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Molecular Endocrinology, doi:10.1210/me.2007-0319
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Molecular Endocrinology 22 (2): 388-402
Copyright © 2008 by The Endocrine Society

Vascular Endothelial Growth Factor Receptor-2 Expression Is Down-Regulated by 17β-Estradiol in MCF-7 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins

Kelly J. Higgins, Shengxi Liu, Maen Abdelrahim, Kathryn Vanderlaag, Xinyi Liu, Weston Porter, Richard Metz and Stephen Safe

Department of Biochemistry and Biophysics (K.J.H., X.L., S.S.), Department of Veterinary Physiology and Pharmacology (K.V., S.S.), and Department of Veterinary Integrated Biosciences (W.P., R.M.), Texas A&M University, College Station, Texas 77843; and Institute of Biosciences and Technology (S.L., M.A., S.S.), Texas A&M Health Science Center, Houston, Texas 77030

Address all correspondence and requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
17β-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at –58 and –44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the –60 to –37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER){alpha}/Sp1 and ER{alpha}/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA in-terference assays we show that the ER{alpha}/Sp protein-promoter interactions are accompanied by recruitment of the corepressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANGIOGENESIS IS A complex biological function that is required for new blood vessel formation and is essential for embryogenesis, wound healing, and many other physiological processes (1, 2, 3). In addition, angiogenic pathways also contribute to disease states including inflammation, diabetes, and cancer where both tumor growth and metastasis are dependent on development of new vasculature in the parent tumor and in distal sites of metastasis (4, 5). Vascular permeability factor or vascular endothelial growth factor (VEGF) is a key angiogenic protein and is a critical activator of this pathway. Several different splice-variant forms of VEGF (or VEGF-A) have been characterized along with VEGF-B, VEGF-C, VEGF-D, VEGF-E, and platelet-induced growth factor (3, 6). The expression of these mitogens is tissue/cell specific, and there is also some specificity in their interactions with VEGF receptors (VEGFRs), which are protein tyrosine kinase transmembrane receptors.

The expression of VEGFRs is cell type specific: the major VEGFRs include VEGFR1(flt-1), soluble VEGFR1(sflt-1), VEGFR2(KDR/flk-1), and VEGFR3(flt-4) (1, 3, 6). Soluble VEGFR1 (sVEGFR1) is a truncated form of VEGFR1 that does not contain the tyrosine kinase domain but expresses the extracellular ligand-binding function of VEGFR1. There is some evidence that sVEGFR1 exhibits antiangiogenic activity by interacting with extracellular VEGF, thereby inhibiting its interactions with other VEGFRs (3, 6). For example, a recent study (7) showed that 17β-estradiol (E2) induced sVEGFR1 (but not VEGFR1) in estrogen receptor {alpha} (ER{alpha})-positive MCF-7 breast cancer cells, the antiestrogen ICI 182,780 inhibited the E2-induced response, and sVEGFR1 levels were increased by the antiestrogen alone. Also, evidence from xenograft studies with MCF-7 cells showed decreased expression of sVEGFR1 after treatment with E2, and this correlated with a decrease in tumor vessel density.

Among the VEGFRs, VEGFR2 is the predominant form that regulates angiogenesis. VEGFR2 is overexpressed in some tumor types (8, 9, 10, 11, 12, 13, 14, 15), and tyrosine kinase inhibitors that block VEGFR signaling have been developed for cancer chemotherapy (16, 17, 18, 19). Regulation of VEGFR2 expression has been investigated in several different cell lines, and analysis of the VEGFR2 promoter has identified several important transacting factors/cis elements (20, 21, 22, 23). The proximal region of the VEGFR2 promoter contains E-boxes, GC-rich, activator protein (AP)-2, and nuclear factor {kappa}B (NF{kappa}B) motifs that are important for VEGFR2 expression in several cell lines, and a recent study showed that transcription factor II (TFII) also modulates endothelial cell expression of VEGFR2 (24). Studies in this laboratory have shown that E2 induced VEGFR2 expression in ER{alpha}-positive ZR-75 breast cancer cells, and this was due to a nonclassical mechanism involving ER{alpha}/Sp3 and ER{alpha}/Sp4 interactions with proximal GC-rich motifs at –58 and –44 (25). However, E2 decreased VEGFR2 mRNA levels in MCF-7 cells, and this further extends the large number of genes that are down-regulated by E2 in this cell line (26, 27, 28, 29, 30). We therefore used VEGFR2 as a model for investigating pathways associated with hormone-dependent gene repression, and analysis of this response showed that the GC-rich sites at –58 and –44 are critical for the decreased response in MCF-7 cells. Results of RNA interference, chromatin immunoprecipitation (ChIP), EMSA, and transient transfection assays suggest that hormone-dependent down-regulation is primarily dependent on ER{alpha}/Sp1 and ER{alpha}/Sp3 promoter interactions that are accompanied by recruitment of the corepressors silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor corepressor (NCoR). RNA interference at the promoter and RNA levels also confirms the role of both NCoR and SMRT in mediating E2-dependent down-regulation of VEGFR2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Down-Regulation of VEGFR2 by E2 in MCF-7 Cells
Treatment of MCF-7 cells with 10 nM E2 significantly decreased VEGFR2 mRNA levels 12 h after treatment, and this persisted for up to 48 h (Fig. 1AGo). These results were observed in replicate experiments and represent an example of hormone-induced down-regulation of gene expression in ER-positive breast cancer cells. pVEGFR2A is a construct containing the –716 to +268 region of the VEGFR2 promoter, and E2 induced transactivation in ZR-75 cells transfected with pVEGFR2A (25). In contrast, E2 decreased luciferase activity in MCF-7 cells transfected with pVEGFR2A (Fig. 1BGo). Transfection of a series of 5'-deletion constructs into MCF-7 cells showed that basal activity was similar after transfection with pVEGFR2A, pVEGFR2B (–225 to +268), pVEGFR2C (–95 to +268), and pVEGFR2D (–77 to +268). A 20–30% loss of activity was observed in cells transfected with pVEGFR2E, suggesting that the GC-rich/AP-2 sites at –77 to –60 play a role in basal expression of VEGFR2 in MCF-7 cells. However, deletion of the proximal GC-rich sites at –58 and –44 resulted in the loss of more than 90% of basal activity, demonstrating the important role for these elements in VEGFR2 expression. E2-dependent down-regulation of luciferase activity was observed in MCF-7 cells transfected with pVEGFR2A, pVEGFR2B, pVEGFR2C, pVEGFR2D, and pVEGFR2E, and deletion of the proximal GC-rich sites (pVEGFR2F) resulted in loss of hormone responsiveness. Thus, the –60 to –37 region of the promoter was critical for both basal and hormone-induced activity. Transfection of a series of constructs containing mutations of a single GC-rich site (pVEGFR2Em1/ pVEGFR2Em2) or mutation of both sites (pVEGFR2Em3) showed that both sites were important for basal luciferase activity of these constructs. However, the low activity observed in cells transfected with the single mutants (pVEGFR2Em1 and pVEGFR2Em2) was further decreased by E2 (Fig. 1CGo). Hormone responsiveness was lost only in cells transfected with pVEGFR2Em3 (mutation of both GC-rich motifs) or pVEGFR2F (deletion of –60 to –37 region of the promoter). Due to the low basal activity associated with these mutant constructs, it is unlikely that the hormonal effects on activity are biologically relevant.


