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Endocrinology Vol. 147, No. 7 3285-3295
Copyright © 2006 by The Endocrine Society

Vascular Endothelial Growth Factor Receptor-2 Expression Is Induced by 17ß-Estradiol in ZR-75 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins

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

Departments of Biochemistry and Biophysics (K.J.H., S.S.), Veterinary Physiology and Pharmacology (K.V., S.S.), and Veterinary Integrated Biosciences (W.P., R.P.M.), Texas A&M University, College Station, Texas 77843; and Institute of Biosciences and Technology (S.L., M.A., K.Y., 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 Texas A&M University, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOGENESIS INVOLVES formation of blood vessels from vascular endothelial cells and preexisting vessels and is a critical process required for neovascularization in normal and cancerous tissues (1, 2, 3). New blood vessel formation is necessary for diverse biological processes including numerous steps in embryogenesis and wound repair, and several diseases including diabetes, cancer, and inflammation are also dependent on angiogenic pathways. Although angiogenesis is dependent on the interplay of many cellular factors, key mediators of this response include vascular endothelial growth factor (VEGF) and its cognate receptors, VEGF receptors (VEGFRs) (4, 5, 6). VEGF or vascular permeability factor belongs to the VEGF platelet-derived growth factor gene family. Several major forms of VEGF are expressed in different tissues and cells based on alternative splicing. VEGFRs are transmembrane tyrosine kinase receptors that are expressed as three major forms, namely VEGFR1 (Flt-1)/soluble VEGFR1 (sFlt-1), VEGFR2 [kinase insert domain receptor (KDR)/Flk-1], and VEGFR3 (Flt-4). Among these three receptors, VEGFR2 is generally recognized as the major form that mediates VEGF-induced responses (6).

VEGFR2 is highly expressed in endothelial cells and has also been detected in tumors and cancer cell lines derived from multiple tissues (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). For example, VEGFR2 expression is increased in prostate cancer samples compared with normal prostate, and there is a switch from VEGFR1 expression to VEGFR2 expression during prostate tumor progression (10). This switch is important because VEGFR1 and VEGFR2 differ considerably in their signaling properties; VEGFR2 is the primary initiator of angiogenesis, whereas VEGFR1 may be an inhibitor of angiogenesis in some tumors (11). VEGFR2 and VEGF are coexpressed in primary breast carcinomas, and their expression is increased when tumors shift to an angiogenic phenotype. In addition, VEGFR2 is constitutively expressed in breast tumor epithelial cultures but exhibits decreased expression in stromal cell cultures (12). Angiogenesis is hormonally regulated in breast cancer cells and other estrogen-responsive tissues, and 17ß-estradiol (E2) induces VEGF expression in many of these cells and tissues (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Hormonal regulation of VEGFR2 has previously been observed in bovine retinal capillary endothelial cells where E2 induces expression of both VEGFR2 and VEGF (29).

Although VEGFR2 is expressed in many different tumor types and has been detected in various cancer cell lines, to our knowledge, little is known about the mechanism of regulation of VEGFR2 in hormonally regulated tissues/cells including various cancer cell lines. The VEGFR2 gene promoter is highly complex with multiple cis-elements; however, consensus or nonconsensus ERE motifs have not been identified in the 5' promoter region of this gene (30). In this study, we show that VEGFR2 is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells and that gene expression is increased after treatment of these cells with E2. Analysis of the VEGFR2 gene promoter shows hormone responsiveness is primarily due to two proximal GC-rich motifs (–60 to –37) that bind Sp proteins, and hormonal activation of VEGRF2 is associated with ER{alpha}/Sp protein-mediated transactivation. These results are similar to those previously observed for hormonal activation of VEGF in the same cell line (27) and suggest a common induction mechanism for both angiogenic factors. However, in contrast to previous reports showing that ER{alpha}/Sp1 is important for activation of hormone-responsive genes in breast cancer cells (31, 32, 33, 34, 35, 36), VEGFR2 is primarily regulated by ER{alpha}/Sp3 and ER{alpha}/Sp4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and plasmids
Dimethyl sulfoxide (Me2SO), E2, 4'-hydroxytamoxifen, 100x antibiotic/antimycotic solution, and PBS were purchased from Sigma (St. Louis, MO). ICI 182,780 was kindly provided by Dr. Alan Wakeling (AstraZeneca, Macclesfield, UK). Lysis buffer, luciferase reagent, restriction enzymes (XhoI and HindIII), and ligase were purchased from Promega (Madison, WI). ß-Galactosidase reagents were purchased from Tropix (Bedford, MA). Taq polymerase and other PCR reagents were purchased from PerkinElmer (Boston, MA). Progesterone (P) 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 [DNA binding domain (DBD) of ER{alpha} deleted] and HE19C [activation function 1 (AF-1) domain of ER{alpha} deleted] were originally obtained from Dr. Pierre Chambon (Instutut 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 Lab, Boston, MA). pGL2 basic luciferase reporter vector was purchased from Promega.

