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Biochemical and Biophysical Research Communications 286, 529–533 (2001)

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itric Oxide Inhibits Selectively the 17b-Estradiol-Inducedene Expression without Affecting Nongenomicvents in HeLa Cells

aria Marino,1 Ramona Ficca, Paolo Ascenzi, and Anna Trentalanceepartment of Biology, University “Roma Tre,” Viale G. Marconi 446, I-00146 Rome, Italy

eceived July 9, 2001

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17b-Estradiol (E2) induces genomic (i.e., pC3-lu-iferase promoter–reporter construct expression) andongenomic (i.e., DNA synthesis and IP3 production)ffects in HeLa cells only after transient transfectionith the human estrogen receptor a (ERa) reporterlasmid. Here the effect of nitric oxide (NO) on both2-induced effects in transiently transfected HeLaells is reported. Remarkably, the E2-dependent generanscription is inhibited dose-dependently by NO. Byontrast, DNA synthesis and IP3 production, repre-enting nongenomic E2-dependent effects, are unaf-ected by NO. The selective NO action on E2-inducedunctions may be related to NO-mediated chemical

odification(s) of the Cys residues present in the DNAecognition domain of ERa impairing DNA binding.2001 Academic Press

Key Words: 17-b-estradiol; human estrogen receptor; transcriptional activity; DNA synthesis; IP3 produc-ion; nitric oxide; gene transcription inhibition; HeLaells.

As occurs for other sex steroids, the cellular effects of7b-estradiol (E2) are mediated through two differentechanisms. The classic genomic mechanism identi-es a pathway, starting with the activation of the cy-osolic estrogen receptor (ER), in both a and b isoformsERa and ERb, respectively), which, in turn, works as

transcription factor by binding the estrogen respon-ive element present in the promoter of hormone-

Abbreviations used: AP-1, activator protein 1; Cor, cordicepyn; E2,7b-estradiol; ER, human estrogen receptor; GSH, glutathione;SNO, S-nitroso-glutathione; IP3, inositoltrisphosphate; MAP ki-ase, mitogen-activated protein kinase; NF-kB, nuclear factor-kB;O, nitric oxide; NOR-3, (6)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-ni-

ro-3-hexenamide; NOR-3*, NO-deprived NOR-3; PBS, phosphate-uffered saline solution; PKC, protein kinase C; PLC, phospholipase; RXR, retinoid X receptor; U7, U-73122; VDR, 1a,25-dihydroxy-itamin D3 receptor.

1 To whom correspondence and reprint requests should be ad-ressed. Fax: 139-06-55176321. E-mail: m.marino@bio.uniroma3.it.

529

ecently, nongenomic estrogen effects, similar to thosevoked by peptide hormones or growth factors (e.g.,LC/PKC activation, intracellular Ca21 release, andAP kinase activation), have been attributed to the

bility of estrogens to interact with a membrane-ssociated ER (for reviews, see Refs. 3 and 4). Theechanism(s) by which E2 may induce either activity

s still unknown. Some reports indicate different struc-ural and functional properties for the membrane es-rogen receptor with respect to ERa and ERb (5, 6).owever, a subpopulation of ERa and ERb is localized

n the plasma membrane and is responsible also ofongenomic function(s) (7–9). Furthermore, this pic-ure is made more obscure by the reported ability of2-induced nongenomic pathways to regulate ER ac-

ivities (10, 11).Among different messengers rapidly induced by es-

rogens, attention has been pointed to nitric oxide (NO)9). This diatomic messenger is known to induce a wideariety of effects in biological systems which can beartially ascribed to the ability of NO to inhibit tran-cription factor (i.e., NF-kB, AP-1, VDR, and RXR)ctivity (12–14) and DNA synthesis (15). Some of theseffects have been associated to chemical modifica-ion(s) of Cys residues present in the DNA-bindingomain of transcription factors (14). The presence ofine Cys residues in the DNA-binding domain of ERa,f which eight are involved in two zinc–thiolate centersnd essential for ERa dimerization and DNA bind-ng (16); and four in the ligand-binding domain (17),

akes ERa susceptible to NO-mediated chemicalodification(s).Here the effect of NO, released from NO donors, on

he ERa functions in HeLa cells is reported. This celline, devoid of endogenous ER (18) and NO synthase19), represents a valuable model to investigate theffect of NO on exogenous ER, properly transfectedith the human ERa-expression plasmid. Remarkably,O inhibits the E2-induced gene expression without

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 286, No. 3, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

n some of the E2-induced functions may be related toO-mediated chemical modification(s) of the Cys resi-ues present in the DNA recognition domain of ERampairing DNA binding.