Figure 1
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  		Fig. 1. Down-Regulation of VEGFR2 by E2 in MCF-7 Cells

A, Down-regulation of VEGFR2 mRNA by E2 in MCF-7 human breast cancer cells. MCF-7 cells were treated with Me2SO or 10 nM E2 for 12, 24, or 48 h. RNA was isolated using the RNeasy Protect Mini Kit (QIAGEN), and samples were analyzed by real-time PCR as described in Materials and Methods. Significant (P < 0.05) down-regulation of VEGFR2 mRNA levels by E2 are indicated by an asterisk. Results are presented as means ± SE for at least three determinations for each treatment group. B, Deletion analysis of the VEGFR2 gene promoter and effects of E2 on luciferase activity in MCF-7 cells. MCF-7 human breast cancer cells were transiently transfected with 500 ng of pVEGFR2A, pVEGFR2B, pVEGFR2C, pVEGFR2D, pVEGFR2E, or pVEGFR2F, 250 ng pCDNA3.1-His-LacZ, and 250 ng ER{alpha}. Cells were treated for 36–48 h with Me2SO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) down-regulation of luciferase reporter activity by E2 is indicated by an asterisk. Results are expressed as means ± SE for at least three determinations for each treatment group. C, Mutation analysis of pVEGFR2E in MCF-7 cells. MCF-7 human breast cancer cells were transiently transfected with 500 ng of pVEGFR2E, pVEGFR2Em1 (mutation of the 5'-GC-rich element), pVEGFR2Em2 (mutation of the 3'-GC-rich element), pVEGFR2Em3 (mutation of both GC-rich elements), or pVEGFR2F, 250 ng pCDNA3.1-His-LacZ, and 250 ng ER{alpha}. Cells were treated for 36–48 h with Me2SO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) down-regulation of luciferase reporter activity by E2 is indicated by an asterisk. Results are expressed as means ± SE for at least three determinations for each treatment group. LUC, Luciferase; β-gal, β-galactosidase.

 
Domain Requirements of ER{alpha} and Hormone Specificity in MCF-7 Cells
We also investigated the receptor specificity of E2-induced inhibition of transactivation in cells transfected with pVEGFR2A and wild-type ER{alpha} or ER{alpha} mutants containing DNA-binding domain (DBD) (HE11C) or A/B domain (HE19C) deletions (Fig. 2AGo). The results showed that both the DBD and C-terminal region of ER{alpha} were required for E2-dependent decreased luciferase expression. Consistent with these observations, the antiestrogen ICI 182,780 also reversed the effects of E2 on luciferase activity in MCF-7 cells transfected with ER{alpha} and pVEGFR2C (Fig. 2BGo), whereas 1 µM ICI 182,780 had no effect on transactivation. Receptor specificity for this response was demonstrated in MCF-7 cells transfected with pVEGFR2C and ER{alpha} or progesterone receptor-B; E2, but not progesterone, decreased transactivation (Fig. 2CGo). These results suggest that E2-dependent down-regulation of VEGFR2 is specific for ER{alpha} and requires the proximal GC-rich motifs at –58 and –44, suggesting a role for Sp proteins in mediating this response.


Figure 2
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  		Fig. 2. ER Domain Requirements and Hormone Specificity in MCF-7 Cells

A, Comparative effects of wild-type and variant ER{alpha} on E2-induced transactivation in MCF-7 cells. MCF-7 cells were transiently transfected with 500 ng of pVEGFR2A and 250 ng of ER{alpha} or variant (HE11C and HE19C) ER{alpha}. Cells were treated with Me2SO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) down-regulation of luciferase activity is indicated by an asterisk. Results are presented as means ± SE for at least three determinations of each treatment group. B, Antiestrogen responsiveness of pVEGFR2C in MCF-7 cells. MCF-7 cells were transiently transfected with 500 ng of pVEGFR2C and 250 ng ER{alpha}. Cells were treated with Me2SO, 10 nM E2, 10 nM E2 + 1 µM ICI 182,780, or 1 µM ICI 182,780 alone. Luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) down-regulation of luciferase activity (*) is indicated. Results are presented as means ± SE for at least three determinations for each treatment group. C, Hormone responsiveness of pVEGFR2C in MCF-7 cells. MCF-7 cells were transiently transfected with 500 ng of pVEGFR2C and 250 ng ER{alpha} or progesterone receptor-B. Cells were treated with Me2SO, 10 nM E2, or 10 nM progesterone. Luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) down-regulation of luciferase activity (*) is indicated. Results are presented as means ± SE for at least three determinations for each treatment group. LUC, Luciferase; β-gal, β-galactosidase; PR-B, progesterone receptor B.

 
Expression of Sp Proteins and Knockdown by RNA Interference
Previous studies have demonstrated expression of Sp1 and Sp3 in breast cancer cells (25, 31, 32, 33, 34), and results in Fig. 3Go, A and B, confirm expression of Sp1, Sp3, and Sp4 in these cells and their binding to the GC-rich region of VEGFR2. Incubation of nuclear extracts from MCF-7 cells with VEGFR2-32P oligonucleotide (–64 5'-CCG GCC CCG CCC CGC ATG GCC CCG CCT CCG-3' –35) gave an intense mobile band and a less intense, more mobile retarded band (Fig. 3AGo) (lane 2) that resemble the patterns previously observed for Sp protein-DNA complexes. Coincubation with antibodies to Sp1 (lane 3), Sp4 (lane 4), or Sp3 (lane 5) gave supershifted bands, whereas nonspecific IgG did not affect the retarded bands (lane 6). Coincubation with 100-fold excess of unlabeled VEGFR2 oligonucleotide reduced intensities of all retarded bands. Further confirmation of Sp1, Sp3, and Sp4 expression in MCF-7 cells was obtained in studies that used short inhibitory RNAs (siRNAs) for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) to knock down all three Sp proteins in MCF-7 cells (Fig. 3BGo) as previously described in other cell lines (31). Western blot analysis of whole-cell lysates from MCF-7 cells transfected with siRNA for Lamin (iLamin) (nonspecific) showed that Sp1, Sp3, and Sp4 are expressed in MCF-7 cells (lane 1). However, in cells transfected with iSp1 (lane 2), iSp3 (lane 3), or iSp4 (lane 4), there was decreased expression of Sp1, Sp3, and Sp4, respectively, in whole-cell lysates, and in replicate experiments (at least three), siRNAs significantly decreased expression of their targeted proteins. The effectiveness of the RNA interference on cellular expression of Sp proteins was also determined by immunofluorescent staining (Fig. 3CGo). Staining for Lamin was clearly decreased in MCF-7 cells transfected with iLamin (panel a) but not in cells transfected with iSp1 (panel b) or iSp3 (panel c). Sp1 exhibited punctate nuclear staining in cells transfected with iLamin (panel d), and this staining was barely visible in cells transfected with iSp1 (panel e). Similarly, Sp3 exhibited a punctate nuclear staining pattern in MCF-7 cells transfected with iLamin (panel g), and transfection with iSp3 (panel h) virtually eliminated the Sp3 staining. In the absence of the primary (panel f) or secondary (panel i) antibodies, no immunofluorescence was detected. The Sp4 antibodies commercially available gave weak immunofluorescent staining patterns and could not be effectively used to confirm Sp4 protein knockdown as observed in the Western blots (Fig. 3BGo).


