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. Author manuscript; available in PMC: 2013 Jun 9.
Published in final edited form as: Cancer Res. 2008 May 15;68(10):3950–3958. doi: 10.1158/0008-5472.CAN-07-2783

Activation of Estrogen Receptor-α by the Anion Nitrite

David J Veselik 1, Shailaja Divekar 1, Sivanesan Dakshanamurthy 2, Geoffrey B Storchan 1, Jasmine MA Turner 2, Kelly L Graham 2, Li Huang 2, Adriana Stoica 2,3, Mary Beth Martin 1,2,3
PMCID: PMC3676890  NIHMSID: NIHMS473775  PMID: 18483281

Abstract

In this study, the ability of nitrite and nitrate to mimic the effects of estradiol on growth and gene expression was measured in the human breast cancer cell line MCF-7. Similar to estradiol, treatment of MCF-7 cells with either 1 μmol/L nitrite or 1 μmol/L nitrate resulted in ~4-fold increase in cell growth and 2.3-fold to 3-fold increase in progesterone receptor (PgR), pS2, and cathepsin D mRNAs that were blocked by the antiestrogen ICI 182,780. The anions also recruited estrogen receptor-α (ERα) to the pS2 promoter and activated exogenously expressed ERα when tested in transient cotransfection assays. To determine whether nitrite or nitrate was the active anion, diphenyleneiodonium was used to inhibit oxidation/reduction reactions in the cell. The ability of diphenyleneiodonium to block the effects of nitrate, but not nitrite, on the induction of PgR mRNA and the activation of exogenously expressed ERα suggests that nitrite is the active anion. Concentrations of nitrite, as low as 100 nmol/L, induced a significant increase in PgR mRNA, suggesting that physiologically and environmentally relevant doses of the anion activate ERα. Nitrite activated the chimeric receptor Gal-ER containing the DNA-binding domain of GAL-4 and the ligand-binding domain of ERα and blocked the binding of estradiol to the receptor, suggesting that the anion activates ERα through the ligand-binding domain. Mutational analysis identified the amino acids Cys381, His516, Lys520, Lys529, Asn532, and His547 as important for nitrite activation of the receptor.

Introduction

Over the past several decades, there has been a continuous increase in the incidence rate of breast cancer in the United States and worldwide (1, 2). Screening practices and access to mammography are thought to have contributed to the increased incidence rate during the 1980s, but changes in screening and mammography do not explain the increasing incidence rate before the 1980s or the increasing rates in countries with low rates of screening. The reason for the continuous increase is not known, but it has been suggested that there is an increased exposure to known risk factors or that there are new unidentified risk factors for the disease (2, 3). Family history is one risk factor (4). However, only 5% of breast cancers are believed to be truly hereditary (4), suggesting that most cases are due to factors such as lifestyle and the environment. Endocrine status is a prominent risk factor for breast cancer and includes early age at menarche and late age at menopause (5), increasing age at first full-term pregnancy (6) and oral contraceptives in specific subgroups of women (7). Postmenopausal obesity is also a risk factor for the disease (3) due to the aromatization of adrenal estrogen precursors in adipose tissue. These well-established risk factors are estimated to account for only ~40% of breast cancer (8). Childhood exposure to ionizing radiation (9) and alcohol consumption (10) are also risk factors. However, these modest risk factors do not account for the remaining cases supporting the hypothesis that there are new unidentified risk factors.

Because estrogens play an important underlying role in the etiology of breast cancer, it has been suggested that environmental exposures that mimic the effects of estrogens may be potential risk factors. However, the role of environmental estrogens is not clear. Early ecological and epidemiologic studies showed that Asian populations that consume diets rich in phytoestrogens (natural estrogens) have a lower incidence of breast cancer, raising the possibility that phytoestrogens may be effective chemo-preventive agents. However, most epidemiologic studies do not show a protective effect (reviewed in ref. 11). While phytoestrogens are thought to be protective, xenoestrogens (synthetic estrogens) are thought to increase the risk of breast cancer (12). However, most exposure studies do not show a clear correlation between xenoestrogens, such as polychlorinated biphenyls and/or p,p'-dichlorodiphenyl dichloroethylene, and the disease (reviewed in ref. 13). In addition to phytoestrogens and xenoestrogens, we have identified a new potent class of environmental estrogens (14–18) called metalloestrogens. These metals fall into two subclasses: metal/metalloid anions that include arsenite, selenite, and vanadate and bivalent cations that include cadmium, cobalt, copper, nickel, chromium, lead, mercury, and tin. In this study, we show that the ubiquitous anions, nitrite and nitrate, mimic the functions of estradiol at environmentally relevant doses. Similar to estradiol, the anions induced the growth and expression of estrogen-regulated genes in breast cancer cells and activated estrogen receptor-α (ERα) in transient transfection experiments. The ability of the anions to mimic the effects of estradiol seems to be due to the interaction of nitrite with the ligand-binding domain of the receptor.