ATERIALS AND METHODS

Reagents. E2, the transcription inhibitor cordycepin (39-deoxy-denosine), gentamicin, glutathione (GSH), (6)-(E)-4-ethyl-2-[(E)-ydroxyimino]-5-nitro-3-hexenamide (NOR-3), Dowex1X-8 resin,enicillin, DMEM (with or without phenol red), fetal calf serum, andharcoal-stripped fetal calf serum were purchased from Sigmahemical Co. (St. Louis, MO). The PLC inhibitor U-73122 was ob-

ained from Calbiochem (San Diego, CA). Methyl-1-[3H]thymidinespecific activity 81 Ci/mmol), myo-2-[3H]inositol (specific activity 23i/mmol), and [3H]IP3 (specific activity 1 mCi/mmol) were purchased

rom Amersham–Pharmacia (Little Chalfont, UK). Lipofectamineeagent was obtained from GIBCO-BRL Life Technologies (Gaith-rsburg, MD). The luciferase kit was obtained from Promega (Mad-son, WI). S-nitroso-glutathione (GSNO) and NO-deprived NOR-3NOR-3*) were prepared as previously reported (20). All the otherroducts were from Sigma Chemical Co. Analytical or reagent graderoducts, without further purification, were used.

Cell culture. HeLa cells were routinely grown in the presence of% CO2 in air atmosphere in modified DMEM phenol red-free me-ium containing 10% (v/v) charcoal-stripped fetal calf serum,-glutamine (2.0 mM), gentamicin (10 mg/ml), and penicillin (100/ml). Cells were passaged every 3 days and media changed everydays.

Transfection and luciferase assay. HeLa cells (2 3 105), ;70%onfluence, were transiently cotransfected with 0.5 mg of the emptylasmid pCMV5 or of the human ERa expression plasmid pCMV5-Ra (21) and 1 mg of the promoter of human complement geneC3-luciferase construct (22), using Lipofectamine Reagent accord-ng to the manufacturer’s instructions. 1 mg of the plasmid express-ng Renilla pRLnull was used to normalize for transfection efficiency.ix hours after transfection, the medium was changed and 24 h later,ells were pre-treated with GSNO, NOR-3, GSH, or NOR-3* (finaloncentration, 10 nM to 1 mM). After 30 min, E2 (final concentration,0 nM) or vehicle (ethanol/PBS 1:10 v/v) was added and the reporterlasmid expression was evaluated 6 h thereafter. Where indicated,ordycepin (final concentration, 30 ng/ml), or U-73122 (final concen-ration, 10 mM), or ICI 182,780 (final concentration, 1 mM) was added5 min before E2. The cell lysis procedure and the subsequent mea-urement of luciferase gene expression were performed using theuciferase kit according to the manufacturer’s instructions, with aerthold luminometer apparatus (Wallack, Italy).

DNA synthesis. DNA synthesis was assayed incubating subcon-uent HeLa cells, transiently transfected with pCMV5 or pCMV5-Ra for different times ranging from 30 min to 24 h, in the absencend presence of E2 (final concentration, 10 nM) with methyl-1-

3H]thymidine (final concentration, 1 mCi/ml). Thymidine incorpora-ion was assayed as previously reported (23). Six hours after trans-ection, the medium was changed and 24 h later, cells were treatedith GSNO, NOR-3, GSH or NOR-3* (final concentration, 10 nM tomM). After 30 min, E2 (final concentration, 10 nM) or vehicle

ethanol/PBS 1:10 v/v) was added. Cordycepin (final concentration,0 ng/ml), or U-73122 (final concentration, 10 mM), or ICI 182,780final concentration, 1 mM) was added 15 min before E2 and methyl--[3H]thymidine.