Figure 3
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  		Fig. 3. Sp Protein Expression in MCF-7 Cells and Knockdown by RNA Interference

A, Sp protein binding to the VEGFR2 promoter-EMSA. Nuclear extracts from MCF-7 cells were incubated with radiolabeled VEGFR2-32P alone or in the presence of unlabeled oligonucleotides and/or antibodies, and DNA-protein complexes were separated by EMSA as described in Materials and Methods. Arrows indicate various retarded and supershifted complexes; F.P., free probe. B, Sp protein knockdown by Western blot analysis. MCF-7 cells were transfected with iSp1, iSp3, or iSp4, and whole-cell lysates were analyzed by Western blot analysis as described in Materials and Methods. The experiments were repeated (3x), and the Sp protein levels were significantly (P < 0.05) decreased by RNA interference (relative to iLamin) as indicated by an asterisk. NS, nonspecific protein used as a loading control. C, Sp protein knockdown-analysis by immunostaining. MCF-7 cells were transfected with iLamin (control) (a, d, and g), iSp1 (b and e), or iSp3 (c and h), and immunostained for Lamin (a–c), Sp1 (d and e), or Sp3 (g and h), as described in Materials and Methods. No primary antibody (f) and no secondary antibody (i) served as controls. Photographs were taken at the magnification of x60. The level of Sp4 expression in these cells was below the detection limit of the assay.

 
Role of Sp Proteins in Hormone-Dependent Down-Regulation of VEGFR2
Previous studies in Hec1A endometrial cancer cells using antisense oligonucleotides showed that hormone-dependent down-regulation of VEGF involved ER{alpha}/Sp3 interactions with GC-rich promoter elements (32), and the role of ER{alpha}/Sp in down-regulation of VEGFR2 was further investigated in this study by RNA interference. MCF-7 cells were cotransfected with pVEGFR2A and iLamin (control), iGL2, iSp1, iSp3, or iSp4, treated with E2, and luciferase activities were determined (Fig. 4AGo). E2 induced down-regulation of luciferase activity in cells transfected with iLamin, and activity was significantly decreased in both Me2SO- and E2-treated groups in cells cotransfected with iGL2 (which targets luciferase mRNA). Basal luciferase activity was decreased in cells transfected with iSp1, iSp3, and iSp4. In MCF-7 cells transfected with iLamin (nonspecific), treatment with E2 decreased luciferase activity by 73% compared with solvent (Me2SO). In contrast, E2-induced decreases were 5, 12, and 49% in cells transfected with iSp1, iSp3, and iSp4, respectively, demonstrating a role for all three Sp proteins in mediating ER{alpha}/Sp-dependent down-regulation of VEGFR2. The role of Sp proteins in mediating E2/ER{alpha}-dependent down-regulation of VEGFR2 was further confirmed by real-time PCR analysis of VEGFR2 mRNA levels in MCF-7 cells cotransfected with either iLamin or iSp1, iSp3, and iSp4 (combined) (Fig. 4BGo). The results showed that the down-regulation of VEGFR2 mRNA levels by E2 was inhibited by cotransfection with iSp1, iSp3, and iSp4 (combined), and these results complement the parallel studies using the pVEGFR2A construct (Fig. 4AGo).


Figure 4
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  		Fig. 4. Role of Sp Proteins in Hormone-Dependent Down-Regulation of VEGFR2

A, Effects of iSp1, iSp3, and iSp4 on basal and E2-dependent activity in MCF-7 cells transfected with pVEGFR2A. MCF-7 human breast cancer cells were transiently transfected with 500 ng of pVEGFR2A and 50 nM of each siRNA, treated with Me2SO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significantly (P < 0.05) decreased reporter activity after treatment with E2 (*) and decreased basal activity by siRNAs (**) are indicated. Results are presented as means ± SE for at least three determinations for each treatment group. B, Effects of iSp1, iSp3, and iSp4 on VEGFR2 mRNA in MCF-7 human breast cancer cells. MCF-7 cells were treated with Me2SO or 10 nM E2 for 24 h. RNA was isolated using the RNeasy Protect Mini Kit (QIAGEN), and samples were analyzed by real-time PCR as described in Materials and Methods. Significant (P < 0.05) down-regulation of VEGFR2 mRNA levels by E2 are indicated (*). Results are presented as means ± SE for at least three determinations for each treatment group. LUC, Luciferase.

