA-3'

Chromatin immunoprecipitation (ChIP) assay

MCF-7 cells (1x107) were treated with Me2SO (time 0) or E2 (10 nM) for different times. Cells were then fixed with 1.5% formaldehyde, and the cross-linking reaction was stopped by addition of 0.125 M glycine. Cells were scraped, pelleted, and hypotonically lysed, and nuclei were collected. Nuclei were then sonicated to the desired chromatin length (~500 bp). The chromatin was precleared by addition of protein A/G-conjugated beads (Pierce, Rockford, IL, USA). The precleared chromatin supernatants were immunoprecipitated with antibodies specific to IgG, ER{alpha}, Sp1, Sp3, and Sp4 (Santa Cruz Biotechnology) at 4 °C overnight. The protein–antibody complexes were collected by addition of protein A/G-conjugated beads for 1 h, and the beads were extensively washed. The protein–DNA crosslinks were eluted and reversed. DNA was purified by Qiaquick Spin Columns (Qiagen) followed by PCR amplification. The pS2 primers are: 5'-CTA GAC GGA ATG GGC TTC AT-3' (forward) and 5'-ATG GGA GTC TCC TCC AAC CT-3' (reverse), which amplify a 209-bp region of the human pS2 promoter containing an estrogen response element (ERE). The CAD primers are: 5'-CTT GGG GTG GGA GGG ACT-3' (forward) and 5'-GCG GCA GCA GCA GAG ACT-3' (reverse), which amplify a 158-bp GC-rich region of the human CAD promoter. The RAR{alpha} primers are: 5'-GCC CTT CCC GAG GTC TAT TA-3' (forward) and 5'-GAG GGT ACG GAG CAG AGG TA-3' (reverse), which amplify a 138-bp GC-rich region of the human RAR{alpha} promoter. The E2F1 primers are: 5'-CTG gta cca tcc gga caa ag-3' (forward) and 5'-TTT TTG CCG CGA AAG AGC-3' (reverse), which amplify a 198-bp region of the human E2F1 promoter containing GC-rich Sp-binding sites. PCR products were resolved on a 2% agarose gel in the presence of 1:10 000 SYBR gold (Molecular Probes).

Statistical analysis

Experiments were repeated two or more times, and data are expressed as mean±S.E.M. for at least three replicates for each treatment group. Statistical differences between treatment groups were determined using Super ANOVA and Scheffé's test. Treatments were considered significantly different from controls if P<0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Role of Sp proteins of hormonal activation of pSp13

Analysis of several gene promoters from E2-responsive genes have identified GC-rich motifs that are required for hormone-induced transactivation in breast cancer and other cell lines (Porter et al. 1996, 1997, Duan et al. 1998, Sun et al. 1998, 2005, Dong et al. 1999, Qin et al. 1999, Wang et al. 1999, 2002, Xie et al. 1999, 2000, Byrne et al. 2000, Salvatori et al. 2000, Saville et al. 2000, Stoner et al. 2000, 2004, Tanaka et al. 2000, Castro-Rivera et al. 2001, Li et al. 2001, Samudio et al. 2001, Bruning et al. 2003, Jacobson et al. 2003, Khan et al. 2003, Ngwenya & Safe 2003, Schultz et al. 2003, 2005, Fujita et al. 2004, Bardin et al. 2005, Zhao et al. 2005, Higgins et al. 2006b). In this study, we have also used RNA interference and small inhibitory RNAs (siRNAs) for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) to investigate the role of individual Sp proteins in ER{alpha}/Sp-mediated transactivation in MCF-7 cells. Previous studies in cancer cell lines have demonstrated the specificity and effectiveness of iSp1, iSp3, and iSp4 in Sp protein knockdown (Abdelrahim et al. 2002, 2004, Higgins et al. 2006a,b), and results in Fig. 1A–C illustrate that transfected iSp1, iSp3, and iSp4 significantly decreased Sp1, Sp3, and Sp4 mRNA levels in whole cell extracts from MCF-7 cells. In parallel studies, transfected iSp1, iSp3, and iSp4 also decreased Sp1, Sp3, and Sp4 protein expression (Fig. 1D–F) in MCF-7 cells.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		Figure 1 siRNAs for Sp1, Sp3, and Sp4. Effects of (A) iSp1, (B) iSp3, and (C) iSp4 on mRNA expression. MCF-7 cells were transfected with iNS1, iSp1, iSp3, or iSp4, treated with Me2SO (D), 10 nM E2 (E), and Sp mRNA levels were determined by real-time PCR as described in the Materials and methods. Results are expressed as means±S.E.M. for three replicated determinations for each treatment group. Significantly (P<0.05) decreased mRNA levels by iSps is indicated by an asterisk. Effects of (D) iSp1, (E) iSp3, and (F) iSp4 on protein expression. MCF-7 cells were transfected with siRNAs as described above, treated with Me2SO (D) or 10 nM E2 (E) and whole cell lysates were analyzed by western blot analysis as described in Materials and methods.

 
The pSp13 construct has been extensively used as a model to investigate the mechanisms of ER{alpha}/Sp-mediated transactivation (Saville et al. 2000, Kim et al. 2003, 2005). This construct contains three tandem consensus GC-rich binding sites linked to a minimal TATA-luciferase and is E2 responsive only after cotransfection with ER{alpha} and this is due, in part, to overexpression of pSp13 in transfected cells and limiting levels of ER{alpha}. This system has been ideal for studying hormone activation of pSp13 in ER-positive or ER-negative cell lines thereby determining the contributions of cell context on the induction response (Saville et al. 2000, Kim et al. 2003, 2005). Results illustrated in Fig. 2A show that E2 induces transactivation in MCF-7 cells transfected with pSp13 and non-specific RNA (iNS1) and similar results were obtained in the presence or absence of transfected iNS1 oligonucleotide (data not shown). The results show that all three siRNAs or their combinations significantly decreased basal luciferase activity, and iSp1 and iSp4 were more effective than iSp3. The fold induction of luciferase by E2 compared with solvent (Me2SO) control shows that the effectiveness of the siRNAs for decreasing fold induction was iSp1>iSp4>iSp3 (Fig. 2B) with only minimal changes in fold induction observed for iSp4 and iSp3. The apparent lack of effectiveness of iSp4 and iSp3 for inhibiting fold induction of luciferase activity when compared with iSp1 was due, in part, to their differential inhibitory effects on basal luciferase activity. In cells transfected with iSps in combination, the maximal inhibition of fold induction was observed in cells transfected with iSp1/iSp3 and iSp1/iSp4, whereas minimal changes were observed in cells transfected with iSp3/iSp4. However, these data suggest that for the pSp13 construct containing three consensus GC-rich sites, Sp1 plays the most important role in E2-dependent activation of ER{alpha}/Sp.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2 Role of Sp proteins in hormonal activation of pSp13. MCF-7 cells were cotransfected with pSp13 and iNS1, iSp1, iSp3, iSp4, or their combinations, treated with Me2SO (D) or 10 nM E2 (E), and (A) luciferase activity or (B) fold induction of luciferase activity by E2 was determined as described in the Materials and methods. Results are expressed as means±S.E.M. for at least three replicate determinations for each treatment group, and significantly (P<0.05) decreased basal or E2-induced activity (compared with iNS1) is indicated by a double asterisk. Significant (P<0.05) induction by E2 compared with Me2SO is also indicated (*).