Cell lines and tissue culture
The human breast cancer cell line ZR-75 was obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (FBS) (Summit Biotechnology, Fort Collins, CO; Intergen, Des Plains, IA; JRH Biosciences, Lenexa, KS; or Atlanta Biologicals, Inc., Norcross, GA). Medium was further supplemented with 1.5 g/liter sodium bicarbonate, 2.38 g/liter HEPES, 0.11 g/liter sodium pyruvate, 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 (Coralville, IA). VEGFR2 promoter deletion constructs pVEGFR2D, pVEGFR2E, pVEGFR2F, and pVEGFR2G were created by PCR amplification using pVEGFR2A as the template. 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 the 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):Go


Figure 1
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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 supplemented with 2.5% charcoal-stripped FBS. After 18–24 h, the appropriate VEGFR2 luciferase reporter plasmid (500 ng), ER{alpha} or ER{alpha} deletion construct expression plasmid (500 ng), and pCDNA3.1-His-LacZ expression plasmid (250 ng) (for normalization of transfection efficiency) were transiently cotransfected into ZR-75 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 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 containing 10 nM E2, 10 nM 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 µl 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 ß-galactosidase activity using Tropix Galacto-Light Plus assay system in a Lumicount microwell plate reader (Packard Instrument Co., Downers Grove, IL). Relative luciferase activity was normalized to relative ß-galactosidase units for each transfection experiment.

Transient transfection of small inhibitory RNA (siRNA)
Cells were cultured in phenol red-free DMEM/F12 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. The amount of siRNA to give a maximal decrease of each target protein was determined experimentally (50 nM final concentration in the well). Oligofectamine reagent (Invitrogen) was used to transfect ZR-75 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 500 ng of the appropriate VEGFR2 luciferase reporter plasmid, 200 ng pCDNA3.1-His-LacZ expression plasmid, and 500 ng ER{alpha} expression plasmid. Four to 8 h later, cells were treated with 10 nM E2 or Me2SO in serum-free, antibiotic-free, phenol red-free DMEM/F12. Cells were harvested 24–48 h after treatment. Cell lysates were assayed for luciferase and ß-galactosidase activity as described earlier.

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, and Sp4 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'; and Sp4, 5'-GCA GUG ACA CAU UAG UGA GCdT dT-3'.

Western blot analysis
Cells (3.0 x 105) were seeded into six-well plates in DMEM/F12 supplemented with 2.5% charcoal-stripped FBS. The next day, cells were transfected with siRNA as described above. 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 phenylmethylsulfonylflouride, 50 mM sodium orthovanadate] on ice for 45–60 min and centrifugation at 20,000 x g for 10 min at 4 C. Sixty micrograms of protein was diluted with Laemmli’s loading buffer, boiled, and loaded onto 7.5% SDS-PAGE. 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 polyvinylidene difluoride membrane (Bio-Rad, 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), and Sp4 (V20) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Sp1 and Sp3 were each diluted 1:1000 and incubated overnight. Sp4 was 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 enhanced chemiluminescence detection system (NEN Life Science Products-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, ZR-75 cells were cultured in serum-free DMEM/F12 for 1–3 d before treatment with 10 nM E2 or Me2SO as a solvent control for 6–24 h. For experiments involving siRNA, ZR-75 breast cancer cells were transfected as described previously. 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 each primer and 2 µl 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 threshold cycle 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'; and 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 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 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 crude nuclear protein, 1 µg poly(deoxyinosine-deoxycytosine), with or without unlabeled competitor oligonucleotide, and 10 fmol 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% PAGE electrophoresis. Specific DNA-protein and antibody-supershifted complexes were observed as retarded bands in the gel.