Materials and Methods

Cell Culture

MCF-7 cells were maintained in improved minimal essential medium (IMEM; Biofluids, Inc.) and 5% fetal bovine serum (FBS). Cells (3 × 104) were then plated into a six-well plate in 4 mL IMEM supplemented with 5% charcoal-stripped calf serum (CCS). At 40% confluence, the medium was changed to phenol red–free IMEM with 5% CCS. Two days later, cells were treated. After treatment, cells were counted with a Coulter counter.

Immunoblots

MCF-7 cells were lysed in 300 μL lysis buffer (400 mmol/L KCl, 50 mmol/L Tris (pH 7.5; 1% NP40, 0.5% Na deoxycholate) in the presence of protease inhibitors (Roche). Fifty micrograms of protein were loaded onto a 7.5% polyacrylamide gel. Gels were run for 1.5 h at 150 V and transferred for 3 h at 100 V. The membranes were blocked, washed, and incubated with the primary antibody progesterone receptor (PgR) antibody 1294, DakoCytomation, Inc., and actin antibody, Santa Cruz Biotechnology). The membrane was then washed and incubated with the secondary antibody (goat anti-mouse IgG + IgM alkaline phosphatase, Amersham Biosciences). After washing, the membrane was incubated with ECF substrate, scanned on the Storm imaging system, and quantified using ImageQuant.

Chromatin Immunoprecipitation

Protein and DNA were cross-linked with 1% formaldehyde for 5 min at 25°C, washed in PBS containing protease inhibitors, collected in lysis buffer [50 mmol/L Tris (pH 8.0), 10 mmol/L EDTA, 1% SDS], and disrupted by sonication. The lysate was precleared by incubating with protein G agarose/salmon sperm DNA and incubated first with ERα antibody (H-184, Santa Cruz Biotech, Inc.) and then with protein G agarose/salmon sperm DNA. After centrifugation, the agarose pellet was washed once with a low salt buffer [20 mmol/L Tris (pH 8.0), 2 mmol/L EDTA, 150 mmol/L NaCl, 0.1% sodium deoxycholate, 1% Triton X-100], twice with a high salt buffer [20 mmol/L Tris (pH 8.0), 2 mmol/L EDTA, 500 mmol/L NaCl, 0.1% sodium deoxycholate, 1% Triton X-100], once with a LiCl buffer [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA, 250 mmol/L LiCl, 0.5% sodium deoxycholate, 05% NP40], and twice with a TE buffer [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA]. The DNA/protein complex was eluted (1% SDS, 0.1 mol/L NaHCO3), the crosslink was reversed with 5 mol/L NaCl, and the protein was digested with proteinase K. DNA was purified with PCR purification kit (Qiagen). For the PCR assay, the forward primer (5′-GGCCATCTCTCACTATGAATCACTTCTGC-3′), the reverse primer (5′-GGCAGGCTCTGTTTGCTTAAAGAGCG-3′; ref. 19), 5 μL of DNA, and 33 cycles of amplification were used.

Real-Time Reverse Transcription–PCR

The RNA was isolated using the Trizol method. DNA was degraded with DNase I. For the reverse transcriptase reaction, each 70 μL reaction contained 7 μL of 10× Taqman RT buffer [500 mmol/L KCl, 100 mmol/L Tris-HCl (pH 8.3)], 15.4 μL 25 mmol/L MgCl2, 14 μL deoxynucleotide triphosphates, 3.5 μL random hexamers, 1.4 μL RNase inhibitor, 1.75 μL MuLV reverse transcriptase, and 1 μg RNA. The mixture was incubated in a thermal cycler for 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C. For the real-time PCR reaction, each 25 μL reaction contained 12.5 μL Universal Master Mix, 1.25 μL of 20× Assay on Demand, and 5 μL cDNA. Samples were run on the 7900HT, and the data were analyzed by the 2−ΔΔCt method using the SDS 2.1 software (Applied Biosystems).