 
Protein Interactions with the Proximal VEGFR2 Promoter
Most studies in MCF-7 and ZR-75 cells show that E2 activates several genes through ER{alpha}/Sp1 complexes, and this is associated with interactions with GC-rich promoter elements (33, 34). The interactions of ER{alpha}, coactivators, and corepressors with the proximal region of the VEGFR2 promoter were further investigated in a ChIP assay. Sp1, Sp3, and Sp4 were associated with the VEGFR2 promoter, and band intensities were similar in the presence or absence of E2 (data not shown). The PCR bands obtained after immunoprecipitation with ER{alpha}, steroid receptor coactivator (SRC)-1, or SRC-3 antibodies also varied less than 2-fold after treatment with 10 nM E2, and these proteins also appeared to be constitutively associated with the VEGFR2 promoter (Fig. 5BGo). In contrast, the nuclear receptor corepressors NCoR and SMRT were minimally associated with the VEGFR2 promoter, and PCR analysis showed a significantly increased association of these proteins with this promoter after treatment with 10 nM E2, and this was replicated over several experiments. Treatment of MCF-7 cells with 10 nM E2 also dramatically increased the association of ER{alpha} with the region of the pS2 promoter containing an estrogen-responsive element (ERE) (Fig. 5BGo), and this was consistent with previous reports of ChIP assays on the pS2 gene promoter (25). In addition, E2 induced recruitment of SRC-3 but not SRC-1 to the pS2 promoter, and association of the corepressors NCoR and SMRT with the pS2 promoter exhibited minimal changes after treatment with E2 for 1 h; however, there was increased association of SMRT after 2 h. However, in replicate experiments this increase in band intensity was minimal, although the increased band intensities for ER{alpha} and SRC-3 after treatment with E2 were consistently observed. We also carried out a parallel experiment in ZR-75 cells where E2 increases VEGFR2 expression (25). The results (Fig. 5CGo) show that treatment with E2 did not affect SMRT or NCoR interactions with the VEGFR2 promoter, whereas ER{alpha} and SRC3 recruitment to the pS2 promoter are E2 dependent as observed in MCF-7 cells (Fig. 5BGo). As a positive control for the ChIP experiment, Fig. 5DGo shows the transcription factor TFIIB was constitutively bound to the proximal region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, and the binding was also observed after treatment with E2 in MCF-7 cells. TFIIB did not interact with exon 1 of the CNAP1 gene, and similar results were observed in ZR-75 cells (data not shown).


Figure 5
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  		Fig. 5. Interaction of Proteins with the Proximal VEGFR2 Promoter

A, Summary of primers (-> <-) and targeted regions of the VEGFR2 and pS2 promoters used in ChIP assays. Analysis of protein interactions with the VEGFR2 and pS2 promoters by ChIP in MCF-7 (B) or ZR-75 (C) cells. The cells were treated with Me2SO (control) or 10 nM E2, harvested after treatment with hormone for up to 2 h, and analyzed in a ChIP assay as described in Materials and Methods. The quantitative effects of E2 on interactions of NCoR and SMRT with the VEGFR2 promoter in MCF-7 cells were determined in three replicate experiments, and results are expressed as means ± SE. Significant (P < 0.05) induction by E2 compared with DMSO control is indicated (*). D, Binding of TFIIB to the GAPDH promoter. The ChIP assay was also used to examine binding of TFIIB to the GAPDH promoter (positive control) and to exon 1 of CNAP1 (negative control) as described in Materials and Methods.

 
Role of Corepressor Proteins in Hormone-Dependent Down-Regulation of VEGFR2
The hormone-dependent repression of VEGFR2 through ER{alpha}/Sp proteins was accompanied by recruitment of SMRT and NCoR to the VEGFR2 promoter (Fig. 5Go) suggesting a possible role for the corepressors in mediating this response. We therefore examined the effects of SMRT and NCoR knockdown by RNA interference in MCF-7 cells transfected with pVEGFR2A and siRNAs for SMRT (iSMRT) and NCoR (iNCoR). Both iSMRT and iNCoR decrease expression of their corresponding proteins in MCF-7 cells (Fig. 6Go, A and B). The results (Fig. 6Go, C and D) showed that, in MCF-7 cells transfected with pVEGFR2A, iSMRT and iNCoR significantly reversed E2-dependent down-regulation of luciferase activity. iSMRT did not decrease basal luciferase activity, whereas a 20–25% decrease in basal activity was observed in cells transfected with iNCoR (Fig. 6Go, C and D). Hormone-dependent decreases in luciferase activity were significantly reversed in cells transfected with iSMRT or iNCoR, and the overall luciferase activity was higher than observed in cells treated with E2 and transfected with pVEGFR2A plus iLamin (nonspecific) We also investigated the effects of iSMRT and iNCoR on E2-dependent down-regulation of VEGFR2 mRNA levels in MCF-7 cells (Fig. 6EGo). In cells transfected with iScr (nonspecific), E2 induced a 60% decrease in VEGFR2 mRNA levels, whereas this E2-dependent response was significantly reversed in cells transfected with iNCoR, iSMRT, or their combination. This demonstrates that both SMRT and NCoR are involved in mediating the E2-dependent down-regulation of VEGFR2 expression in MCF-7 cells.


Figure 6
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  		Fig. 6. Roles of SMRT and NCoR in Hormonal Down-Regulation of VEGFR2

A, SMRT protein knockdown by Western blot analysis. MCF-7 cells were transfected with 40 or 60 nM iSMRT, and whole-cell lysates were analyzed by Western blot analysis as described in Materials and Methods. The experiments were done in triplicate. NS, nonspecific protein used as a loading control. B, NCoR protein knockdown by Western blot analysis. MCF-7 cells were transfected with 5, 10, or 20 nM iNCoR, and whole-cell lysates were analyzed by Western blot analysis as described in Materials and Methods. The experiments were done in triplicate. Effects of iSMRT (C) and iNCoR (D) on basal and E2-dependent activity in MCF-7 cells transfected with pVEGFR2A. MCF-7 human breast cancer cells were transiently transfected with 400 ng of pVEGFR2A and 40–60 nM of iSMRT, or 2.5 to 20 nM iNCoR, treated with Me2SO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) reversal of down-regulated reporter activity by siRNAs is indicated (*). Results are presented as means ± SE for at least three determinations for each treatment group. E, Effects of iNCoR and iSMRT on VEGFR2 mRNA levels. MCF-7 cells were transfected with iNCoR, iSMRT or their combination, treated with 10 nM E2 for 24 h, and VEGFR2 mRNA was determined by real-time PCR as described in Materials and Methods. Significant (P < 0.05) reversal of E2-dependent down-regulation of VEGFR2 mRNA by the siRNAs is indicated (*). Results are presented as means ± SE for at least three determinations for each treatment group. LUC, Luciferase.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Estrogen regulation of gene expression is highly complex and dependent on multiple factors including the structure of the ligand and the relative tissue/cell-specific expression of ER{alpha}, ERβ, and various coregulatory proteins (35, 36). The classical mechanism of E2-dependent up-regulation of many genes involves ligand-induced ER homodimerization and interaction of the nuclear ER homodimer with EREs in target gene promoters. Ligand-induced formation of the ER homodimer-DNA complex is accompanied by recruitment of coactivators and other nuclear factors and by interactions with the basal transcription machinery to activate gene transcription (37, 38, 39). Studies on the molecular biology of ER action have subsequently revealed more complex mechanisms, which involve DNA-bound ER interacting with other transcription factors such as Sp1, and ER-transcription factor interactions where the latter protein(s) but not ER binds its cognate response element (33, 40). For example, ER{alpha}/Sp1-, ER{alpha}/AP-1-, and ERβ/AP-1-mediated transactivation through binding GC-rich and AP-1 motifs have been extensively investigated (33, 40, 41, 42).