Immunofluorescence
Rabbit polyclonal antibodies for VEGFR2/KDR, Lamin, Sp1, Sp3, Sp4, and normal rabbit IgG were purchased from Santa Cruz Biotechnology. Fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) or Santa Cruz Biotechnology.

ZR-75 cells were seeded in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) at 0.75–1.0 x 105 cells/well in phenol red-free DMEM/F12 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 previously. For experiments involving E2 treatment, ZR-75 cells were treated with 10 nM E2 or Me2SO in serum-free media for 7 h and fixed with cold methanol at –20 C for 5 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 VEGFR2/KDR (1:25), 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 x 10 min, the samples were incubated with FITC-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. In some experiments, ZR-75 cells were stained with propidium iodide for nuclear counterstaining.

Chromatin immunoprecipitation (ChIP) assay
ZR-75 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 (approximately 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 (transcription factor IIB), Sp1, Sp3, Sp4, and ER{alpha} (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 estrogen response element (ERE). The VEGF primers are: 5'-GGT CGA GCT TCC CCT TCA-3' (forward) and 5'-GAT CCT CCC CGC TAC CAG-3' (reverse), which amplify a 202-bp region of human VEGF promoter containing GC-rich/Sp1 binding sites. 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 glyceraldehyde-3-phosphate dehydrogenase (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 (Invitrogen).

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 Scheffé’s test or Fisher’s protected least significant difference (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of VEGFR2 by E2 in ZR-75 cells
The effect of E2 on VEGFR2 mRNA expression in ZR-75 human breast cancer cells was investigated using real-time PCR, and preliminary studies showed that 10 nM E2 gave optimal induced responses. VEGFR2 mRNA expression was significantly up-regulated by E2 in ZR-75 cells 6 h after treatment but decreased to background levels 12 and 24 h after treatment (Fig. 1AGo). We also investigated the effects of E2 on VEGFR2 expression by immunofluorescence staining. ZR-75 cells were treated with Me2SO or 10 nM E2 for 7 h. IgG (nonspecific) and VEGFR2 antibodies were used to visualize protein expression (green), and nuclei were stained with propidium iodide (Fig. 1BGo). The results show that in Me2SO-treated cells, weak VEGFR2 staining was observed (Fig. 1BGo, e and f), and after treatment with 10 nM E2, there was enhanced cytoplasmic VEGFR2 staining (green). Thus, both VEGFR2 mRNA and protein are induced by E2 in ZR-75 cells.


Figure 1
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  		FIG. 1. A, Up-regulation of VEGFR2 mRNA by E2 in ZR-75 human breast cancer cells. ZR-75 cells were treated with Me2SO or 10 nM E2 for 6, 12, or 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) induction of VEGFR2 mRNA levels by E2. Results are presented as means ± SE for at least three determinations for each treatment group. B, Immunofluorescence detection of VEGFR2/KDR in ZR-75 cells treated with E2. ZR-75 cells were treated with 10 nM E2 (a–c and g–i) or Me2SO (d–f) for 7 h and incubated with normal rabbit IgG (a–c) or rabbit anti-KDR (d–f and g–i) and FITC (green)-conjugated secondary antibody as shown in b, e, and h. Nuclei were counterstained with propidium iodide (red) as shown in a, d, and g. Photographs were taken at the magnification of x200. Two respective photos were merged and shown in c, f, and i. VEGFR2/KDR staining (green) was increased in ZR-75 cells treated with E2.

 
Hormonal regulation of VEGFR2 promoter constructs: deletion and mutation analysis
The VEGFR2 promoter does not contain EREs; however, there are multiple cis-elements within the –716 to +268 region of the promoter. The results in Fig. 2AGo show that E2 induced activity in ZR-75 cells transfected with pVEGFR2A, pVEGFR2B, and pVEGFR2C, which contain –716 to +268, –225 to +268, and –95 to +268 VEGFR2 promoter inserts. Basal activity was essentially unchanged in the transfected cells, and hormone inducibility ranged from 2.4- to 4.5-fold. Further deletion of the –95 to –78 and –77 to –61 sequences resulted in lower (approximately 35%) basal activity (i.e. after transfection of pVEGFR2C, pVEGFR2D, and pVEGFR2E); however, hormone inducibility was slightly enhanced in cells transfected with these VEGFR2 deletion constructs (Fig. 2BGo). In contrast, both basal and hormone-induced activity decreased dramatically in ZR-75 cells transfected with pVEGFR2F, suggesting that the two GC-rich sites between –60 and –38 were critical elements for regulating VEGFR2 expression. Fold-inducibility was also increased in cells transfected with pVEGFR2F; however, because absolute activity was low, the fold-inducibility was highly variable; thus, hormone-responsive elements in the –37 to +268 region of the VEGFR2 promoter were not further investigated. Mutation of one or both GC-rich sites at –58 and –44 in the VEGFR2 promoter resulted in loss of basal and hormone-induced activity (Fig. 2CGo). These results demonstrate the importance of the two proximal GC-rich motifs in mediating hormonal activation of VEGFR2 in ZR-75 cells.