 
ER{alpha}/Sp-mediated activation of E2F1

E2 induces E2F1 gene expression in MCF-7 cells (Wang et al. 1999, Ngwenya & Safe 2003), and promoter analysis has identified critical GC-rich sites at between –169 and –111 in the proximal region of the E2F1 promoter. Figure 3A illustrates luciferase activity in MCF-7 cells transfected with pE2F1 (–169/–54) and various iSps or their combinations. The results show that individual siRNAs for Sp1, Sp3, and Sp4 decreased luciferase by 70–90% with iSp3 as the most effective siRNA. These results indicate that Sp proteins play the major role in regulating basal luciferase activity in MCF-7 cells transfected with pE2F1 (–169/–54). Moreover, the effectiveness of iSp1, iSp3, and iSp4 in decreasing activity suggests that the loss of any single Sp protein was important for transcription, indicating cooperative interactions among these transcription factors for regulating luciferase activity. The effects of individual and combined Sp protein knockdown in MCF-7 cells transfected with pE2F1 (–169/–54) and treated with Me2SO or E2 were also determined, and the effects on fold induction by E2 are illustrated in Fig. 3B. With the exception of the results observed for iSp3, the fold induction by E2 was decreased 50–65% by iSp1, iSp4, and their combinations (including iSp3+iSp1 and iSp3+iSp4). iSp3 only slightly decreased the fold induction by E2 and was less effective than either iSp1 or iSp4. These results were similar to those observed for the effects of iSps on hormone inducibility in MCF-7 cells transfected with pSp13 (Fig. 2A).


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Figure 3 Role of Sp proteins in hormonal activation of E2F1. (A) Luciferase activity and (B) fold induction by E2 in MCF-7 cells cotransfected with ER{alpha} expression plasmid (150 ng) and pE2F1 (–161/+54). MCF-7 cells were transfected with pE2F1 (–161/+54) and iNS1, iSp1, iSp3, iSp4, or their combinations, and luciferase activity determined as described in the Materials and methods. Hormonal activation of pE2F1 requires cotransfection with ER{alpha}. Results are expressed as means±S.E.M. for three replicate determinations for each treatment group, and significantly (P<0.05) decreased basal or hormone-induced activity (compared with iNS1) is indicated (**). Role of Sp proteins in hormonal activation of (C) E2F1 mRNA and (D) protein. Cells were transfected with iNS1, iSp1, iSp3, or iSp4, and then treated with Me2SO (D) or 10 nM E2 (E) for 12 h. Whole cell extracts were analyzed for E2F1 mRNA or protein as described in the Materials and methods. E2F1 mRNA data were determined in three separate experiments and results (normalized to TBP mRNA) are means±S.E.M.. Significant (P<0.05) inhibition of E2-induced mRNA levels by iSp1, iSp2, and iSp3 are indicated (**). Western blot analysis of whole cell lysates was determined as described in the Materials and methods. The immunoblot shown in Fig. 3D was one of two replicates that gave similar results. Significant induction by E2 compared with Me2SO (D) is indicated (*).

 
The effects of siRNAs for Sp proteins on induction of E2F1 mRNA levels in MCF-7 cells were also investigated. Cells were transfected with iNS1 (non-specific RNA), iSp1, iSp3 or iSp4 for 55–60 h, then treated with Me2SO or 10 nM E2 for an additional 12 h and mRNA levels were determined in whole cell extracts by real-time PCR. Previous studies show that within this time period, there is a maximal decrease of Sp proteins using these siRNAs (Abdelrahim et al. 2002, 2004, Higgins et al. 2006a,b). Basal E2F1 mRNA levels were significantly decreased by transfecting iSp1, iSp3, and iSp4 by 40, 86, and 77% respectively, demonstrating the role of Sp proteins in mediating E2F1 mRNA expression. E2 induced an approximately threefold increase in E2F1 mRNA expression and in cells transfected with iSp1, iSp3, and iSp4, there were significant decreases in hormone-induced E2F1 mRNA levels and cells transfected with iSp4 gave the lowest inducibility. However, the results suggest that all three Sp proteins are involved in ER{alpha}/Sp-dependent activation of E2F1. iSp3 was more effective in decreasing induction of E2F1 mRNA by E2 (Fig. 3C) than decreasing induction of luciferase activity by E2 in cells transfected with pE2F1h (Fig. 3B). This suggests that other regulatory regions of E2F1 that are affected by Sp3 are important for induction of E2F1 mRNA levels by E2. We also observed in a separate experiment that induction of E2F1 protein by E2 and basal E2F1 protein expression were also decreased in MCF-7 cells transfected with iSp1, iSp3, and iSp4 (Fig. 3D). These results demonstrate the hormone inducibility of E2F1 is dependent on Sp transcription factors. Thus, although Sp3 plays a minimal role in hormone-induced activation of pE2F1 (–169/–54), this transcription factor is important for mediating hormone-dependent induction of E2F1 mRNA and protein.