Several studies have examined more global gene expression profiles of estrogen-responsive genes in breast cancer and other cell lines (26, 27, 28, 29, 30). Frasor and co-workers (26) reported that more than 400 genes "showed a robust pattern of regulation" by E2, and more than 70% of these genes were down-regulated. Thus, although E2 plays a major role in decreasing gene expression in MCF-7 cells, mechanisms associated with this response have not been extensively investigated. The mechanisms of E2-dependent inhibition of genes regulated by NF{kappa}B have been studied, and the results show that these effects are complex and dependent on the gene, cell context, and ligand structure (43, 44, 45, 46, 47, 48, 49, 50). At least one mechanism involves direct binding of ER{alpha} to nuclear NF{kappa}B, and this results in inhibition of coactivator recruitment and decreased NF{kappa}B binding to promoter elements and decreased transactivation. Several nuclear factors can repress ER{alpha} function through different pathways, and these effects are ligand-, cell context-, and gene-dependent (51, 52, 53, 54). For example, scaffold attachment factors B1 and B2 and nuclear receptor corepressors suppress ER{alpha}-mediated transcription (51, 52) in breast cancer cells. Both repressors constitutively interact with the E2-responsive region of the pS2 gene promoter, and E2 decreases but the antiestrogen tamoxifen increases this association. In contrast, many genes such as VEGFR2 are down-regulated in MCF-7 cells treated with E2 (25) and therefore, we further investigated this response in MCF-7 cells as a model for understanding the molecular mechanisms of E2-dependent down-regulation of gene expression.

The deletion and mutation analysis of the VEGFR2 promoter (Fig. 1Go, B and C), coupled with the effects of antiestrogens and the requirement for wild-type ER{alpha} or HE11C (Fig. 2Go), indicate that ER{alpha} interactions with Sp proteins are required for down-regulation of VEGFR2 by E2 in MCF-7 cells. The critical GC-rich sites at –58 and –44 were required for E2-dependent up-regulation of VEGFR2 mRNA or promoter constructs in ZR-75 cells (25) and down-regulation of these same responses in MCF-7 cells. The major difference between the two cell lines was associated with the domains of ER{alpha} required for these responses. In MCF-7 cells, deletion of the N-terminal A/B region did not affect transactivation in cells transfected with pVEGFR2 constructs, whereas deletion of the DBD resulted in loss of transactivation (Fig. 2AGo). This suggested that decreased transactivation in MCF-7 cells treated with E2 was dependent on the C-terminal C-F domains of ER{alpha} containing both the DBD and AF-2. In contrast, the DBD of ER{alpha} was not required for induction of transactivation in ZR-75 cells transfected with pVEGFR2 constructs (25), and similar results were obtained for induction of many other E2-responsive genes by both ER{alpha}/Sp1 and HE11C/Sp1 (33, 40). Both AF-1 and AF-2 in the C- and N-terminal regions of ER{alpha} were required for up-regulation of ER{alpha}/Sp-dependent gene expression by E2 (25, 33, 40); however, E2-dependent inhibition of transactivation in cells transfected with the pVEGFR2A construct required the DBD and AF-2 domain but was AF-1 independent (Fig. 2AGo).

The pattern of retarded bands associated with Sp-DNA (VEGFR2 oligonucleotide) interactions in MCF-7 cells (Fig. 3AGo) was similar to that observed in ZR-75 cells (25) and was associated with binding of Sp1, Sp3, and Sp4 proteins, which are expressed in both cell lines. ER{alpha} enhances the on-rate of Sp binding to GC-rich motifs (55); yet, ternary ER{alpha} -Sp-DNA complexes were not detected in EMSAs in this study or in previous reports (33, 40). However, using a ChIP assay, we have shown that ER{alpha} was constitutively bound to the GC-rich promoter (56) (Fig. 5BGo), and treatment with E2 did not appreciably enhance the PCR bands associated with ER{alpha}. The results are consistent with the fact that ER{alpha} binds Sp1 and Sp3 in the presence or absence of ligand (32, 55), and nuclear colocalization of ER{alpha} and Sp proteins is observed in breast cancer cells in the presence or absence of ligand (data not shown).

Studies on hormonal regulation of genes through ER{alpha}/Sp proteins have shown that ER{alpha}/Sp1 is involved in induced expression of several genes, whereas down-regulation of VEGF in Hec1A endometrial cancer cells was due to ER{alpha}/Sp3 interactions with proximal GC-rich motifs (32). The role of Sp3 in mediating decreased VEGF expression in Hec1A cells treated with E2 was supported by studies with dominant-negative Sp3, which reversed the effects of E2. The relative contributions of Sp1 and Sp4 were not determined. Results of RNA interference studies showed that knockdown of Sp1, Sp3, or Sp4 blocked E2-dependent repression of luciferase activity in MCF-7 cells transfected with pVEGFR2A (Fig. 4AGo). Moreover, transfection of these siRNAs also inhibited E2-dependent repression of VEGFR2 mRNA (Fig. 4BGo). These results demonstrate that E2-dependent down-regulation of VEGFR2 expression in MCF-7 cells involves all three Sp proteins.

As indicated above, ER{alpha} decreases NF{kappa}B-dependent transactivation through multiple pathways (43, 44, 45, 46, 47, 48, 49, 50), and ER and other nuclear receptors decrease expression of genes/reporter genes by modulating the activity of other DNA-bound transcription factors (55, 56). Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma})-dependent suppression of thromboxane receptor expression in vascular smooth muscle cells is dependent on a GC-rich promoter sequence and may be due to decreased Sp-1 promoter (DNA) interactions (57). Ligands for PPAR{gamma} and PPAR{alpha} also decrease VEGFR2 expression in retinal capillary endothelial and human umbilical vein endothelial cells, respectively, and these responses were associated with decreased Sp1/Sp3 ratios and Sp1-DNA (promoter) binding, respectively (58, 59). In contrast, decreased VEGFR2 expression in MCF-7 cells treated with E2 was not accompanied by decreased association of Sp proteins to the VEGFR2 promoter in ChIP (data not shown) or EMSAs (Fig. 3AGo). Moreover, studies with recombinant ER{alpha} plus Sp1 showed that ER enhances Sp1 binding to GC-rich oligonucleotides (55), and nuclear extracts from E2- and solvent (Me2SO)-treated cells gave retarded bands with similar intensities (56, 60). Similar results were observed using treated nuclear extracts from MCF-7 cells and the VEGFR2-32P oligonucleotide (Fig. 3AGo), suggesting that other factors were responsible for decreased VEGFR2 expression in MCF-7 cells treated with E2.