Figure 2
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  		FIG. 2. Deletion analysis of the VEGFR2 gene promoter and effects of E2 on luciferase activity in ZR-75 cells. ZR-75 human breast cancer cells were transiently transfected with 500 ng pVEGFR2A, pVEGFR2B, or pVEGFR2C (A), and pVEGFR2C, pVEGFR2D, pVEGFR2E, or pVEGFR2F (B), 250 ng pCDNA3.1-His-LacZ, and 500 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) induction of luciferase reporter activity by E2. a, No significant difference from E2-treated pGL2 (control). b, No significant difference from Me2SO-treated pGL2 (control). Results are expressed as means ± SE for at least three determinations for each treatment group. C, Mutation analysis of pVEGFR2E in ZR-75 cells. ZR-75 human breast cancer cells were transiently transfected with 500 ng 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, 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) induction of luciferase reporter activity by E2. a, No significant difference from E2-treated pGL2 (control). b, No significant difference from Me2SO pGL2 (control). Results are expressed as means ± SE for at least three determinations for each treatment group.

 
Previous studies have demonstrated that ER/Sp1-mediated transactivation of E2-responsive GC-rich promoter did not require the DBD of ER{alpha} (31, 32, 33, 34, 35, 36). In ZR-75 cells transfected with wild-type ER{alpha} and variants containing deletions in the DBD (HE11C) or AF-1 (HE19), E2 induced transactivation in cells cotransfected with the two former expression plasmids (Fig. 3AGo). These results demonstrate the requirement of the AF-1 but not the DBD of ER{alpha} for transactivation, and these results are similar to those observed for other hormone-induced genes activated by ER{alpha}/Sp proteins (31, 32, 33, 34, 35, 36). The results in Fig. 3BGo demonstrate the hormone receptor specificity of hormonal activation of VEGFR2. E2 induced activity in ZR-75 cells transfected with ER{alpha} and pVEGFR2C, and the antiestrogen ICI 182,780 inhibited hormone-induced transactivation. In contrast, P did not affect activity in ZR-75 cells transfected with PR-B, and similar results on the hormone receptor specificity of this response were observed in studies with VEGF in the same cell line (27).


Figure 3
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  		FIG. 3. A, Comparative effects of wild-type and variant ER{alpha} on E2-induced transactivation in ZR-75 cells transfected with pVEGFR2C. ZR-75 cells were transiently transfected with 500 ng pVEGFR2C and 500 ng 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) induction of luciferase activity. Results are presented as means ± SE for at least three determinations of each treatment group. B, Hormone and antiestrogen responsiveness of pVEGFR2C in ZR-75 cells. ZR-75 cells were transiently transfected with 500 ng pVEGFR2C and 500 ng ER{alpha} or PR-B. Cells were treated with Me2SO, 10 nM E2, 10 nM E2 + 1 µM ICI 182,780, 1 µM ICI 182,780 alone, or 10 nM P. Luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) induction of luciferase activity (*) and inhibition of induced activity by the antiestrogen ICI 182,780 (**) are indicated. Results are presented as means ± SE for at least three determinations for each treatment group.