ER{alpha}/Sp-mediated activation of CAD and RAR{alpha}

The –90 to +25 region of the CAD gene promoter is also E2 responsive in transfection assays, and deletion and mutation analyses have identified three proximal GC-rich sites which are required for hormone responsiveness (Khan et al. 2003). Results in Fig. 4A show that in MCF-7 cells transfected with pCAD (–90/+25), cotransfection with iSp1, iSp3, iSp4, or their combinations significantly decreased activity (>50%); however, the magnitude of the decreased activity was significantly less than observed for pE2F1 (–169/–54; Fig. 3A). Since the magnitude of the decreased response was similar after transfection with iSp1, iSp3, or iSp4, it is likely that all three transcription factors are required for basal activity. In MCF-7 cells transfected with pCAD (–90/+25) plus iNS1 and treated with Me2SO or E2, cotransfection with iSp1, iSp3, iSp4, or their combinations significantly decreased hormone-induced transactivation (Fig. 4B). Fold induction was decreased only slightly more effectively in cells transfected with iSp1 and iSp4 when compared with iSp3; however, all three Sp transcription factors were important for hormone responsiveness and combinations of iSps inhibited hormone inducibility more than individual siRNAs. Similar results were observed for induction of CAD mRNA levels by E2 (Fig. 4C) where the induction response was significantly decreased in MCF-7 cells transfected with iSp1, iSp3, and iSp4. In contrast, iSps had only a minimal effect of CAD mRNA levels (data not shown).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Figure 4 Role of Sp proteins in hormonal activation of CAD. (A) Luciferase activity and (B) fold induction by E2 in MCF-7 cells cotransfected with ER{alpha} expression plasmid (150 ng) and pCAD (–90/+25). MCF-7 cells were transfected with pCAD (–90/+25) and iNS1, iSp1, iSp3, iSp4, or their combinations, and luciferase activity determined as described in the Materials and methods. The requirement of ER{alpha} for hormonal activation of this construct is also indicated (B). Results are expressed as means±S.E.M. for three replicate determinations for each treatment group, and significantly (P<0.05) decreased basal or hormone-induced activity (compared with iNS1) is indicated (**). The effects of ER expression plasmid on hormone inducibility is also indicated. (C) Role of Sp proteins in hormonal activation of CAD mRNA. MCF-7 cells were transfected with iNS, iSp1, iSp3, or iSp4, treated with Me2SO (D) or 10 nM E2 (E) for 6 h, and mRNA levels determined as described in the Materials and methods. CAD mRNA levels (normalized to TBP mRNA) are means±S.E.M. for at least three determinations and significantly (P<0.05) decreased hormone-induced CAD mRNA is indicated (**). Significant induction by E2 compared with ME2SO is indicated (*).

 
We also investigated the role of Sp proteins in hormonal activation of RAR{alpha} since previous studies have identified proximal GC-rich motifs at –68 to –62 and –59 to –52 required for hormonal activation of promoter constructs (Sun et al. 1998). In contrast to the pCAD (–90/+25) and pE2F1 (–169/–54) constructs, basal luciferase activity was not decreased in MCF-7 cells transfected with pRAR{alpha} (–79/–49) and iSp1 or iSp4 but was significantly decreased only after cotransfection with iSp3 (Fig. 5A). Cotransfection with iSp combinations (except iSp1+iSp3) decreased activity; however, it was evident that despite the presence of GC-rich motifs in pRAR{alpha} (–79/–49), Sp proteins were not the predominant transcription factors required for basal activity. In contrast, fold inducibility of luciferase activity in MCF-7 cells treated with Me2SO or 10 nM E2 and transfected with pRAR{alpha} (–79/–49) was primarily decreased in cells transfected with iSp1 and iSp4 but not iSp3 (Fig. 5B). Since both iSp1 and iSp4 significantly decreased hormone responsiveness, any combination of these siRNAs inhibited E2-induced transactivation. Thus, hormone-dependent activation of pRAR{alpha} (–79/–49) was primarily dependent on ER{alpha}/Sp1 and ER{alpha}/Sp4, and this was similar to results obtained with pSp13 (Fig. 2B) and pE2F1 (–169/–54; Fig. 3B). Basal expression of RAR{alpha} mRNA was only minimally affected by iSps (data not shown); however, induction of RAR{alpha} mRNA levels by E2 was decreased after cotransfection with iSp1, iSp3, and iSp4 with iSp3 being the most effective siRNA (Fig. 5C). iSp3 completely abrogated induction of RAR{alpha} by E2, whereas in transfection studies with pRAR{alpha}2, which contains a –79 to –49 RAR{alpha} promoter insert, iSp3 did not significantly decrease hormone responsiveness (Fig. 5B). These results suggest that GC-rich sites in addition to those at –79 to –49 may be important for ER{alpha}/Sp-mediated induction of RAR{alpha} mRNA by E2.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Figure 5 Role of Sp proteins in hormonal activation of RAR{alpha}. (A) Luciferase activity and (B) fold induction by E2 in MCF-7 cells cotransfected with ER{alpha} expression plasmid (150 ng) and pRAR{alpha} (–79/–49). MCF-7 cells were transfected with pRAR{alpha} (–79/–49) and iNS1, iSp1, iSp3, iSp4, or their combinations, and luciferase activity determined as described in the Materials and methods. Hormonal activation of pRAR{alpha} requires cotransfection with ER{alpha}. Results are expressed as means±S.E.M. for three replicate determinations for each treatment group, and significantly (P<0.05) decreased basal or hormone-induced activity (compared with iNS1) is indicated (**). (C) Role of Sp proteins in hormonal activation of RAR{alpha} mRNA. MCF-7 cells were transfected with iNS, iSp1, iSp3, or iSp4, treated with Me2SO (D) or 10 nM E2 (E) for 12 h, and mRNA levels determined as described in the Materials and methods. RAR{alpha} mRNA levels (normalized to TBP mRNA) are expressed as means±S.E.M. for at least three determinations and significantly (P<0.05) decreased induction (fold) of RAR{alpha} mRNA by E2 is indicated (**). Significant induction by E2 compared with ME2SO is indicated (*).