Transfection Assays

COS-1 cells (75 × 103) were plated into a 12-well plate in 1 mL of phenol red–free IMEM supplemented with 5% CCS. At 50% confluence, the cells were transfected with Fugene 6 and 2 μg of DNA (1 ER:10 reporter:0.4 β-galactosidase). A/B-GAL and GAL-ER (20, 21) and the ER mutants C381A, C417A, C447A, H516A, N519A, K520A, E523Q, K529A, C530A, N532D,and D538N (22–24) are described elsewhere. The ERα mutant H547A was generated using a QuikChange site-directed mutagenesis kit. After treatment, the amount of CAT activity was measured using the CAT enzyme assay (Promega). Heat-inactivated cell lysate (100 μL) was incubated with [14C]chloramphenicol and n-butryl CoA for 1 h at 37°C. The reaction product was extracted with mixed xylenes, and the amount of radioactivity was measured. For the β-galactosidase assay, 8.0 mmol/L O-nitrophenyl-b-d-galactopyranosidase were added to the cell lysate and incubated at 37°C for 2 h. The absorbance was read at 414 nm.

Binding Assay

Human recombinant ERα (4 × 10−9 mol/L; PanVera Corp.) was incubated on ice for 1 h with sodium nitrite. [3H]Estradiol (10−8 mol/L) was added in the presence or absence of a 200-fold molar excess of diethylstilbestrol and incubated for 2 h at 37°C. Dextran-coated charcoal was added to each sample and incubated for 15 min at room temperature. The samples were centrifuged, and the supernatant was counted in a liquid scintillation counter. Specific binding was calculated by subtracting nonspecific binding from total binding.

Molecular Modeling of the Nitrite Ion into Potential Binding Sites

Model construction, visualization, and analysis were performed with Insight II (Accelrys, Inc.) and Sybyl 7.2 (Tripos, Inc.) in conjunction with the molecular mechanics/dynamics package DISCOVER (Accelrys, Inc.). Molecular mechanics was used to minimize the energy of the molecule and to approach the global minimum energy state. Molecular dynamics was then performed on these molecules until they reached dynamic equilibrium. Both techniques treat the molecules as a collection of interaction centers (atoms) held together by parameterized intramolecular forces and are used to represent the bonds between atoms and the van der Waals forces between nonbonded atoms. Within the molecular dynamics simulation, the molecules adjust their conformations to optimize the bond lengths and angles to achieve a near “minimum energy” least strained structure, attained by a compromise minimum distortion. The force field is the sum of the various contributions derived from a potential. The force field used was the all-atom “consistent valence force field,” which includes explicit hydrogens.

Model building

The potential binding sites of nitrite were analyzed based on the crystal structure of the ligand-binding domain of ERα (PDB: 3ERD; ref. 25), the chemical properties of nitrite, and the mutational data. The oxyanion motif and the histidine, lysine, arginine, and cysteine side-chain residues were used in the identification of the binding sites. Models were constructed with nitrite docked manually into different binding sites. The quality of the models was checked by comparison of coordination geometry with the representative models of nitrite-binding proteins from the PDB database. Binding models were energy minimized followed by 25 ps molecular dynamics simulations using DISCOVER. In vacuo molecular dynamics simulations followed by energy minimizations on such dimolecular complex produced trigonal coordination geometries. The hydrogen bond distances between nitrite and the side chains ranged from 2.5 to 3.0 Å.

Energy minimization

A series of algorithms was used for minimization, including an initial steepest descent stage followed by a conjugate gradient stage converging to within the final mean square derivative threshold of 0.001 kcal mol−1 Å−1. The efficient Newton-Raphson algorithm was avoided because of instability when applied to the adopted energy expression. Periodic boundary conditions were applied using the explicit image convention.

Molecular dynamics

A dynamic simulations protocol, consisting of a molecular dynamics run at different temperatures followed by minimization, was found to be most effective for the dynamic behavior and binding mode prediction of the nitrite ion. Crystallographic coordinates of ERα were used as starting points, but no constraints were imposed during the simulation, so that the simulated structures depend only on the applied force field and not on the initial geometries. Insight II/DISCOVER, using the consistent valence force field, was used in the molecular dynamics simulations. Periodic boundary conditions, with bonds across the boundaries in the chain direction, were used in these dynamic simulations. The Ewald summation method was used in the calculation of nonbonded interactions. The simulations were performed at 298 K under constant pressure and temperature assembly conditions and consisted of 10,000 molecular dynamics steps in 1 fs intervals. The effect of cutoff distances was examined at rc = 12.0 Å. All other simulation variables were kept on default. All systems were equilibrated under the simulation conditions for at least 10 ps before collecting the trajectories for subsequent analysis.

Results

Effects of nitrite and nitrate on estrogen responses in MCF-7 cells

To determine whether nitrite and/or nitrate have estrogen-like effects, the ability of the anions to replace estradiol in the hormone-dependent proliferation of breast cancer cells was tested. MCF-7 cells, in hormone-free medium, were treated with 1 nmol/L estradiol, 1 μmol/L nitrite, or 1 μmol/L nitrate in the presence or absence of the antiestrogen ICI 182,780 (500 nmol/L). The number of cells was counted on day 0 and day 4 (Fig. 1A). As expected, treatment with estradiol resulted in ~4-fold increase in cell growth by day 4 that was blocked by the antiestrogen. Similar to estradiol, treatment with either nitrite or nitrate increased cell number by ~4-fold, and the increase was blocked by the antiestrogen, suggesting that the growth effects of the anions are mediated by ERα.