Several studies report that corepressors NCoR and SMRT bind promoters in E2-responsive genes and play a role in modulation of nuclear receptor-mediated transactivation (56, 60, 61, 62, 63, 64, 65). ChIP analysis of the proximal region of the VEGFR2 promoter indicates constitutive binding of NCoR, SMRT, SRC-1, and SRC-3. However, after treatment with E2, there was increased binding of NCoR and SMRT but minimal changes in SRC-1 and SRC-3 binding to the VEGFR2 promoter in MCF-7 cells (Fig. 5BGo). In contrast, both ER and SRC-3 were recruited to the E2-responsive region of the pS2 gene promoter, whereas minimal changes were observed in binding of SRC-1, NCoR, and SMRT to the pS2 gene promoter (Fig. 5BGo). NCoR and SMRT are also associated with the VEGFR2 promoter in ZR-75 cells (Fig. 5CGo) where E2 induced VEGFR2 expression (25). As a positive control for the ChIP assay in ZR-75 cells, we observed E2-dependent recruitment of SRC-3 to E2-responsive pS2 and VEGFR2 gene promoters (Fig. 5CGo). However, results of ChIP assays indicated that treatment with E2 had minimal effects on NCoR/SMRT interactions with the VEGFR2 promoter in ZR-75 cells, and this clearly differed from the same interactions in MCF-7 cells (Fig. 5BGo). These differences in SMRT/NCoR interactions with the VEGFR2 gene promoter in MCF-7 and ZR-75 cells may explain, in part, the cell context-dependent effects of E2 on VEGFR2 expression. However, it is apparent that other factors must also be involved, and these are currently being investigated. The recruitment of corepressors has previously been linked to ligand-dependent repression of genes (51, 66, 67, 68), and we therefore further examined the effects of SMRT and NCoR knockdown on luciferase activity in cells transfected with pVEGFR2A and treated with E2 (Fig. 6Go, C and D). The results showed that E2-dependent down-regulation of activity was reversed, in part, after cotransfection with iSMRT and iNCoR, and similar results were observed for E2-dependent repression of VEGFR2 mRNA where combined knockdown of both corepressors blocked the effects of E2 (Fig. 6EGo).

These observations suggest a possible model for the mechanism of E2-dependent down-regulation of VEGFR2 gene expression (Fig. 7Go). ER{alpha} forms an ER/Sp complex on the VEGFR2 promoter in the absence of ligand; however, after treatment with E2, the nuclear corepressors SMRT and NCoR are recruited, and ER{alpha}/SMRT or ER{alpha}/NCoR acts to depress transactivation. In the absence of E2, knockdown of SMRT does not decrease Sp-dependent transactivation associated with the VEGFR2 promoter (Fig. 6CGo), suggesting the repressed transactivation is associated with the liganded ER{alpha}-SMRT complex. In contrast, decreased NCoR expression after transfection with iNCoR resulted in decreased basal activity and hormone responsiveness (Fig. 6DGo); however, the loss of basal activity was variable and dependent on the amount of transfected iNCoR. Although this model (Fig. 7Go) does not fully define the mechanism of E2-dependent down-regulation of gene expression, we have demonstrated that SMRT and NCoR play a role in mediating this response. Current studies are focused on identifying other key elements (51) involved in E2-dependent down-regulation of VEGFR2 and other genes, thereby providing insights on an important pathway of estrogen action involving gene repression that is not well understood. In addition, the VEGFR2 gene will also be used as a model in future studies for determining the critical cell context-dependent factors that regulate E2-dependent repression or induction in MCF-7 and ZR-75 cells, respectively.


Figure 7
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  		Fig. 7. Proposed Model for the Mechanism of E2-Dependent Down-Regulation of VEGFR2 Gene Expression in MCF-7 Cells

Role of ER{alpha}/Sp proteins and involvement of the corepressors SMRT and NCoR.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals, Plasmids, and Gifts
Dimethyl sulfoxide (Me2SO), E2, 4'-hydroxytamoxifen, 100x antibiotic/antimycotic solution, and PBS were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 182,780 was kindly provided by Dr. Alan Wakeling (AstraZeneca, Macclesfield, UK). Lysis buffer (5x), luciferase reagent, restriction enzymes (XhoI and HindIII), and ligase were purchased from Promega Corp. (Madison, WI). β-Galactosidase reagents were purchased from Tropix, Inc. (Bedford, MA). Taq polymerase and other PCR reagents were purchased from PerkinElmer (Boston, MA). Progesterone and other chemicals were obtained from commercial sources of the highest quality possible.

Human ER{alpha} expression plasmid was provided by Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). ER{alpha} deletion constructs HE11C (DBD of ER{alpha} deleted) and HE19C (AF-1 domain of ER{alpha} deleted) were originally obtained from Dr. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France) and inserted into vectors pCDNA3 and pCDNA3.1/His C. pCDNA3.1-His-LacZ expression plasmid was obtained from Invitrogen (Carlsbad, CA). VEGFR2 promoter luciferase constructs pVEGFR2A, pVEGFR2B, and pVEGFR2C (previously named pKDR–716/+268, pKDR–225/+268, and pKDR–95/+268) were provided by Dr. Arthur Mu-EnLee (deceased) and Dr. Koji Maemura (Cardiovascular Biology Laboratory, Boston, MA). pGL2 basic luciferase reporter vector was purchased from Promega.

Cell Lines and Tissue Culture
The human breast cancer cell line MCF-7 was obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in DMEM/F12 (Sigma) supplemented with 5 or 10% fetal bovine serum (FBS) (Summit Biotechnology, Fort Collins, CO; Intergen, Des Plains, IL; JRH Biosciences, Lenexa, KS; or Atlanta Biologicals, Inc., Norcross, GA). Medium was further supplemented with 2.2 g/liter sodium bicarbonate and 100x antibiotic/antimycotic solution (Sigma). Cells were maintained at 37 C with a humidified CO2-air (5:95) mixture.

Cloning and Oligonucleotides
VEGFR2 promoter-derived oligonucleotides, PCR primers, and primers employed in plasmid construction were synthesized by Genosys/Sigma (The Woodlands, TX) or Integrated DNA Technologies (IDT) (Coralville, IA). VEGFR2 promoter deletion constructs pVEGFR2D, pVEGFR2E, pVEGFR2F, and pVEGFR2G were created by PCR amplification using pVEGFR2A as the template (25). Forward primers were designed with XhoI restriction enzyme sites at the 5'-end. A reverse luciferase primer was used for PCR. PCR products were digested with XhoI and HindIII and subsequently ligated into the pGL2 basic vector. All constructs were in pGL2 basic luciferase reporter vector, and all constructs were sequenced to verify their identity. Mutation constructs pVEGFR2Em1, pVEGFR2Em2, and pVEGFR2Em3 were constructed by PCR amplification using the reverse luciferase primer paired with the forward primer containing the desired mutations. Forward primers are as follows (mutated bases are underlined).