 
Colocalization of ER{alpha} with Sp1 and Sp4

Previous studies showed that ER{alpha} interacts with Sp1 and Sp3 (Porter et al. 1996, 1997, Saville et al. 2000, Stoner et al. 2000); however, the results of RNA interference assays now demonstrate that, in MCF-7 cells, Sp4 plays an important role in E2-dependent activation of ER{alpha}/Sp. Therefore, we have investigated colocalization of ER{alpha} and Sp1 in MCF-7 cells, which exhibit endogenous expression of both proteins (Fig. 6A). ER{alpha} and Sp1 proteins alone exhibit nuclear staining and the colocalized proteins exhibit a punctate staining pattern, and these results complement previous studies showing direct interactions of both proteins (Porter et al. 1997). Colocalization experiments with ER{alpha} and Sp4 were examined in COS-7 cells transfected with ER{alpha} and Sp4 expression plasmids (Fig. 6B). Immunostaining of endogenous Sp4 in MCF-7 cells was weak due to the relative insensitivity of the commercially available antibodies; however, in transfected COS-7 cells, Sp4 staining is observed and is more intense in the central part of the nucleus. ER{alpha} alone is uniformally observed in the nucleus and, after treatment with E2, a more punctate staining is observed and the colocalized ER{alpha}/Sp4 also exhibited punctate staining. Several attempts to investigate colocalization of ER{alpha} and Sp3 were unsuccessful, due to the failure of available Sp3 antibodies to detect Sp3 by this method. However, these results demonstrate that ER{alpha} colocalizes with Sp1 and Sp4 and these transcription factors play an important role in ER{alpha}/Sp-mediated expression of E2F1, CAD, and RAR{alpha} in MCF-7 cells. However, the effects of E2 on colocalization are minimal, suggesting interactions of ER{alpha} with Sp proteins in the presence or absence of hormone, and this is consistent with previous ChIP assays (Higgins et al. 2006a) and the reported coimmunoprecipitation of ER{alpha} and Sp1 (±hormone; Saville et al. 2000).


Figure 6
View larger version (31K):
[in this window]
[in a new window]

  		Figure 6 Colocalization of ER{alpha} with Sp1 and Sp4 in (A) MCF-7 and (B) COS-7 cells. MCF-7 and COS-7 cells were seeded in two-well chamber slides. The latter cells were transfected with ER{alpha} and Sp4 expression plasmids. Both cell lines were treated with Me2SO or 10 nM E2 for 1 h, and cells were immunostained as described in the Materials and methods.

 
Interactions of ER{alpha} and Sp proteins with the pS2, E2F1, CAD, and RAR{alpha} gene promoters

Previous studies show that the E2-responsive region of the pS2 gene promoter contains GC-rich motifs and ChIP analysis of the pS2 promoter (Fig. 7A) shows constitutive binding of Sp1, Sp3, and Sp4. The band intensities exhibit minimal changes after treatment with 10 nM E2 for up to 2 h. However, as previously reported (Shao et al. 2002, Metivier et al. 2003, Acevedo et al. 2004, Krieg et al. 2004, Sun et al. 2005), treatment of MCF-7 cells recruits ER{alpha} to the pS2 promoter, and this is consistent with interactions with the ERE motif at –405 to –393. Results in Fig. 7B–D summarize ChIP assays, which examine basal and hormone-induced interactions of ER{alpha}, Sp1, Sp3, and Sp4 with the GC-rich regions of the RAR{alpha}, E2F1, and CAD gene promoters respectively. Sp1, Sp3, and Sp4 are constitutively bound to all three promoters and treatment with E2 does not increase or decrease Sp interactions with these promoters. Moreover, similar results were also observed for ER{alpha} which was bound to the GC-rich RAR{alpha}, E2F1, and CAD gene promoters in the presence or absence of hormone treatment. Since Sp1, Sp3, and Sp4 are important for basal expression of the genes examined in this study, it is not surprising that these proteins are associated with these GC-rich promoters. These results are consistent with the known ligand-independent interactions of ER{alpha} with Sp proteins (Porter et al. 1997, Saville et al. 2000, Stoner et al. 2000), and the colocalization of ER{alpha} with Sp1 and Sp4 in Me2SO (and E2)-treated cells (Fig. 6). Moreover, we have recently reported similar ER{alpha} and Sp interactions with the VEGFR2 promoter in ZR-75 cells using a ChIP assay (Higgins et al. 2006b). Our results demonstrate for the first time that hormonal activation of genes containing E2-responsive GC-rich promoters in MCF-7 cells requires ER{alpha} interactions with Sp1, Sp3, and Sp4.


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Figure 7 ChIP assay of ER{alpha} and Sp protein interactions with E2-responsive gene promoters. MCF-7 cells were treated with Me2SO (D) or 10 nM E2 for different time points, and the interaction of ER{alpha} and Sp proteins to the (A) pS2, (B) RAR{alpha}, (C) E2F1, and (D) CAD gene promoters was determined in a ChIP assay as described in the Materials and methods. Similar results were obtained in duplicate experiments. We also observed specific binding of transcription factor IIB to the glyceraldehyde 3-phosphate dehydrogenase promoter and not to exon 1 of the CNAP1 gene (data not shown). This serves as an additional positive control for the ChIP assay (Higgins et al. 2006b).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The mechanism of ligand-dependent activation of nuclear receptors involves a simple concept in which receptor ligands induce formation of a transcriptionally active receptor complex bound to a cognate response element in the target gene promoter. However, transactivation mediated by nuclear receptors such as steroid hormone receptors is highly complicated and is dependent on ligand structure, hormone receptor subtype, tissue-specific expression of coactivators, other nuclear cofactors, and promoter context (Katzenellenbogen et al. 1996, Lemon & Freedman 1999, Klinge 2000, Rosenfeld & Glass 2001, Smith & O'Malley 2004). For example, although E2 activates hormone-responsive gene expression in multiple tissues, other ER agonists such as the antiestrogen tamoxifen exhibit tissue-specific ER agonist and antagonist activities (Jordan 2003a,b). Differences between the activities of E2 and tamoxifen and other antiestrogens are due, in part, to their ligand-induced conformational changes in the ER and their subsequent differences in binding affinities for specific nuclear coactivators (Schwabe et al. 1993, Brzozowski et al. 1997, Shiau et al. 1998, 2002, Pike et al. 1999, Jordan 2003a).