Figure 1.

Figure 1

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Effects of nitrite and nitrate on estrogen responses in MCF-7 cells. MCF-7 cells were grown in IMEM supplemented with 5% FBS. Two days before treatment, the medium was changed to phenol red–free IMEM supplemented with 5% CCS. Cells were then treated with 1 nmol/L estradiol, 1 μmol/L sodium nitrite, or 1 μmol/L sodium nitrate in the presence or absence of 500 nmol/L ICI 182,780. A, effects of nitrite and nitrate on the growth of MCF-7 cells. The number of cells was counted on day 0 and day 4. Columns, mean; bars, SD (n = 4; *, P < 0.005). □, day 0; ■ day 4. B, effects of nitrite and nitrate on the expression of estrogen regulated genes. MCF-7 cells were treated for 24 h. The amount of PgR protein was determined by Western blot analysis and normalized to the amount of actin. The amounts of PgR, pS2, and cathepsin D mRNA were determined by real-time RT-PCR and normalized to the amount of GAPDH mRNA. Data are expressed as percentage control. Columns, mean; bars, SD (n = 3; *, P < 0.05). ■ PgR protein; □ PgR mRNA; Inline graphic, pS2 mRNA; Inline graphic, cathepsin D mRNA. C, effects of nitrite and nitrate on the expression of PgR. MCF-7 cells were treated for 24 h. A representative Western blot analysis is shown. D, effects of nitrite on the recruitment of ERα to the pS2 promoter. MCF-7 cells were treated for 45 min, and the occupancy of ERα on the pS2 promoter was examined using a chromatin immunoprecipitation assay (n = 3). A representative PCR reaction is shown.

To test whether nitrite and nitrate also mimic the effects of estradiol on gene expression, the ability of the anions to induce PgR, pS2, and cathepsin D was measured (Fig. 1B and C). MCF-7 cells were treated for 24 h with 1 nmol/L estradiol, 1 μmol/L nitrite, or 1 μmol/L nitrate in the presence or absence of the antiestrogen ICI 182,780 (500 nmol/L). The amount of PgR protein was determined by Western blot analysis, normalized to the amount of actin, and expressed as percentage control. The amount of PgR, pS2, and cathepsin D mRNA was determined by real-time reverse transcription–PCR (RT-PCR), normalized to the amount of glyceraldehyde 3-phopshate dehydrogenase (GAPDH) mRNA, and also expressed as percentage control. As expected, treatment of cells with estradiol resulted in ~4-fold increase in PgR protein and ~2.5-fold, 4-fold, and 2.6-fold increase in PgR, pS2, and cathepsin D mRNA, respectively, that were blocked by the antiestrogen. Treatment with nitrite and nitrate also resulted in ~3.3-fold increase in PgR protein, as well as ~2.5-fold, 3-fold, and 2.3-fold increase in PgR, pS2, and cathepsin D mRNA, respectively, that were blocked by the antiestrogen. Treatment with estradiol and nitrite had no additional effect. To determine whether treatment with the anions results in the recruitment of ERα to estrogen-responsive promoters, cells were incubated with estradiol or nitrite for 45 min and the occupancy of the pS2 promoter was examined using a chromatin immunoprecipitation assay. As shown in Fig. 1D, treatment with estradiol and nitrite induced a significant increase in ERα occupancy of the pS2 promoter, suggesting that the anions activate ERα.

Effects of nitrite on the expression of PgR

In cells, nitrite and nitrate are readily interconverted by oxidoreductases, such as xanthine oxidoreductase (26). To ask whether nitrite or nitrate is the active anion, the oxidoreductase inhibitor diphenyleneiodonium was used. MCF-7 cells were treated with 1 nmol/L estradiol, 1 μmol/L nitrite, or 1 μmol/L nitrate in the presence or absence of 20 μmol/L diphenyleneiodonium and the effects on PgR mRNA were measured. The data were normalized to the amount of GAPDH mRNA and expressed as percentage control (Fig. 2A). As expected, treatment with estradiol, nitrite, or nitrate resulted in ~3-fold induction of PgR mRNA. Treatment with diphenyleneiodonium did not block the effects of estradiol or nitrite but blocked the effects of nitrate on the induction of PgR, suggesting that nitrite is the anion that activates ERα.

Figure 2.