 
ER{alpha} and Sp protein interactions with the VEGFR2 promoter
Interaction of Sp proteins with the proximal GC-rich sequences in the VEGFR2 promoter was investigated using EMSA with nuclear extracts from ZR-75 cells and VEGFR232P that contain the –64 to –35 proximal GC-rich sequence from the VEGFR2 promoter (Fig. 4AGo). The results show a pattern of retarded bands comparable with those observed using cancer cell nuclear extracts and other GC-rich oligonucleotides (34, 35, 36). Sp1 and Sp4 complexes form an overlapping retarded band, and a more mobile Sp3-DNA complex is also observed (lane 2). Coincubation with antibodies for Sp1, Sp3, and Sp4 resulted in formation of supershifted complexes (lanes 3–5, respectively), and coincubation with 100-fold excess of unlabeled oligonucleotide decreased intensity of all the retarded bands (lane 7). However, nonspecific IgG did not affect retarded band intensities (lane 6). These results clearly show that Sp1, Sp3, and Sp4 are expressed in ZR-75 cells, and all three proteins bind the GC-rich VEGFR2 oligonucleotide. We did not observe direct interactions of ER{alpha} with the Sp1-DNA complex in the EMSA, and this was consistent with results of previous studies with GC-rich oligonucleotides that did not observe formation of a ternary ER{alpha}/Sp1-DNA complex (31).


Figure 4
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  		FIG. 4. Sp protein binding to the VEGFR2 promoter. A, EMSA. Nuclear extracts from ZR-75 cells were incubated with radiolabeled VEGFR232P 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, Various retarded and supershifted complexes. B, Summary of primers (-> <-) and targeted regions of the pS2, VEGF, and VEGFR2 promoters used in ChIP assays. C, Analysis of protein interactions with the pS2, VEGF, and VEGFR2 promoter by ChIP. ZR-75 cells were treated with Me2SO (control) or 10 nM E2, and cells were harvested after treatment with hormone for up to 2 h and analyzed in a ChIP assay as described in Materials and Methods. 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.

 
Interactions of ER{alpha}, Sp1, Sp3, and Sp4 with the proximal GC-rich region of the VEGFR2 promoter in ZR-75 cells were also investigated using a ChIP assay (Fig. 4Go, B and C). In untreated cells (0 time), Sp1, Sp3, Sp4, and ER{alpha} were bound to the VEGFR2 promoter, and similar results were observed for binding to the E2-responsive GC-rich region of the VEGF promoter. VEGF was used as a comparative reference for studying the VEGFR2 promoter because both proteins/genes are induced by E2 in ZR-75 cells (27). The pS2 gene was also used as a control because previous studies show that treatment of MCF-7 cells with E2 enhances binding of ER{alpha} to the nonconsensus ERE in the pS2 promoter (37, 38, 39, 40). The results obtained in this study also show that E2 induces ER{alpha} binding to the pS2 promoter and that Sp1, Sp3, and Sp4 are constitutively bound to the promoter in the presence or absence of E2. GC-rich sites that bind Sp proteins have been identified previously in the ERE region of the pS2 promoter (41). In contrast, after treatment of ZR-75 cells with E2 for 15, 60, or 120 min, there were minimal changes in ER{alpha} or Sp protein binding to the VEGF or VEGFR2 promoter. Thus, in contrast to the results obtained for protein assembly on the pS2 promoter, ER{alpha} and Sp1, Sp3, and Sp4 are constitutively bound to the VEGFR2 (and VEGF) promoter. Results in Fig. 4DGo show that TFIIB binds to the GAPDH promoter (positive control) but not to exon 1 of the CNAP1 promoter (negative control).

RNA interference studies
Sp proteins play a critical role in regulating genes involved in growth and angiogenesis. Recent RNA interference studies in pancreatic cancer cells showed that Sp1, Sp3, and Sp4 are important for VEGF expression (27, 42). Initial studies showed that after transfection of ZR-75 cells with small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4), there was 35–50% knockdown of Sp proteins as determined by Western blot analysis of whole-cell lysates (Fig. 5AGo). Transfection efficiencies were 40–60%, indicating that the siRNAs were highly active, and this was confirmed in immunostaining of transfected cells, which indicated that in transfected cells, more than 90% of the targeted protein was degraded (Fig. 5BGo). In Fig. 5BGo, a–d, cells were stained for Lamin, and decreased staining was observed in cells transfected with iLamin (a); however, Lamin staining was observed in cells transfected with small inhibitory RNAs for Sp proteins (b–d). Sp1 (e), Sp3 (g), and weak Sp4 (i) immunostaining was observed in ZR-75 cells transfected with iLamin (nonspecific control), but transfection with iSp1 (f), iSp3 (h), and iSp4 (j) decrease staining of Sp1, Sp3, and Sp4 proteins, respectively. Staining with IgG or the secondary antibody (k and l) is also shown. The decreases observed with iSp1 and iSp3 are consistent with results of previous studies (42); the antibody available for Sp4 was less efficient, but iSp4 decreased the overall immunostaining for this protein. Results in Fig. 5Go, C and D, show that iGL2 (siRNA for luciferase) decreased activity by more than 90% in ZR-75 cells transfected with pVEGFR2A and pVEGFR2C; however, the effects of RNA interference of Sp protein expression were surprising. iSp3 and iSp4 significantly decrease hormone responsiveness, yet iSp1 did not affect basal or inducible luciferase activity. These results suggest that hormonal regulation of VEGFR2 in ZR-75 cells is primarily due to ER{alpha}/Sp3 and ER{alpha}/Sp4 but not ER{alpha}/Sp1.