ER/Sp1-dependent transactivation is also complex and dependent on multiple variables. For example, E2 activates ER{alpha}/Sp but not ERß/Sp in several different cell lines and in HeLa cells, E2 decreases ERß/Sp1-dependent transactivation and this may be a pathway for decreased gene expression by hormones (Saville et al. 2000). In addition, there is also evidence that activation of ER{alpha}/Sp1 involves multiple domains of ER{alpha} and domain requirements are also dependent on the structure of the ER agonist (Saville et al. 2000, Kim et al. 2003). Previous studies have identified multiple gene promoters activated by ER{alpha}/Sp and Sp1 knockdown by RNA interference inhibits hormone-induced G0/G1 to S-phase progression in MCF-7 cells (Abdelrahim et al. 2002). However, the role of individual Sp1, Sp3, and Sp4 proteins in mediating ER{alpha}/Sp-induced gene expression in MCF-7 cells has not been determined. In this study, we have used RNA interference to investigate the effects of Sp protein knockdown (individual and combined) on induction of E2F1, RAR{alpha}, and CAD gene expression by E2 since all three genes contain GC-rich promoters that are E2-responsive in transfection studies (Wang et al. 1999, Khan et al. 2003, Ngwenya & Safe 2003).

The siRNAs for Sp proteins are highly specific and have been used in several studies (Abdelrahim et al. 2002, 2004, Higgins et al. 2006a,b), and they efficiently decrease expression of both Sp1, Sp3, and Sp4 mRNA and protein (Fig. 1). The effects of iSps on basal luciferase activity in MCF-7 cells transfected with pSp13, pE2F1 (–169/–54), pCAD (–90/+25), and pRAR{alpha} (–79/–49) demonstrated that despite common GC-rich sites in all three promoters, decreased Sp protein expression differentially affected activity. Luciferase activity in cells transfected with pE2F1 (–169/–54) was decreased ~70–90% after cotransfection with iSp1, iSp3, and iSp4, whereas in cells transfected with pRAR{alpha} (–79/–49), only iSp3 decreased activity and iSp1 actually increased activity. Although basal activity of the four promoters were differentially affected by Sp protein knockdown, E2-induced transactivation in cells transfected with these constructs, and fold induction was decreased in cells transfected with iSp1 or Sp4 (and their combinations), whereas iSp3 was the least effective or ineffective at decreasing fold induction. However, this may be, in part, due to the effects of iSp3 on decreasing basal activity in cells transfected with iSp13, pRAR{alpha} (–79/–49), and pE2F1 (–169/–54). All three iSps induced a comparable decrease in basal activity in MCF-7 cells transfected with pCAD (–90/+25) and iSp3 also decreased hormone-induced activity with this construct. Transfected iSps significantly decreased induction of E2F1, RAR{alpha}, and CAD mRNA levels by E2 in MCF-7 cells (Figs 3–5GoGo), and the order of effectiveness was iSp4≥iSp3>iSp1. Knockdown of Sp3 resulted in a complete loss of hormone-induced RAR{alpha} mRNA, whereas in cells transfected with pRAR{alpha} (–79/–49), iSp3 did not affect E2-induced transactivation. This suggests that other GC-rich sites in the RAR{alpha} promoter are required for ER{alpha}/Sp-mediated expression of this gene. Previous studies demonstrate that both Sp1 and Sp3 interact with ER{alpha} (Porter et al. 1996, 1997, Saville et al. 2000, Stoner et al. 2000) and we now show that both Sp1 and Sp4 colocalize with ER{alpha} in MCF-7 cells (Fig. 6). Thus, at least for the RAR{alpha}, CAD, and E2F1 genes, all three Sp proteins play a role in ER{alpha}/Sp-induced transactivation and, for E2F1, we also showed decreased protein expression (Fig. 3).

The ChIP assay has been used extensively to investigate the ligand-induced assembly of ER{alpha} and other coactivators and nuclear cofactors on hormone-responsive gene promoters, and pS2 has been used extensively as a model for these studies (Shao et al. 2002, Metivier et al. 2003, Acevedo et al. 2004, Krieg et al. 2004, Sun et al. 2005). This gene has a well-characterized E2-responsive non-consensus ERE motif, and several studies have reported that treatment of MCF-7 cells with E2 recruits ER{alpha} to the pS2 promoter. Similar results were observed in this study (Fig. 7A). Moreover, Sp1, Sp3, and Sp4 were also bound to the pS2 promoter, and PCR bands associated with the Sp protein-promoter interactions were not appreciably changed after treatment with E2. Sun et al. (2005)) also reported interactions of Sp1 and Sp3 with the pS2 promoter. Band intensities associated with Sp3-promoter interactions were unchanged after treatment with E2, whereas Sp1-promoter interactions were decreased; however, these changes were not observed in this study. We further investigated Sp protein interactions with the E2-responsive GC-rich regions of the E2F1, CAD, and RAR{alpha} promoters in a ChIP assay and observed interactions of Sp1, Sp3, and Sp4 with these promoters in untreated MCF-7 cells (Fig. 7B and D). After treatment of MCF-7 cells with E2, Sp protein-promoter interactions did not appreciably change, and this was similar to that observed for interactions of Sp proteins with the pS2 promoter (Fig. 7A). Binding of Sp1, Sp3, and Sp4 to the CAD, E2F1, and RAR{alpha} promoters was similar in solvent and E2-treated cells, and this is consistent with the role of all three proteins in mediating basal expression and hormonal activation of ER{alpha}/Sp-dependent CAD, E2F1, and RAR{alpha} mRNAs (Figs 3C, 4C, and 5C).

ER{alpha} was also constitutively bound to the GC-rich promoters and, in contrast to observations with the pS2 promoter (Fig. 7A; Shao et al. 2002, Metivier et al. 2003, Acevedo et al. 2004, Krieg et al. 2004, Sun et al. 2005), E2 did not enhance ER{alpha} interactions with the CAD, E2F1, and RAR{alpha} promoters in MCF-7 cells. Similar results were reported for the VEGFR2 promoter in ZR-75 cells (Higgins et al. 2006a) suggesting that there are fundamental differences in hormone-dependent interactions of ER{alpha} with E2-responsive promoters containing GC-rich or ERE motifs. We have carried out ChIP assays on several GC-rich promoters under a variety of conditions but invariably find that ER{alpha}, Sp1, Sp3, and Sp4 are associated with these promoters in the presence or absence of E2. These results suggest that there may also be differences in the recruitment of other nuclear cofactors to these gene promoters, and promoter-dependent differences in coactivator recruitment are currently being investigated.