Figure 2

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Effects of nitrite on estrogen responses in MCF-7 cells. MCF-7 cells were grown and treated as described in Fig. 1. Total mRNA was isolated, and the amount of PgR mRNA was determined by real-time RT-PCR. Data were normalized to the amount of GAPDH mRNA and expressed as percentage control. A, effects of diphenyleneiodonium on PgR induction by nitrite and nitrate. MCF-7 cells were treated for 24 h with 1 nmol/L estradiol, 1 μmol/L sodium nitrite, or 1 μmol/L sodium nitrate in the presence or absence of 20 μmol/L diphenyleneiodonium (DPI). Columns, mean; bars, SD (n = 3; *, P < 0.05). B, dose effect of nitrite on the expression of PgR. MCF-7 cells were treated with 0.01 nmol/L, 0.1 nmol/L, 1 nmol/L, 10 nmol/L, 0.1 μmol/L, 1 μmol/L, or 10 μmol/L sodium nitrite for 24 h. Points, mean; bars, SD (n = 3; P < 0.05 for 0.1–10 μmol/L).

To determine the concentrations of nitrite that induce the expression of PgR, MCF-7 cells were treated for 24 h with nitrite doses ranging from 0.01 nmol/L to 10 μmol/L. The effects on PgR mRNA were measured using the real-time RT-PCR assay (Fig. 2B). Treatment with 100 nmol/L, 1 μmol/L, or 10 μmol/L nitrite resulted in ~2.3-fold, 3.7-fold, and 4.1-fold induction, respectively, indicating that, at physiologically and environmentally relevant concentrations, nitrite has estrogenic activity.

Effects of nitrite and nitrate on the activity of ERα

To test the ability of nitrite and nitrate to activate ERα, COS-1 cells were transiently cotransfected with wild-type ERα and an estrogen response element-CAT reporter construct. The transfected cells were treated for 24 h with 1 nmol/L estradiol, 1 μmol/L nitrite, or 1 μmol/L nitrate in the presence or absence of the antiestrogen ICI 182,780 (500 nmol/L) or the oxidoreductase inhibitor diphenyleneiodonium (20 μmol/L).CAT activity was measured, normalized to the amount of β-galactosidase activity, and expressed as percentage control. Treatment with estradiol resulted in ~3-fold to 4-fold increase in CAT activity that was blocked by the antiestrogen. Similar to estradiol, treatment with either nitrite or nitrate resulted in ~3-fold increase in CAT activity that was also blocked by the antiestrogen (Fig. 3A), demonstrating the ability of the anions to activate exogenously expressed ERα. The oxidoreductase inhibitor had no effect on the induction of CAT activity by estradiol or nitrite but blocked the induction of CAT activity by nitrate (Fig. 3B) providing further support that nitrite is the anion that activates ERα.

Figure 3.

Figure 3

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Effects of nitrite and nitrate on the activity of ERα. COS-1 cells were grown in phenol red–free IMEM supplemented with 5% CCS and transiently cotransfected with wild-type ERα and an estrogen response element-CAT reporter construct. The transfected cells were treated for 24 h with 1 nmol/L estradiol, 1 μmol/L sodium nitrite, or 1 μmol/L sodium nitrate in the presence or absence of 500 nmol/L ICI 182,780 (A) or 20 μmol/L diphenyleneiodonium chloride (B). CAT activity was measured, normalized to the amount of β-galactosidase activity, and expressed as percentage control. Columns, mean; bars, SD (n = 3; *, P < 0.05).

Effects of nitrite and nitrate on the activation of ERα mutants

To identify the region of ERα activated by nitrite, transient transfection assays were conducted using chimeric receptors (20, 21). To determine whether the anion activates the receptor through the NH2 terminus, the A/B-GAL chimera containing the A/B domain of ERα fused to the DNA-binding domain of the yeast transcription factor GAL4 was used. To establish whether the anions activate through the COOH terminus, the GAL-ER chimera was used. The latter chimera contains the DNA-binding domain of GAL4 fused to the ligand-binding domain of ERα. In these assays, the chimeric receptors were transiently cotransfected with a GAL4-CAT reporter construct into COS-1 cells and the cells were treated for 24 hours with 1 nmol/L estradiol, 1 μmol/L nitrite, or 1 μmol/L nitrate in the presence or absence of the antiestrogen ICI 182,780 (500 nmol/L). The amount of CAT activity was measured, normalized to the amount of β-galactosidase activity, and expressed as percentage control. When cells were transfected with A/B-GAL and treated with estradiol, nitrite, or nitrate, there was no increase in CAT activity (data not shown). As a positive control, cells transfected with A/B-GAL were treated with insulin-like growth factor (IGF-I; 40 ng/mL). As expected, treatment with IGF-I induced ~2-fold increase in CAT activity (data not shown). In contrast to the effects on A/B-GAL, when cells were transfected with GAL-ER and treated with estradiol, nitrite, or nitrate, there was ~3-fold increase in CAT activity that was blocked by the antiestrogen (Fig. 4B). The ability of nitrite to activate GAL-ER suggests that the anion activates ERα through the ligand-binding domain.