IP3 production. Subconfluent cells, grown in DMEM medium con-aining 0.2 mM myo-inositol, were exposed, after transfection, toyo-2-[3H]inositol (1 mCi/ml) for 24 h. Then, medium was discarded

nd, after three washes with PBS, cells were incubated with fresh,

530

final concentration, 10 nM to 1 mM). After 30 min, E2 (final concen-ration, 10 nM), or vehicle (ethanol/PBS 1:10 v/v), was added for 5in. Where indicated, cordycepin (final concentration, 30 ng/ml), or-73122 (final concentration, 10 mM), or ICI 182,780 (final concen-

ration, 1 mM) was added 15 min before E2. At the end of incubationime, cells were rinsed with PBS and harvested in 1 ml of 10%richloroacetic acid (w/v) containing 2.0 mM EDTA. The trichloro-cetic acid soluble fraction was washed with diethyl ether, and thennalyzed by anionic exchange chromatography on Dowex1X-8 resinformate form). IP3 was eluted from small glass columns with 0.8 Mmmonium formate in formic acid (0.1 M), as previously reported24), and radioactivity was counted with a liquid scintillation counterPackard-Camberra, The Netherlands). Under the same experimen-al conditions, [3H]IP3 standard was eluted from small glass columnsith 15 ml ammonium formate (0.8 M) in formic acid (0.1 M). The

richloroacetic acid-insoluble fraction was dissolved in 1.0 M NaOHnd the protein content was measured (25).

ESULTS AND DISCUSSION

E2 induced the expression of the reporter gene pC3-uciferase, IP3 production and DNA synthesis in HeLaells only after the transient transfection of ERa ex-ression plasmid. No E2 effect occurred after transfec-ion of HeLa cells with the empty plasmid. Further-ore, the E2 antagonist ICI 182,780 completely

revented all E2-induced effects. The transcription in-ibitor cordycepin (10, 26) inhibited the expression ofhe reporter gene pC3-luciferase, without affectingoth nongenomic activities (i.e., IP3 production andNA synthesis). Moreover, the PLC inhibitor U-73122locked both IP3 production and DNA synthesis, with-ut affecting genomic activity, as previously reported10) (Fig. 1).

These results indicate that nongenomic/membraneesponse(s) is ERa-dependent. Although in most caseshe plasma membrane E2-binding sites have not iso-ated and characterized, different categories of puta-ive receptors have been described (5–9, 27). Presentata show that IP3 is rapidly produced in HeLa cellsransiently transfected with expression vector for sol-ble ERa, thereby indicating that E2 effects are re-

ated to a membrane protein corresponding to the cog-ate nuclear receptor (7–9). Furthermore, the rate ofP3 production and DNA synthesis are increased by E2ven in the presence of the transcription inhibitor,ndicating that nongenomic activities of ERa are notelated to the genomic functions. Consistently withreviously reported data (11, 28), this result may beonsidered a new paradigm for E2 action.As shown in Fig. 2, both NO donors NOR-3 andSNO inhibited dose-dependently the E2-inducedC3-luciferase gene expression, without affecting IP3

roduction and DNA synthesis. Notably, the NO donoroncentration used (ranging between 10 nM and 1 mM)ell in the physiological NO concentration range (29).s expected, NO-deprived GSNO and NOR-3 (i.e., GSHnd NOR-3*, respectively) did not affect genomic andongenomic ERa actions. Therefore, NO selectively

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531

locks the E2-induced transcriptional activity of ERa.emarkably, NO blocks also the transcriptional activ-

ty of the nuclear transcription factors NF-kB and AP-130), as well as of the nuclear receptors VDR and RXR14, 30).

The NO(-donor)-mediated chemical modification(s)f (macro)molecular targets, such as Cys-containingroteins, occurs via (trans)nitrosylation reactions andixed disulfide bridge formation. The NO-mediated

hemical modification(s) of Cys residues is facilitatedy neighboring amino acid side chains, acting as basend/or acid, as well as by metal centers, stabilizing theeactive anionic form of thiols (31–33). In particular,he great propensity for nitrosothiol and mixed disul-de bridge formation represents a modulation mecha-ism of (macro)molecules containing NO-reactive Cys

FIG. 2. Dose-dependent effect of NOR-3 and GSNO (i.e., NO) onhe E2-induced ERa genomic (i.e., pC3 transcription) and non-enomic (i.e., IP3 production and DNA synthesis) functions in HeLaells transiently transfected with pCMV5-ERa. After 24 h, the cellsere pretreated (30 min) with NO donors in the presence of 10 nM2. Then the expression of the pC3-luciferase reporter gene, the

hymidine incorporation into DNA, and the myo-inositol incorpora-ion into IP3 were evaluated. NO-deprived NOR-3 (NOR-3*) andSNO (GSH) did not affect the E2-induced ERa functions. Each bar

epresents the mean 6 SD of five independent experiments, eacherformed in duplicate. *Significantly different from cells stimulatedith 17b-estradiol (Control) (P , 0.001). For further experimentaletails, see the text.