In summary, results of this study demonstrate that Sp1, Sp3, and Sp4 transcription factors are important for hormone-induced E2F1, CAD, and RAR{alpha} mRNA expression. This is the first report showing the different contributions of ER{alpha}/Sp1, ER{alpha}/Sp3, and ER{alpha}/Sp4 on hormone-induced transactivation in MCF-7 cells. The relative effects of these individual proteins on ER{alpha}/Sp-mediated transactivation were dependent on the specific gene and gene promoter. Basal activity of the GC-rich proximal promoter regions of the E2F1, CAD, and RAR{alpha} were differentially effected by Sp protein knockdown (Figs 3A, 4A, and 5A) and activity in cells transfected with pRAR{alpha}-2 was only minimal decreased by iSps. Despite these differences, results of ChIP assays show that Sp1, Sp3, Sp4, and ER{alpha} were associated with all three promoters in the absence or presence of E2 (Fig. 7). In ongoing studies, we are further analyzing interactions of ER{alpha} and Sp proteins with the E2F1, RAR{alpha}, CAD, and other gene promoters and comparing their binding to E2-responsive and non-responsive promoter sequences. We are also investigating the effects of ligand structure, coactivators, and cell context on this important genomic pathway of estrogen action using both cell culture and in vivo models.


   Acknowledgements
 
The financial assistance of the National Institutes of Health (ES09106, ES04917, and CA104116 [GenBank] ), the Department of the Defense (DAMD17-03-1-0341), and the Texas Agricultural Experiment Station is gratefully acknowledged. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Abdelrahim M, Samudio I, Smith R, Burghardt R & Safe S 2002 Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells. Journal of Biological Chemistry 277 28815–28822.[Abstract/Free Full Text]

Abdelrahim M, Smith R III, Burghardt R & Safe S 2004 Role of Sp proteins in regulation of vascular endothelial growth factor expression and proliferation of pancreatic cancer cells. Cancer Research 64 6740–6749.[Abstract/Free Full Text]

Acevedo ML, Lee KC, Stender JD, Katzenellenbogen BS & Kraus WL 2004 Selective recognition of distinct classes of coactivators by a ligand-inducible activation domain. Molecular Cell 13 725–738.[CrossRef][Web of Science][Medline]

Bardin A, Moll F, Margueron R, Delfour C, Chu ML, Maudelonde T, Cavailles V & Pujol P 2005 Transcriptional and posttranscriptional regulation of fibulin-1 by estrogens leads to differential induction of messenger ribonucleic acid variants in ovarian and breast cancer cells. Endocrinology 146 760–768.[CrossRef][Web of Science][Medline]

Blobel GA & Orkin SH 1996 Estrogen-induced apoptosis by inhibition of the erythroid transcription factor GATA-1. Molecular and Cellular Biology 16 1687–1694.[Abstract/Free Full Text]

Bruning JC, Lingohr P, Gillette J, Hanstein B, Avci H, Krone W, Muller-Wieland D & Kotzka J 2003 Estrogen receptor-{alpha} and Sp1 interact in the induction of the low density lipoprotein-receptor. Journal of Steroid Biochemistry and Molecular Biology 86 113–121.[CrossRef][Web of Science][Medline]

Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA & Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389 753–758.[CrossRef][Medline]

Byrne IM, Flanagan L, Tenniswood MP & Welsh J 2000 Identification of a hormone-responsive promoter immediately upstream of exon 1c in the human vitamin D receptor gene. Endocrinology 141 2829–2836.[Abstract/Free Full Text]

Castro-Rivera E, Samudio I & Safe S 2001 Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements. Journal of Biological Chemistry 276 30853–30861.[Abstract/Free Full Text]

Chow J, Tobias JH & Colston KW 1992 Estrogen maintains trabecular bone volume in rats not only by suppression of bone resorption but also by stimulation of bone formation. Journal of Clinical Investigation 89 74–78.[Web of Science][Medline]

Clark JH Female reproduction and toxicology of estrogensKS Korach Reproductive and Developmental Toxicology 1998 Marcel Dekker New York 259–275.

Cowley SM, Hoare S, Mosselman S & Parker MG 1997 Estrogen receptors {alpha} and ß form heterodimers on DNA. Journal of Biological Chemistry 272 19858–19862.[Abstract/Free Full Text]

Delfino F & Walker WH 1999 Hormonal regulation of the NF-{kappa}B signaling pathway. Molecular and Cellular Endocrinology 157 1–9.[CrossRef][Web of Science][Medline]

Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, Kladde M, Vyhlidal C & Safe S 1999 Mechanisms of transcriptional activation of bcl-2 gene expression by 17ß-estradiol in breast cancer cells. Journal of Biological Chemistry 174 32099–32107.

Duan R, Porter W & Safe S 1998 Estrogen-induced c-fos protooncogene expression in MCF-7 human breast cancer cells: role of estrogen receptor Sp1 complex formation. Endocrinology 139 1981–1990.[Abstract/Free Full Text]

Farhat MY, Lavigne MC & Ramwell PW 1996 The vascular protective effects of estrogen. FASEB Journal 10 615–624.[Abstract]

Fujita N, Kajita M, Taysavang P & Wade PA 2004 Hormonal regulation of metastasis-associated protein 3 transcription in breast cancer cells. Molecular Endocrinology 18 2937–2949.[Abstract/Free Full Text]

Hall JM, Couse JF & Korach KS 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. Journal of Biological Chemistry 276 36869–36872.[Free Full Text]

Higgins KJ, Abdelrahim M, Liu S, Yoon K & Safe S 2006a Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins. Biochemical and Biophysical Research Communications 345 292–301.[CrossRef][Web of Science][Medline]

Higgins KJ, Liu S, Abdelrahim M, Yoon K, Vanderlaag K, Porter W, Metz RP & Safe S 2006b Vascular endothelial growth factor receptor-2 expression is induced by 17ß-estradiol in ZR-75 breast cancer cells by estrogen receptor {alpha}/Sp proteins. Endocrinology 147 3285–3295.[CrossRef][Web of Science][Medline]