M1 = 5'-GAT GAT CTC GAG CCA AGC CCC GCA TGG CCC CGC C-3'

M2 = 5'-GAT GAT CTC GAG CCC CGC CCC GCA TGG CCA AGC CTC CGC GC-3'

M3 = 5'-GAT GAT CTC GAG CCA AGC CCC GCA TGG CCA AGC CTC CGC GC-3'

Transient Transfection Assays
Cells were seeded in 12-well plates at a concentration of 1.5–3.0 x 105 cells per well in phenol red-free DMEM/F12 media supplemented with 2.5% charcoal-stripped FBS. After 18–24 h, the appropriate VEGFR2 luciferase reporter plasmid (500 ng), 250 or 500 ng ER{alpha}, or ER{alpha} deletion constructs expression plasmid, and 250 ng pCDNA3.1-His-LacZ expression plasmid (for normalization of transfection efficiency) were transiently cotransfected into MCF-7 cells using the calcium phosphate-DNA coprecipitation method. pCDNA3.1 empty vector was transfected to maintain DNA mass balance among different transfection groups. An estrogen-responsive pC3-Luc construct, containing the mouse complement-3 (C3) gene promoter insert, was kindly provided by Dr. Donald P. McDonnell (Duke University Medical School, Durham, NC) and was used as a positive control in most experiments to confirm hormone responsiveness of the transfected cells.

After transfection (4–8 h), cells were shocked with 25% glycerol in PBS to increase transfection efficiency. Then cells were washed with PBS and treated for 24–48 h with fresh serum-free DMEM/F12 medium containing 10 nM E2, 10 nM progesterone (P), 10 nM E2 + 1 µM ICI 182,780, 1 µM ICI 182,780 dissolved in Me2SO, or Me2SO alone as a solvent control. Cells were harvested by scraping the plates in 100–200 ml of 1x lysis buffer (Promega). An aliquot of soluble protein was obtained by one cycle of freezing/thawing the cells, vortexing (30 sec), and centrifuging at 12,000 x g (1 min). Cell lysates (30 µl) were assayed for luciferase activity using Luciferase Assay Reagent (Promega) and β-galactosidase activity using Galacto-Light Plus assay system (Tropix) in a Lumicount microwell plate reader (Packard Instrument Co., Downers Grove, IL). Luciferase activity was normalized relative to β-galactosidase units for each transfection experiment.

Transient Transfection of siRNA
Cells were cultured in phenol red-free DMEM/F12 medium supplemented with 2.5% charcoal stripped FBS in 12-well plates until 50–70% confluent. Cells were washed once with serum free, antibiotic free, phenol red-free DMEM/F12 media. The amount of siRNA to give a maximal decrease of each target protein was determined experimentally (2.5–60 nM final concentration in the well). Oligofectamine reagent (Invitrogen) was used to transfect MCF-7 cells with siRNA according to the manufacturer’s protocol. The next day, following the manufacturer’s instructions, Lipofectamine 2000 reagent (Invitrogen) was used to transfect cells with 400 ng of the appropriate VEGFR luciferase reporter plasmid and 200 ng of pCDNA3.1-His-LacZ, as well as 400 ng ER{alpha}. Cells were treated 4–8 h later with 10 nM E2 or Me2SO in serum free, antibiotic free, phenol red-free DMEM/F12 media. Cells were harvested 24 h after treatment. Cell lysates were assayed for luciferase and β-galactosidase activity as described above.

The Lamin A/C duplex (target sequence: 5'-CTG GAC TTC CAG AAG AAC A-3') and the Luciferase GL2 duplex (target sequence: 5'-CGT ACG CGG AAT ACT TCG A-3') RNA from Dharmacon (Lafayette, CO) were used for controls in siRNA transfections. The siRNA oligonucleotides for Sp1, Sp3, Sp4 (31), NCoR, and SMRT were also ordered from Dharmacon as follows: Sp1: 5'-AUC ACU CCA UGG AUG AAA UGA dTdT-3'; Sp3: 5'-GCG GCA GGU GGA GCC UUC ACU dTdT-3'; Sp4: 5'-GCA GUG ACA CAU UAG UGA GCdT dT-3'; NCoR: 5'-AAG AAG GAU CCA GCA UUC GGA dTdT-3'; SMRT: 5'-AAA GUC UAA ACU GAG CUC GCA dTdT-3'.

Western Blot Analysis
Cells were seeded into six-well plates in DMEM/F12 medium supplemented with 2.5% charcoal stripped FBS. The next day, cells were transfected with siRNA as described earlier. Protein was extracted from the tissue culture cells by harvesting in a high-salt lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (vol/vol) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml aprotinin, 50 mM phenylmethylsulfonylfluoride, 50 mM sodium orthovanadate] on ice for 45–60 min and centrifugation at 20,000 x g for 10 min at 4 C. Protein (30–60 µg) was diluted with Laemmli’s loading buffer, boiled, and loaded onto a 7.5% sodium dodecyl sulfate-polyacrylamide gel. Samples were resolved using electrophoresis at 150–180 V for 3–4 h and transferred (transfer buffer: 48 mM Tris-HCl, 29 mM glycine, and 0.025% sodium dodecyl sulfate) to a polyvinylidinedifluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA) by electrophoresis at 0.2 A for approximately 12–16 h.

Membranes were blocked with excess protein and then probed with polyclonal primary antibodies for Sp1 (PEP2), Sp3 (D20), Sp4 (V20), and SMRT (N20) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibody to NCoR was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Sp1 and Sp3 were each diluted 1:1000 and Sp4 was diluted 1:250 or 1:500 and incubated overnight. NCoR and SMRT were diluted 1:250 and incubated overnight as well. Membranes were probed with a horseradish peroxidase-conjugated secondary antibody (1:5000) for 3–6 h. Blots were visualized using the chemiluminescent substrate ECL detection system (NEN-DuPont, Boston, MA) and exposure on Kodak X-O Mat autoradiography film (Eastman Kodak Co., Rochester, NY). Band intensity values were obtained by scanning the film on a Sharp JX-330 scanner (Sharp Electronics, Mahwah, NJ) and by densitometry using the Zero-D Scanalytics software package (Scanalytics, Sunnyvale, CA).

Real-Time PCR
For experiments involving hormonal regulation, MCF-7 cells were cultured in serum-free DMEM/F12 media for 1–3 d before treatment with 10 nM E2 or Me2SO as a solvent control for 6–48 h. For experiments involving siRNA, MCF-7 breast cancer cells were transfected as described above. Total RNA was isolated using the RNeasy Protect Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. RNA was eluted with 30 µl RNase-free water and stored at –80 C. RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol.