Figure 5
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  		FIG. 5. Role of Sp proteins in hormonal regulation of VEGFR2. A, Sp protein knockdown, Western blot analysis. ZR-75 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). *, Sp protein levels were significantly (P < 0.05) decreased by RNA interference (relative to iLamin). B, Sp protein knockdown, analysis by immunostaining. ZR-75 cells were transfected with iLamin (control) (a, e, g, and i), iSp1 (b and f), iSp3 (c and h), or iSp4 (d and j) and immunostained for Lamin (a–d), Sp1 (e and f), Sp3 (g and h), or Sp4 (i and j) as described in Materials and Methods. IgG (k) and mouse secondary antibody (l) served as controls. Photographs were taken at the magnification of x60. Effects of iSp1, iSp3, and iSp4 on basal and E2-dependent activity in ZR-75 cells transfected with pVEGFR2A (C) and VEGFR2C (D). ZR-75 human breast cancer cells were transiently transfected with 500 ng pVEGFR2A or VEGFR2C 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 basal reporter activity by siRNAs (*) and decreased activity after treatment with E2 (**) compared with nonspecific control (iNS) are indicated. Results are presented as means ± SE for at least three determinations for each treatment group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
VEGFR2 is a key mediator of angiogenesis in normal and cancerous tissues, and this receptor is up-regulated in many cancer cell lines and tumors (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Tyrosine kinases such as VEGFR2 are targets for development of antiangiogenic drugs, and several studies have characterized VEGFR inhibitors that block tyrosine kinase activities (43, 44, 45, 46). For example, CEP-7055 (or N,N-dimethylglycine 3-[5,6,7,18-tetrahydro-9-[(1-methyleneoxy)methyl]-5-oxo-12H-indeno(2,1-9)pyrrolo(3,4-c) carbazol-12-yl] propyl ester) is a pan-VEGFR inhibitor that inhibits angiogenesis in both in vitro and in vivo models (44). Moreover, in athymic nude mouse xenograft studies, CEP-7055 inhibits growth of multiple tumor types including tumors in mice bearing MCF-7 breast cancer cell xenografts where a dose of 23.8 mg/kg·d (for 26 d) resulted in a 65% inhibition of tumor growth. Results of these inhibitor studies suggest that VEGFR2 plays a role in tumor growth, progression, and metastasis. Previous reports also showed that VEGFR2 was expressed in mammary tumors and both ER-negative and -positive breast cancer cells lines including T47D and MCF-7 cells (14). Results of this study confirm that VEGFR2 is also expressed in ZR-75 cells (Fig. 1Go).

Regulation of VEGFR2 expression is dependent on a number of factors including cell context. Initial studies by Patterson et al. (30) using VEGFR2 promoter constructs showed that basal activity in bovine aortic endothelial cells was primarily associated with the GC-rich –95 to –60 region of the promoter, which contains Sp, AP-2, and NF{kappa}B motifs. This analysis was also supported by DNA footprinting studies showing protected sequences between –110 and –25 in human umbilical vein endothelial cells (HUVECs). Interestingly, comparable interactions were not observed in fibroblasts or HeLa cells (47). Hata et al. (48) also showed that the GC-rich –79 to –68 region of the promoter was essential for activity in endothelial cells. This region bound both Sp1 and Sp3; however, their results suggested that Sp1 expression enhanced VEGFR2 expression but that Sp3 attenuated this response. In contrast, Urbich et al. (49) showed that basal and shear stress-induced activation of VEGFR2 promoter constructs in HUVECs was primarily dependent on two more proximal GC-rich sites at –58 and –44 bp. Results of this study using epithelial-derived ZR-75 cells show a remarkable similarity to the results reported for shear stressed HUVECs where the –58 and –44 sites in the VEGFR2 promoter are essential for high basal expression of VEGFR2 (Fig. 2Go). In ZR-75 cells, we have also confirmed, by both EMSA and ChIP assays, that Sp1, Sp3, and Sp4 constitutively bind regions of the VEGFR2 promoter encompassing the two proximal GC-rich sites (Fig. 4Go). The potential role of Sp3 in activating VEGF (27) and VEGFR2 expression in ZR-75 cells is in contrast to the inhibitory effects of the protein in endothelial cells, and this illustrates the important cell and promoter context-dependent effects of Sp3 on transactivation observed in other studies (50, 51, 52).