Figure 4.

Figure 4

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Effects of nitrite and nitrate on the activation of ERα mutants. COS-1 cells were transiently cotransfected with ERα mutants and a CAT reporter construct. Cells were treated for 24 h with 1 nmol/L estradiol, 1 μmol/L sodium nitrite, or 1 μmol/L sodium nitrate in the presence or absence of 500 nmol/L ICI 182,780. CAT activity was measured, normalized to the amount of β-galactosidase activity, and expressed as percentage control (mean ± SD; n = 3; *, P < 0.05). A, diagram of ERα expression vectors. B, ERα ligand-binding domain chimera (GAL-ERα). C, ERα mutants C381A, C417A, C447A, H516A, N519A, K520A, E523Q, K529A, C530A, N532D, D538N, and H547A.

Because helix H11 and helix H12 undergo conformational changes when ERα is activated, amino acids on helices H10 and H11, in the loop structure between helix H11 and helix H12, and on helix H12, that can potentially interact with nitrite, were examined. The ability of the anions to activate the mutant H516A (helix H10), mutants N519A and K520A (helix H11), mutants K529A, C530A, and N532D (loop 11–12), and mutant H547A (helix H12) were tested. As negative controls, E523Q (helix H11) and D538N (loops 11–12) were tested. In addition to the repositioning of helices H11 and H12, several other movements are thought to occur upon activation of the ligand-binding domain. To identify amino acids that can potentially facilitate these movements, cysteine mutants C381A, C417A, and C447A located on helix H4, helix H6/H7, and helix H8, respectively, were tested due to the ability of nitrite to interact with thiol groups. As shown in Fig. 4C all of the mutants were activated by estradiol. Treatment with the hormone resulted in a 2.5-fold to 4-fold increase in CAT activity. Treatment with nitrite and nitrate also activated the mutants C417A, C447A, N519A, E523Q, C530A, and D538N; there was ~3-fold increase in CAT activity. However, treatment with the anions failed to activate the mutants C381A, H516A, K520A, K529A, N532D, and H547A suggesting that these amino acids are involved in nitrite activation of the receptor.

Effects of nitrite on estradiol binding to ERα

To determine whether nitrite blocks the binding of estradiol to ERα, purified human recombinant ERα was incubated with various concentrations of nitrite (10−12 to 10−6 mol/L) for 1 hour. [3H]Estradiol (10 nmol/L) was then added in the presence or absence of a 200-fold molar excess of diethylstilbestrol for 2 hours at 37°C. Specific binding of [3H]estradiol was determined and expressed as percentage control. As shown in Fig. 5, increasing concentrations of nitrite blocked the binding of estradiol to ERα suggests that the anion interacts directly with the ligand-binding domain.

Figure 5.

Figure 5

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Effects of nitrite on estradiol binding to ERα. Purified human recombinant ERα was incubated with various concentrations of sodium nitrite (10−12 to 10−6 mol/L) for 1 h. [3H]Estradiol (10 nmol/L) was added in the presence or absence of a 200-fold molar excess of diethylstilbestrol for 2 h at 37°C. The amount of specific binding of [3H]estradiol was determined and expressed as percentage control. Columns, mean; bars, SD (n = 3).

Molecular modeling of the nitrite ion into potential binding sites of ERα

The mutational analysis described above identified cys381 on helix H4, his516 on helix H10, lys520 and lys529 on helix H11, asn532 in the loop between H11 and H12, and his547 on helix H12 as potential interaction sites of nitrite with ERα. Based on these data, the crystal structure of the ligand-binding domain of ERα (PDB: 3ERD; ref. 25), and the chemical properties of nitrite, potential binding sites of the anion with the receptor were identified using molecular modeling. For the construction, visualization, and analysis of the model, Insight II and Sybyl 7.2 were used. Three potential binding sites were identified for nitrite on the solvent accessible surface of the ligand-binding domain (Fig. 6A–C). Site 1 was formed by a direct interaction of nitrite with Lys529 on helix H11 and Asn532 in the loop between helices H11 and H12 and by an indirect interaction through water with Val534 also in the loop between helices H11 and H12. Site 2 was formed by a direct interaction of the anion with His516 on helix H10 and lys520 on helix H11 and an indirect interaction through water with Asn519 at the interface of helices H10 and H11 and Glu523 on helix H11. The third site, site 3, was formed by the direct interaction of nitrite with Cys381 on helix H4, Arg515 on helix H10, and His547 on helix H12. The identification of three potential binding sites formed by amino acids on different helices suggests that nitrite promotes a conformational change in the ligand-binding domain, thereby activating the receptor.