PCR was carried out using SYBR Green PCR Master Mix from PE Applied Biosystems (Warrington, UK) on an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The 25 µl final volume contained 0.5 µM of each primer and 2 µL of cDNA template. TATA binding protein (TBP) was used as an exogenous control to compare the relative amount of target gene in different samples. The PCR profile was as follows: one cycle of 95 C for 10 min, then 40 cycles of 95 C for 15 sec and 60 C for 1 min. The comparative CT method was used for relative quantitation of samples. Primers were purchased from Integrated DNA Technologies. The following primers were used: KDR (forward): 5'-cac cac tca aac gct gac atg ta-3'; KDR (reverse): 5'-cca act gcc aat acc agt gga t-3'; TBP (forward): 5'-tgc aca gga gcc aag agt gaa-3'; TBP (reverse): 5'-cac atc aca gct ccc cac ca-3'.

Preparation of Nuclear Extracts
Cells were cultured in phenol red-free medium supplemented with 2.5% charcoal-stripped FBS. The next day, cells were switched to serum free, phenol red-free media for 1–3 d. Cells were treated with Me2SO or 10 nM E2 for 30 min before harvesting. Cells were washed in PBS (2x), scraped in 1 ml of 1x lysis buffer, incubated at 4 C for 15 min, and centrifuged 1 min at 14,000 x g. Cell pellets were washed in 1 ml of lysis buffer (3x). Lysis buffer supplemented with 500 mM KCl was then added to the cell pellet and incubated for 45 min at 4 C with frequent vortexing. Nuclei were pelleted by centrifugation at 14,000 x g for 1 min at 4 C, and aliquots of supernatant were stored at –80 C until needed.

EMSA
VEGFR2 oligonucleotide (–64 5'-CCG GCC CCG CCC CGC ATG GCC CCG CCT CCG-3' –35) was synthesized and annealed, and 5-pmol aliquots were 5'-end labeled using T4 kinase and [{gamma}-32P]ATP. A 30-µl EMSA reaction mixture contained approximately 100 mM KCl, 3 µg of crude nuclear protein, 1 µg polydeoxyinosinic deoxycytidylic acid, with or without unlabeled competitor oligonucleotide, and 10 fmol of radiolabeled probe. After incubation for 20 min on ice, antibodies against Sp1, Sp3, or Sp4 proteins were added and incubated another 20 min on ice. Protein-DNA complexes were resolved by 5% polyacrylamide gel electrophoresis. Specific DNA-protein and antibody-supershifted complexes were observed as retarded bands in the gel.

ChIP Assay
MCF-7 cells (1.0 x 107) were treated with Me2SO (time 0) or 10 nM E2 for 15, 60, and 120 min. 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 desired chromatin length (~500 bp). The chromatin was precleared by addition of protein A-conjugated beads (Pierce Biotechnology, Rockford, IL). The precleared chromatin supernatants were immunoprecipitated with antibodies specific to IgG, TFIIB, Sp1, Sp3, Sp4, ER{alpha}, SRC-1, SRC-3, NCoR, and SMRT (Santa Cruz Biotechnology) at 4 C overnight. The protein-antibody complexes were collected by addition of protein A-conjugated beads for 1 h, and the beads were extensively washed. The protein-DNA cross-links were eluted and reversed. DNA was purified by Qiaquick Spin Columns (QIAGEN) and 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 ERE. The VEGFR2/KDR primers are: 5'-GTC CAG TTG TGT GGG GAA AT-3' (forward) and 5'-GAG CTG GAG CCG AAA CTC TA-3' (reverse), which amplify a 169-bp region of human VEGFR2/KDR promoter containing GC-rich/Sp1 binding sites. The positive control primers are: 5'-TAC TAG CGG TTT TAC GGG CG-3' (forward) and 5'-tcg aac agg agg agc aga gag cga-3' (reverse), which amplify a 167-bp region of human GAPDH gene. The negative control primers are: 5'-atg gtt gcc act ggg gat ct-3' (forward) and 5'-tgc caa agc cta ggg gaa ga-3' (reverse), which amplify a 174-bp region of genomic DNA between the GAPDH gene and the CNAP1 gene. PCR products were resolved on a 2% agarose gel in the presence of 1:10,000 SYBR gold (Molecular Probes-Invitrogen, Carlsbad, CA).

Immunofluorescence
Rabbit polyclonal antibody for Lamin, Sp1, Sp3, Sp4, and normal rabbit IgG were purchased from Santa Cruz Biotechnology. Fluorescein isothiocyanate-conjugated goat antirabbit IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) or Santa Cruz Biotechnology. MCF-7 cells were seeded in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) at 75,000–100,000 cells per well in phenol red-free DMEM/F12 medium supplemented with 2.5 or 5% charcoal-stripped FBS. The next day cells were either washed with PBS, changed to serum free medium and incubated for 24 h, or were transfected with siRNAs as described above. For experiments involving E2 treatment, MCF-7 cells were treated with 10 nM E2 or Me2SO in serum free media for 4–7 h and fixed with cold methanol at –20 C for 5 or 10 min. After washing with PBS, cells were blocked with 4% goat serum at room temperature for 1 h and incubated with the primary rabbit polyclonal antibodies against Lamin (1:200), Sp1 (1:200), Sp3 (1:200), Sp4 (1:100), or normal rabbit IgG (1:1000) at 4 C overnight. After washing with PBS/0.3% Tween 3 for 10 min, the samples were incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:500 or 1:1000) at room temperature for 1 h. After PBS/Tween rinsing, glass coverslips were mounted over the samples with mounting medium (Vector Laboratories, Burlingame, CA) or ProLong Gold (Invitrogen), and cells were examined with a fluorescence microscope.

Statistical Analysis
Results of transient transfection studies are presented as means ± SE for at least three replicates for each treatment group. All other experiments were carried out at least two times to confirm a consistent pattern of responses. Significant statistical differences between treatment groups were determined by analysis using SuperANOVA and Scheffe’s test or Fisher’s Protected LSD (P < 0.05).


   FOOTNOTES
 
This work was supported by National Institutes of Health (Grants ES04917 and CA104116) and the Texas Agricultural Experiment Station.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 15, 2007

Abbreviations: AP, Activator protein; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iLamin, siRNA Lamin; iNCoR, siRNA NCoR; iSMRT, siRNA SMRT; iSpl, siRNA Spl; iSp3, siRNA Sp3; iSp4, siRNA Sp4; NCoR, nuclear receptor corepressor; NF{kappa}B, nuclear factor-{kappa}B; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; siRNA, short interfering RNA; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; sVEGFR1, soluble VEGFR1; TBP, TATA binding protein; TFII, transcription factor II; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Received for publication June 25, 2007. Accepted for publication November 5, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   NCOR  |  SMRT
Ligands:   17β-Estradiol  |  Fulvestrant



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