Hormone-dependent activation of VEGFR2 also primarily involves the proximal GC-rich sites in the VEGFR2 promoter (Fig. 2Go), and the results with PR and ER{alpha} variants (Fig. 3Go) are similar to those observed for other hormone-responsive genes activated through interactions of ER{alpha}/Sp with GC-rich cis-elements (52). Most previous studies in ZR-75 and MCF-7 breast cancer cells indicate that other E2-responsive genes regulated through GC-rich promoter sequences are primarily dependent on ER{alpha}/Sp1-mediated transactivation (27, 31, 32, 33, 34, 35, 36, 52). All of these genes, including VEGF, c-fos, adenosine deaminase, DNA polymerase {alpha}, E2F-1, and cad, contain the same consensus GGCGGG motifs that are present in the VEGFR2 promoter. However, RNA interference studies and selective knockdown of Sp1, Sp3, and Sp4 demonstrate that ER{alpha}/Sp1 plays a minimal role in activation of VEGFR2 and that both ER{alpha}/Sp3 and ER{alpha}/Sp4 are the critical factors required for this response (Fig. 5Go). We conclude that the gene promoter-selective use of one or more Sp proteins must be dependent not only on consensus GC-rich sites but also on flanking sequences, and current studies are investigating the role of these sequences in ER{alpha}/Sp-mediated transactivation. Moreover, unlike the pS2 gene where E2 enhances recruitment of ER{alpha} to the ERE promoter site (37, 38, 39, 40, 41), ER{alpha} and the Sp proteins are constitutively bound to the proximal GC-rich VEGFR2 and VEGF promoters (Fig. 4Go), and treatment with hormone has minimal effects on these interactions. Previous studies have confirmed that ER{alpha} interacts with Sp proteins in the absence of ligand (31, 53), and the ChIP results suggest that in ZR-75 cells, unliganded ER{alpha} is associated with Sp protein bound to E2-responsive GC-rich promoters and that addition of E2 does not significantly alter Sp or ER promoter interactions. Presumably, hormone induces recruitment of coregulatory proteins required for transactivation, and current studies in this laboratory are focused on identification and characterization of ER{alpha}/Sp coactivators.

In summary, our results show that ER{alpha}/Sp3 and ER{alpha}/Sp4 are involved in hormone-dependent activation of VEGFR2 in ZR-75 cells. Studies in several laboratories have demonstrated an important role for DNA-independent activation of genes through nuclear receptor interactions with DNA-bound Sp transcription factors (54, 55, 56, 57, 58, 59, 60, 61, 62, 63). In contrast to results of this study, peroxisome proliferator-activated receptor {gamma} differentially activated VEGFR2 through Sp1 but not Sp3 in retinal capillary endothelial cells (62). Peroxisome proliferator-activated receptor {alpha} agonists inhibited VEGFR2 in HUVECs, and this response was linked to interactions with Sp1 bound to the proximal –58 and –44 GC-rich sites (63). Thus, expression of VEGFR2 and other genes with GC-rich promoters can be up- or down-regulated by ER and other nuclear receptors, and current studies in this laboratory are focused on further understanding this pivotal gene regulatory pathway involving nuclear receptors and Sp proteins in breast cancer cells and other hormone-responsive tissues.


   Footnotes
 
This work was supported by the National Institutes of Health (Grants ES09106 and CA104116) and by the Texas Agricultural Experiment Station.

The authors have no conflicts of interest.

First Published Online March 30, 2006

Abbreviations: AF, Activation function; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; KDR, kinase insert domain receptor; P, progesterone; siRNA, small inhibitory RNA; TBP, TATA binding protein; TFIIB, transcription factor IIB; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Received January 20, 2006.

Accepted for publication March 22, 2006.


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 Discussion
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