Figure 6.

Figure 6

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Molecular model of the interaction of nitrite with ERα. Three potential nitrite-binding sites on the solvent accessible surface of the ligand-binding domain of ERα were identified by computational analysis as described in Materials and Methods. A shows site 1 which is formed by Lys529 on helix H11 and Asn532 and Val534 in the loop between helices H11 and H12. B displays site 2 which is formed by His516 on helix H10 and Asn519, Lys520, and Glu523 on helix H11. C shows site 3 which is composed of Cys381 on helix H4, Arg515 on helix H10, Asn519 on helix H11, and His547 on helix H12. D shows the proposed model of ERα activation by nitrite. In step 1, nitrite interacts with site 1 on helix H11 and in the loop between helices H11 and H12, resulting in the movement of helix H12 toward the ligand-binding pocket. In step 2, the anion interacts with site 2 on helix H10 and on helix H11 resulting in the formation of a continuous bent helix between helices H10 and H11. In step 3, nitrite interacts with site 3 on helix H4 and helix H12 pulling helix H12 under helix H4 resulting in the displacement of the ω loop and the repositioning of helix H3 to form the coactivator binding site. Water molecules are represented by balls (red); nitrite and amino acid side chains are presented as ball and stick model.

Discussion

This study shows the ability of physiologically and environmentally relevant doses of nitrite and nitrate to mimic the effects of estradiol in human breast cancer cells. The ability of low doses of nitrite and nitrate to mimic estradiol suggests that the anions may play a role in the etiology and progression of breast cancer due, in part, to their ability to activate ERα. Human exposure to these anions occurs through both exogenous and endogenous sources as a result of the environment, diet, and medicinal drugs and as the breakdown product of nitric oxide. In surface and drinking water, nitrate is generally low but has increased over the last few decades (27) due to the use of nitrogen-based fertilizers, contamination from refuse dumps, oxidation of ammonia from human and animal waste, and treatment of drinking water with chloramines (28). High amounts of nitrate are also present in some fruits and vegetables as a result of cultivation in greenhouses and in cured and processed meats due to their addition as preservatives and color enhancers (29). Medications, including antimalarials, antidiarrheals, diuretics, and vasodilators, also contribute to exposure. Our total estimated daily exposure to exogenous nitrites and nitrates ranges from 1.2 to 3 mg and from 39 to 268 mg, respectively (30). Exposure to the anions also occurs as a result of the endogenous oxidation of nitric oxide to nitrites and nitrates. Nitric oxide is synthesized by a family of nitric oxide synthases (NOS). In serum, the normal concentration of nitrite is ~18.0 to 43.5 μmol/L (31–34) whereas, in breast cancer patients, serum concentrations range from 246 to 363 μmol/L (35). The amount of nitrite is also elevated in breast tissue from breast cancer patients (745 nmol/g wet weight) compared with the amount in benign breast disease (442 nmol/g wet weight; ref. 36). In breast tumors, the expression of NOS is associated with increased proliferation and tumor grade and the expression of PgR and nitric oxide is associated with invasive disease (37). Similarly, in animal models, nitric oxide promotes the progression of mammary tumors (38–40). Interestingly, epidemiologic studies suggest a link between postmenopausal breast cancer and farming (41, 42). There is also evidence that nitric oxide and/or its metabolites have other endocrine-like effects. In older women, intermittent use of nitrates is associated with increased bone mineral density (43), and in the neuronal NOS knockout mouse, more aggressive and inappropriate sexual behavior is observed (44). Taken together, these observations suggest a possible endocrine-like role for nitrite and nitrates in breast cancer.

The ability of nitrite and nitrate to mimic the effects of estradiol is due to an interaction of nitrite with the ligand-binding domain of ERα. Similar to other nuclear receptors (25, 45–47), the ligand-binding domain of ERα contains 11 α helices (H1–H12 minus H2) folded into a three-layered antiparallel α helical sandwich. The central core layer contains three α-helices (H5/6, H9, and H10) sandwiched between two additional layers of helices composed of H1–H4, H7, H8, and H11 and flanked by helix H12 (46, 47). Upon binding, the ligand induces a conformational change that results in a transcriptionally active receptor. Based upon the crystal structure of apo-RXR-α and holo-RXR-α (45, 48), several major structural changes are thought to occur as a result of ligand binding; helix H12 is repositioned over the ligand-binding pocket; helix H11 rotates ~180° around its helical axis and tilts away from the pocket; the NH2 terminal end of helix H3 rotates ~90° around its axis and bends toward helix H4 and the core of the pocket; the adjacent ω loop relocates under helix H6; and the β turns, located between helices H4/H5 and H6, shift away from the core of the pocket (45, 48). To account for these structural changes, it has been proposed (49, 50) that the retinoid is initially attracted into the ligand-binding pocket by electrostatic interactions. Once inside the pocket, the retinoid induces the rearrangement of helix H3 which, in turn, causes helix H11 to rotate and to form a continuous bent helix with helix H10. The repositioning of helix H11 pulls helix H12 under helix H4 freeing the ω loop from its interaction with helix H12. The repositioning of helices H3 and H12 results in the formation of a shallow hydrophobic groove that constitutes the AF-2 domain, the binding site for steroid receptor coactivators. In the case of ERα, it has been suggested that, in addition to inducing a conformational change, estradiol functions inside the pocket as a scaffold for the structure of the ligand-binding domain (47). The hydroxyl group on the A ring of the steroid hydrogen bonds with Glu353 on helix H3, Arg394 on helix H5, and a water molecule, whereas the hydroxyl group on the D ring hydrogen bonds with His524 on helix H11. The steroid backbone forms van der Waals interactions with hydrophobic amino acids on helices H3, H5, H7, and H11 and in the β-turn (46, 47). In contrast to estradiol, nitrite seems to interact with amino acids on the solvent accessible surfaces of the ligand-binding domain. Mutational analysis identified Cys381 on helix H4, His516 on helix H10, Lys520 and Lys529 on helix H11, Asn532 in the loop between H11 and H12, and His547 on helix H12 as potential sites of interaction between nitrite and the receptor. Molecular modeling suggests that these amino acids form three potential binding sites on the surface of the activated receptor. Site 1 is formed by the interaction of nitrite with Lys529 on helix H11 and Asn532 in the loop between helices H11 and H12; site 2 results from the interaction of the anion with His516 on helix H10 and Lys520 on helix H11, whereas site 3 is due to an interaction of nitrite with Cys381 on helix H4 and His547 on helix H12. Molecular modeling also identified Val534 in the loop between helices H11 and H12 as a potential amino acid in site 1; Asn519 and Glu523 on helix H11 as potential amino acids in site 2; and Arg515 on helix H10 as a potential amino acid in site 3. The interactions of nitrite with Val534, Asn519, and Glu523 seem to be indirect interactions through water and may explain the ability of nitrite to activate the N519A mutant. Although the precise mechanism by which nitrite interacts with these sites to activate ERα remains to be defined, it is proposed that the interaction of the anion with these sites induces structural changes that mimic the structural changes induced upon the binding of estradiol (Fig. 6D). In step 1, the interaction of nitrite with Lys529 on helix H11 and Asn532 in the loop between helices H11 and H12 (site 1) may result in the movement of helix H12 over the ligand-binding pocket; in step 2, the interaction with His516 on helix H10 and Lys520 on helix H11 (site 2) would result in the formation of a continuous bent helix between helices H10 and H11; and in step 3, the interaction of nitrite with Cys381 on helix H4 and His547 on helix H12 (site 3) would pull helix H12 under helix H4 resulting in the displacement of the ω loop that causes the repositioning of helix H3 to form the coactivator binding site. It is not known whether nitrite interacts with these sites in a sequential manner or whether the anion occupies all three sites simultaneously. In a simultaneous model, three molecules of nitrite bind to the ligand-binding domain resulting in the formation of the active conformation of the receptor. In a sequential model (Fig. 6D), site 1 is postulated as the initial entrant site located in the flexible region near the COOH terminal end of the ligand-binding domain. As a consequence of the flexibility of the ligand-binding domain and the conformational changes that occur upon binding of nitrite, the anion is displaced from site 1 and moves into the second binding site and subsequently into the third binding site. Because the molecular modeling is based on the static, activated receptor, not all of the amino acids in transient interaction sites will be identified. For example, if sites 1 and 2 are transient binding sites, it is possible that additional amino acids may be involved in the interaction of the anion with these sites and between the sites. These models remain to be tested. The amino acids involved in nitrite activation of ERα are conserved in ERβ, suggesting that the anion may also activate other steroid receptors.

Acknowledgments

Grant support: Komen Foundation, AICR, NIH ES11745, NIH P30-CA51008, P50-CA58185, and U54 CA0100970.

We thank Prof. P. Chambon and Dr. B. Katzenellenbogen for estrogen receptor mutants, Dr. S. Byers, Dr. B. Katzenellenbogen, and Dr. A.T. Reigel for helpful discussion, National Cancer Institute for allocation of computing time, and staff support at the Advanced Biomedical Computing Center.

Footnotes

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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