FIG. 1. E2-induced genomic (i.e., pC3 transcription) and non-enomic (i.e., IP3 production and DNA synthesis) functions in tran-iently transfected HeLa cells. The cells were cotransfected withammalian expression vectors for empty or pCMV5-ERa (ERa) plas-ids together with pC3-luciferase (pC3). After 24 h, the cells were

retreated (15 min) with 30 ng/ml cordycepin (E2 1 Cor), 10 mM U3,122 (E2 1 U7), or 1 mM ICI 182,780 (E2 1 ICI) in the absenceEtOH) or presence of 10 nM 17b-estradiol (E2). Then the expressionf the pC3-luciferase reporter gene, the thymidine incorporation intoNA, and the myo-inositol incorporation into IP3 were evaluated.ach bar represents the mean 6 SD of five independent experi-ents, each performed in duplicate. *Significantly different fromonstimulated cells (EtOH) (P , 0.001). °Significantly differentrom cells stimulated with 17b-estradiol (E2) (P , 0.001). Forurther experimental details, see the text.

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nd/or allosteric site(s) (31–36). Notably, human ge-atinase-A and the nuclear receptors VDR and RXR

ay be considered as paradigms for the NO-mediatedhemical modification(s) of zinc–thiolate centers (30, 37).

The prevalence and high reactivity of thiols overther nucleophiles (31–36) suggest that Cys residue(s)ay represent NO targets also in ERa. The ligand-

inding domain and the DNA-binding domain of ERaontain four and nine Cys residues, respectively (16,7, 38). Although the Cys residues present in theigand-binding domain (Cys381, Cys417, Cys447, andys530) are solvent exposed, they are unlikely to un-ergo NO-mediated chemical modification(s). In fact,2 still induces nongenomic ERa functions in the pres-nce of GSNO and NOR-3 (i.e., NO) (see Fig. 2). Re-arkably, mutation(s) of the Cys residues present in

he ERa ligand-binding domain significantly reduces2 affinity (38). Eight of nine Cys residues present inRa DNA-binding domain are involved in two zinc–

hiolate centers (Cys7, Cys10, Cys24, Cys27, andys43, Cys49, Cys59, Cys62, respectively), Cys67 be-

ng completely buried in the protein matrix. Within theys residues forming the two zinc–thiolate centers of

he ERa DNA-binding domain, only the two solvent-xposed Cys24 and Cys43 residues are the most likelyandidates for NO-mediated chemical modification(s).otably, the exposition of the ERa DNA-binding do-ain to thiol-reacting oxidants prevents ERa dimer-

zation and DNA binding (39). NO-mediated chemicalodification(s) of Cys residues forming zinc–thiolate

enters induces metal release with the concomitantoss of DNA binding capabilities (30, 37). However,long-range) allosteric effects cannot be excluded. Fi-ally, present data indicate a new selective regulatoryole for NO on E2-induced-cell functions.

CKNOWLEDGMENTS

The generous gifts of pCMV5-ERa from Dr. Carolyn L. SmithDepartment of Molecular and Cellular Biology, Baylor College of

edicine, Houston, TX), C3 promoter construct from Professor D.cDonnell (Department of Pharmacology and Cancer Biology, Dukeniversity Medical Center, Durham, NC), and ICI 182,780 fromrofessor A. Weisz (Istituto di Patologia Generale, II Universita diapoli, Federico II, Napoli, Italy) are gratefully acknowledged. Theuthors thank Dr. Fabio Polticelli (Department of Biology, Univer-ity Roma Tre, Rome, Italy) for helpful discussions. This work wasartially supported by grants from target-oriented projects “1999niversita Roma Tre Fondi per lo Sviluppo” to A.T. and “2001niversita Roma Tre Fondi ex-60%” to M.M.

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