Hyder SM, Chiappetta C & Stancel GM 1999 Interaction of human estrogen receptors alpha and beta with the same naturally occurring estrogen response elements. Biochemical Pharmacology 57 597–601.[CrossRef][Web of Science][Medline]

Jacobson D, Pribnow D, Herson PS, Maylie J & Adelman JP 2003 Determinants contributing to estrogen-regulated expression of SK3. Biochemical and Biophysical Research Communications 303 660–668.[CrossRef][Web of Science][Medline]

Jordan VC 2003a Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. Receptor interactions. Journal of Medicinal Chemistry 46 883–908.[CrossRef][Web of Science][Medline]

Jordan VC 2003b Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 2. Clinical considerations and new agents. Journal of Medicinal Chemistry 46 1081–1111.[CrossRef][Web of Science][Medline]

Katzenellenbogen JA, O'Malley BW & Katzenellenbogen BS 1996 Tripartite steroid hormone receptor pharmacology – interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones. Molecular Endocrinology 10 119–131.[Free Full Text]

Kelly MJ & Wagner EJ 1999 Estrogen modulation of G-protein-coupled receptors. Trends in Endocrinology and Metabolism 10 369–374.[CrossRef][Web of Science][Medline]

Khan S, Abdelrahim M, Samudio I & Safe S 2003 Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17ß-estradiol in breast cancer cells. Endocrinology 144 2325–2335.[Abstract/Free Full Text]

Kim K, Thu N, Saville B & Safe S 2003 Domains of estrogen receptor {alpha} (ER{alpha}) required for ER{alpha}/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells. Molecular Endocrinology 17 804–817.[Abstract/Free Full Text]

Kim K, Barhoumi R, Burghardt R & Safe S 2005 Analysis of estrogen receptor {alpha}-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer. Molecular Endocrinology 19 843–854.[Abstract/Free Full Text]

Klein-Hitpass L, Schorpp M, Wagner U & Ryffel GU 1986 An estrogen-responsive element derived from the 5'-flanking region of the xenopus vitellogenin A2 gene functions in transfected human cells. Cell 46 1053–1061.[CrossRef][Web of Science][Medline]

Klinge CM 2000 Estrogen receptor interactions with co-activators and corepressors. Steroids 65 227–251.[CrossRef][Web of Science][Medline]

Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA & Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. PNAS 95 15677–15682.[Abstract/Free Full Text]

Krieg AJ, Krieg SA, Ahn BS & Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. Journal of Biological Chemistry 279 5025–5034.[Abstract/Free Full Text]

Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM & Webb P 2000 Estrogen receptor pathways to AP-1. Journal of Steroid Biochemistry and Molecular Biology 74 311–317.[CrossRef][Web of Science][Medline]

Lemon BD & Freedman LP 1999 Nuclear receptor cofactors as chromatin remodelers. Current Opinion in Genetics and Development 9 499–504.[CrossRef][Web of Science][Medline]

Levin ER 2005 Integration of the extranuclear and nuclear actions of estrogen. Molecular Endocrinology 19 1951–1959.[Abstract/Free Full Text]

Li C, Briggs MR, Ahlborn TE, Kraemer FB & Liu J 2001 Requirement of Sp1 and estrogen receptor {alpha} interaction in 17ß-estradiol-mediated transcriptional activation of the low density lipoprotein receptor gene expression. Endocrinology 142 1546–1553.[Abstract/Free Full Text]

Matthews J & Gustafsson JA 2003 Estrogen signaling: a subtle balance between ER{alpha} and ERß. Molecular Interventions 3 281–292.[Abstract/Free Full Text]

Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M & Gannon F 2003 Estrogen receptor-{alpha} directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115 751–763.[CrossRef][Web of Science][Medline]

Ngwenya S & Safe S 2003 Cell context-dependent differences in the induction of E2F-1 gene expression by 17ß-estradiol in MCF-7 and ZR-75 cells. Endocrinology 144 1675–1685.[Abstract/Free Full Text]

Nilsson S & Gustafsson JA 2002 Biological role of estrogen and estrogen receptors. Critical Reviews in Biochemistry and Molecular Biology 37 1–28.[CrossRef][Web of Science][Medline]

Olefsky JM 2001 Nuclear receptor minireview series. Journal of Biological Chemistry 276 36863–36864.[Free Full Text]

Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, Ljunggren J, Gustafsson JA & Carlquist M 1999 Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO Journal 18 4608–4618.[CrossRef][Web of Science][Medline]

Porter W, Wang F, Wang W, Duan R & Safe S 1996 Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression. Molecular Endocrinology 10 1371–1378.[Abstract/Free Full Text]

Porter W, Saville B, Hoivik D & Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Molecular Endocrinology 11 1569–1580.[Abstract/Free Full Text]

Qin C, Singh P & Safe S 1999 Transcriptional activation of insulin-like growth factor binding protein 4 by 17ß-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes. Endocrinology 140 2501–2508.[Abstract/Free Full Text]

Razandi M, Pedram A, Merchenthaler I, Greene GL & Levin ER 2004 Plasma membrane estrogen receptors exist and functions as dimers. Molecular Endocrinology 18 2854–2865.[Abstract/Free Full Text]

Rosenfeld MG & Glass CK 2001 Coregulator codes of transcriptional regulation by nuclear receptors. Journal of Biological Chemistry 276 36865–36868.[Free Full Text]

Safe S & Kim K 2004 Nuclear receptor-mediated transactivation through interaction with Sp proteins. Progress in Nucleic Acid Research and Molecular Biology 77 1–36.[Web of Science][Medline]

Salvatori L, Ravenna L, Felli MP, Cardillo MR, Russo MA, Frati L, Gulino A & Petrangeli E 2000 Identification of an estrogen-mediated deoxyribonucleic acid-binding independent transactivation pathway on the epidermal growth factor receptor gene promoter. Endocrinology 141 2266–2274.[Abstract/Free Full Text]

Samudio I, Vyhlidal C, Wang F, Stoner M, Chen I, Kladde M, Barhoumi R, Burghardt R & Safe S 2001 Transcriptional activation of DNA polymerase {alpha} gene expression in MCF-7 cells by 17ß-estradiol. Endocrinology 142 1000–1008.[Abstract/Free Full Text]

Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson J-A & Safe S 2000 Ligand-, cell- and estrogen receptor subtype ({alpha}/ß)-dependent activation at GC-rich (Sp1) promoter elements. Journal of Biological Chemistry 275 5379–5387.[Abstract/Free Full Text]

Schultz JR, Petz LN & Nardulli AM 2003 Estrogen receptor {alpha} and Sp1 regulate progesterone receptor gene expression. Molecular and Cellular Endocrinology 201 165–175.[CrossRef][Web of Science][Medline]

Schultz JR, Petz LN & Nardulli AM 2005 Cell- and ligand-specific regulation of promoters containing activator protein-1 and Sp1 sites by estrogen receptors {alpha} and ß. Journal of Biological Chemistry 280 347–354.[Abstract/Free Full Text]

Schwabe JW, Chapman L, Finch JT & Rhodes D 1993 The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75 567–578.[CrossRef][Web of Science][Medline]

Shao W, Halachmi S & Brown M 2002 ERAP140, a conserved tissue-specific nuclear receptor coactivator. Molecular and Cellular Biology 22 3358–3372.[Abstract/Free Full Text]

Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA & Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95 927–937.[CrossRef][Web of Science][Medline]

Shiau AK, Barstad D, Radek JT, Meyers MJ, Nettles KW, Katzenellenbogen BS, Katzenellenbogen JA, Agard DA & Greene GL 2002 Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism. Nature Structural Biology 9 359–364.[Web of Science][Medline]

Smith CL & O'Malley BW 2004 Coregulator function: a key to understanding tissue specificity of selected receptor modulators. Endocrine Reviews 25 45–71.[Abstract/Free Full Text]

Stoner M, Wang F, Wormke M, Nguyen T, Samudio I, Vyhlidal C, Marme D, Finkenzeller G & Safe S 2000 Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor {alpha} and Sp3 proteins. Journal of Biological Chemistry 275 22769–22779.[Abstract/Free Full Text]

Stoner M, Wormke M, Saville B, Samudio I, Qin C, Abdelrahim M & Safe S 2004 Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor {alpha} and Sp proteins. Oncogene 23 1052–1063.[CrossRef][Web of Science][Medline]

Sun G, Porter W & Safe S 1998 Estrogen-induced retinoic acid receptor {alpha}1 gene expression: role of estrogen receptor-Sp1 complex. Molecular Endocrinology 12 882–890.[Abstract/Free Full Text]

Sun JM, Spencer VA, Li L, Yu CH, Yu J & Davie JR 2005 Estrogen regulation of trefoil factor 1 expression by estrogen receptor {alpha} and Sp proteins. Experimental Cell Research 302 96–107.[CrossRef][Web of Science][Medline]

Tanaka N, Yonekura H, Yamagishi S, Fujimori H, Yamamoto Y & Yamamoto H 2000 The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-{alpha} through nuclear factor-{kappa}B, and by 17ß-estradiol through Sp-1 in human vascular endothelial cells. Journal of Biological Chemistry 275 25781–25790.[Abstract/Free Full Text]

Turner RT, Riggs BL & Spelsberg TC 1994 Skeletal effects of estrogen. Endocrine Reviews 15 275–300.[Abstract/Free Full Text]

Wang W, Dong L, Saville B & Safe S 1999 Transcriptional activation of E2F1 gene expression by 17ß-estradiol in MCF-7 cells is regulated by NF-Y – Sp1/estrogen receptor interactions. Molecular Endocrinology 13 1373–1387.[Abstract/Free Full Text]

Wang F, Samudio I & Safe S 2002 Transcriptional activation of rat creatine kinase B by 17ß-estradiol in MCF-7 cells involves an estrogen responsive element and GC-rich sites. Journal of Cellular Biochemistry 84 156–172.[CrossRef][Web of Science]

Watson CS, Campbell CH & Gametchu B 2002 The dynamic and elusive membrane estrogen receptor-{alpha}. Steroids 67 429–437.[CrossRef][Web of Science][Medline]

Weihua Z, Saji S, Makinen S, Cheng G, Jensen EV, Warner M & Gustafsson JA 2000 Estrogen receptor (ER) ß, a modulator of ER{alpha} in the uterus. PNAS 97 5936–5941.[Abstract/Free Full Text]

Xie W, Duan R & Safe S 1999 Estrogen induces adenosine deaminase gene expression in MCF-7 human breast cancer cells: role of estrogen receptor–Sp1 interactions. Endocrinology 140 219–227.[Abstract/Free Full Text]

Xie W, Duan R, Chen I, Samudio I & Safe S 2000 Transcriptional activation of thymidylate synthase by 17ß-estradiol in MCF-7 human breast cancer cells. Endocrinology 141 2439–2449.[Abstract/Free Full Text]

Zhao YL, Han WD, Li Q, Mu YM, Lu XC, Yu L, Song HJ, Li X, Lu JM & Pan CY 2005 Mechanism of transcriptional regulation of LRP16 gene expression by 17-ß estradiol in MCF-7 human breast cancer cells. Journal of Molecular Endocrinology 34 77–89.[Abstract/Free Full Text]

Received in final form 3 July 2007
Accepted 7 August 2007



This article has been cited by other articles:


Home page
 J EndocrinolHome page
R C M Simmen, J M P Pabona, M C Velarde, C Simmons, O Rahal, and F A Simmen
The emerging role of Kruppel-like factors in endocrine-responsive cancers of female reproductive tissues
J. Endocrinol., March 1, 2010; 204(3): 223 - 231.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
F. Wu, I. Ivanov, R. Xu, and S. Safe
Role of SP transcription factors in hormone-dependent modulation of genes in MCF-7 breast cancer cells: microarray and RNA interference studies
J. Mol. Endocrinol., January 1, 2009; 42(1): 19 - 33.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
S. Safe and K. Kim
Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways
J. Mol. Endocrinol., November 1, 2008; 41(5): 263 - 275.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
G. Chadalapaka, I. Jutooru, S. Chintharlapalli, S. Papineni, R. Smith III, X. Li, and S. Safe
Curcumin Decreases Specificity Protein Expression in Bladder Cancer Cells
Cancer Res., July 1, 2008; 68(13): 5345 - 5354.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, S.
Right arrow Articles by Safe, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, S.
Right arrow Articles by Safe, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS