Cellular Signalling 26 (2014) 1466–1475

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Cellular Signalling

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Niacin activates the G protein estrogen receptor(GPER)-mediated signalling

Maria Francesca Santolla a, Ernestina Marianna De Francesco a, Rosamaria Lappano a, Camillo Rosano b,Sergio Abonante c, Marcello Maggiolini a,⁎a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Cosenza, Italyb U.O.S. Biopolymers and Proteomics, Irccs AOU San Martino, IST-National Institute for Cancer Research, Genova, Italyc Regional Hospital, Cosenza, Italy

Abbreviations:GPER, G-protein estrogen receptor; CAFCTGF, connective tissue growth factor; EGFR, epidermal gtracellular signal-regulated kinase; TNF-α, tumor necrosis⁎ Corresponding author at: Dept. of Pharmacy, Hea

University of Calabria, 87036 Rende, Cosenza, Italy. Tel0984493458.

E-mail address: marcellomaggiolini@yahoo.it (M. Mag

http://dx.doi.org/10.1016/j.cellsig.2014.03.0110898-6568/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 January 2014Accepted 17 March 2014Available online 21 March 2014

Keywords:GPERNicotinic acidNicotinamideBreast cancer cellsCancer-associated fibroblasts

Nicotinic acid, also known as niacin, is the water soluble vitamin B3 used for decades for the treatment of dyslip-idemic diseases. Its action is mainly mediated by the G protein-coupled receptor (GPR) 109A; however, certainregulatory effects on lipid levels occur in a GPR109A-independent manner. The amide form of nicotinic acid,named nicotinamide, acts as a vitamin although neither activates the GPR109A nor exhibits the pharmacologicalproperties of nicotinic acid. In the present study, we demonstrate for the first time that nicotinic acid and nico-tinamide bind to and activate the GPER-mediated signalling in breast cancer cells and cancer-associated fibro-blasts (CAFs). In particular, we show that both molecules are able to promote the up-regulation of wellestablishedGPER target genes through the EGFR/ERK transduction pathway. As a biological counterpart, nicotinicacid and nicotinamide induce proliferative and migratory effects in breast cancer cells and CAFs in a GPER-dependent fashion. Moreover, nicotinic acid prevents the up-regulation of ICAM-1 triggered by the pro-inflammatory cytokine TNF-α and stimulates the formation of endothelial tubes through GPER in HUVECs.Together, our findings concerning the agonist activity for GPER displayed by both nicotinic acid and nicotinamidebroaden themechanisms involved in the biological action of thesemolecules and further support the potential ofa ligand to induce different responses mediated in a promiscuous manner by distinct GPCRs.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

G protein-coupled receptors (GPCRs) are cell-surface proteins in-volved in multiple pathophysiological responses including cancer [1].Increasing evidence has demonstrated that theGprotein-coupled estro-gen receptor, namedGPER/GPR30mediates diverse biological effects in-duced by a wide number of compounds like estrogens, anti-estrogensand environmental contaminants in normal and malignant cells [2–6].GPER mediates rapid events that activate diverse transduction path-ways leading to a characteristic gene signature involved in important bi-ological functions [7]. Notably, GPER couples these biological responsesin tumor cells and in cancer-associated fibroblasts (CAFs) that play astimulatory role within the tumor microenvironment [8–10]. Consider-ing the involvement of GPER in cancer progression, a great interest iscurrently addressed towards the identification of novel compounds

s, cancer-associated fibroblasts;rowth factor receptor; ERK, ex-factor-α.lth and Nutritional Sciences,.: +39 0984493076; fax: +39

giolini).

that binding to GPER promote relevant activities in different cell con-texts [11–17].

Niacin, also known as vitamin B3, refers to nicotinic acid, nicotin-amide and derivatives that display comparable physiological functions[18]. The greatest contribution to the niacin intake comes from mixeddishes high in meat, fish, or poultry, enriched and whole-grain breadsand bread products, fortified ready-to-eat cereals. The recommendeddaily intake of niacin is 2–12 mg/day for children and 14–16 mg/dayfor adult [19]. Nicotinic acid can be also synthesized by the liver fromthe essential amino acid tryptophan; however 60 mg of tryptophan isrequired to make 1 mg of nicotinic acid [20]. As therapeutic drug, nico-tinic acid has been used for decades in various dyslipidemic conditionsas it lowers circulating triglycerides (TGs), very low-density lipoprotein(VLDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol andraises high-density lipoprotein (HDL)-cholesterol [21]. Moreover, nico-tinic acid used alone or in combination with statins exhibited beneficialcardiovascular effects in several clinical trials [22]. Given the importantpharmacological features elicited by nicotinic acid, a great interest hasfocused on the molecular mechanisms involved in its beneficial effects.

The biological action of nicotinic acid is mediated, at least in part,by the G protein-coupled receptor (GPR) 109A (also referred to asHM74A) and the GPR109B that display a high and low binding

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affinity, respectively [23]. In particular, the activation of theGPR109A by nicotinic acid triggers a Gi-mediated inhibition ofadenylyl cyclase and a subsequent decrease in intracellular cyclicAMP levels leading to antilipolytic effects [24]. As it concerns nicotin-amide, it neither activates GPR109A nor exhibits the pharmacologicalactions exerted by nicotinic acid, even though nicotinamide acts as a vi-tamin like nicotinic acid [23,25,26]. In this regard, it has been shownthat nicotinamide serves as a precursor in the generation of NAD+,which plays a critical role inmany physiological functions [27]. Nicotin-amide may be potentially transformed to nicotinic acid; however thelevels available after this conversion are not sufficient to produce eitherundesirable effects like flushing or the beneficial changes in plasmalipids [28]. Nevertheless, nicotinamide is effective in fulfilling the nutri-tional requirement for nicotinic acid and in preventing vitamin defi-ciency [18].

Here, we demonstrate that nicotinic acid and nicotinamide acting asGPER ligands induce important biological responses in breast cancercells and CAFs. Hence, our data broaden the mechanisms involved inthe various actions exerted by thesemolecules through the engagementof a further GPCR family member distinct from the cognate receptorsGPR109A and GPR109B.

2. Materials and methods

2.1. Materials

Tyrphostin AG1478 (AG) was purchased from Biomol ResearchLaboratories, Inc (Milan, Italy). PD98059 (PD) was obtained fromCalbiochem (Milan, Italy). 1-[4-(-6-Bromobenzol [1,3] diodo-5-yl)-3a,4,5,9btetrahydro-3H-cyclopenta[c-] quinolin8yl] ethanone (G-1)was purchased from Merck KGaA (Frankfurt, Germany). Ethyl 3-[5-(2 ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1hindol-3-yl]but-2-enoate (MIBE) was synthesized as previously described [29]. Pyridine-3-carboxylic acid (nicotinic acid) and pyridine-3-carboxylic acidamide (nicotinamide) were purchased from Sigma-Aldrich Srl. (Milan,Italy). PD was dissolved in ethanol, G-1 and AG1478 were dissolved indimethyl sulfoxide (DMSO), nicotinic acid (pyridine-3-carboxylicacid) and nicotinamide (piridina-3-carbossammide) were solubilizedin water.

2.2. Molecular modelling and docking simulations

GPER three dimensional structure was modelled as previously de-scribed and molecular docking simulations were performed using theprogram GOLD following a procedure adopted and validated in our re-cent studies [29–31]. In order to find novel GPER ligands, we screened“in silico” a small “home-made” library of chemical fragments usingthe program “GOLD”. By analysing the ranking scores, we noticed a rel-atively good affinity for nicotinic acid to GPER; therefore we focused thecurrent investigation on this moiety. In order to evaluate the bindingmodes of nicotinic acid as well as nicotinamide within GPER andGPR109A receptors, we performed a blind docking simulation usingthe three dimensional model of these two proteins as targets. The 3Dmodel of GPR109A was built by homology using as target the crystallo-graphic structure of the bovine Rhodopsin and was retrieved by theSwiss Model Repository databank [32–34].

2.3. Cell culture

SkBr3 breast cancer cells were maintained in RPMI 1640 withoutphenol red supplementedwith 10% fetal bovine serum (FBS) (Life Tech-nologies,Milan, Italy) and100 μg/ml penicillin/streptomycin (Life Tech-nologies, Milan, Italy). Human embryonal kidney Hek293 cells weremaintained in DMEM with phenol red supplemented with 10% FBSand 100 μg/ml penicillin/streptomycin. HUVECs human umbilical veinendothelial cells (kindly provided by Dr. Caruso, University of Brescia,

Italy) were seeded on collagen-coated flasks (Sigma-Aldrich, Srl,Milan, Italy) and cultured in endothelial growth medium (EGM)(Lonza, Milan, Italy), supplemented with 5% FBS (Lonza, Milan,Italy). Cancer-associated fibroblasts (CAFs) were obtained frombreast cancer patients as previously described [8]. Informed consentwas obtained from each patient. CAFs were maintained in a mixtureof MEDIUM 199 and HAM'S F-12 (1:1) supplemented with 10% FBS.LoVo colorectal adenocarcinoma cells were maintained in RPMI-1640 with phenol red, supplemented with 10% FBS and 100 μg/mlpenicillin/streptomycin and used as positive control for the expres-sion of both isoforms (GPR109A and GPR109B) of the nicotinic acidreceptor (Supplemental Fig. 1). All cell lines were grown in a 37 °Cincubator with 5% CO2. Primary cell cultures of breast fibroblastswere characterized by immunofluorescence. Briefly cells were incu-bated with human anti-vimentin (V9) and human anti-cytokeratin14 (LL001) all antibodies from Santa Cruz Biotechnology, DBA(Milan, Italy). In addition, we used anti-fibroblast activated protein α(FAPα) antibody (H-56), also purchased from Santa Cruz Biotechnology,DBA (Milan, Italy), for fibroblast activation characterization (datanot shown). All procedures conformed to the Helsinki Declarationfor the research on humans. The experimental research has beenperformed with the ethical approval provided by the Ethics Commit-tee of the Regional Hospital in Cosenza (Italy). Informed consent wasobtained from each patient.

2.4. Gene expression studies

Total RNA was extracted using Trizol commercial kit (Life Technolo-gies, Milan, Italy) according to the manufacturer's protocol. RNA wasquantified spectrophotometrically, and its quality was checked by elec-trophoresis through agarose gels stained with ethidium bromide. Onlysamples that were not degraded and showed clear 18S and 28S bandsunder ultraviolet light were used for real-time PCR. Total cDNA wassynthesized from the RNA by reverse transcription using the murineleukaemia virus reverse transcriptase (Life Technologies, Milan, Italy)following the protocol provided by the manufacturer. The expressionof selected gene was quantified by real-time PCR using Step One (TM)sequence detection system (Applied Biosystems Inc., Milano, Italy),following the manufacturer's instructions. Gene-specific primerswere designed using Primer Express version 2.0 software (AppliedBiosystems. Inc.). For GPR190A, GPR109B, c-fos, CTGF, ICAM-1 and theribosomal protein 18S, which was used as a control gene to obtain nor-malized values, the primers were: 5′-GTCTTTGTCATCTGCTTCCTTC-3′(GPR109A forward), 5′-CTTGGCATGGTTATTTAAGGAG-3′ (GPR109Areverse), 5′-CATCTGAAACTTGCTTCATCTCT-3′ (GPR109B forward),5′-TAACCAAAGCATGTGAGTCTCTT-3′ (GPR109B reverse), 5′-CGAGCCCTTTGATGACTTCCT-3′ (c-fos forward), 5′-GGAGCGGGCTGTCTCAGA-3′ (c-fos reverse); 5′-ACCTGTGGGATGGGCATCT-3′ (CTGF forward),5′-CAGGCGGCTCTGCTTCTCTA-3′ (CTGF reverse), 5′-CCCGAGCTCAAGTGTCTAAA-3′ (ICAM-1 forward), 5′-ATTATGACTGCGGCTGCTAC-3′(ICAM-1 reverse), 5′-GGCGTCCCCCAACTTCTTA-3′ (18S forward) and5′-GGGCATCACAGACCTGTTATT-3′ (18S reverse). Assays were per-formed in triplicate and the results were normalized for 18S expressionand then calculated as fold induction of RNA expression.

2.5. Gene silencing experiments

For gene silencing experiments cells were plated onto 10-cm dishes,maintained in a serum-free medium for 24 h and then transfected foradditional 24 h before treatments with a control vector or an indepen-dent shRNA sequence for each target gene using X-treme GENE9(Roche Molecular Biochemicals, Milan, Italy). Short hairpin constructsagainst human GPER (shGPER) and CTGF (shCTGF) were obtained andused as previously described [7].

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2.6. Ligand binding assays

In ligand binding assay for GPER, SkBr3 and Hek293 cells weregrown in 10-cm cell culture dishes, washed two times and incubatedwith 50 nM [5,6-3H] nicotinic acid (50–60 Ci/mmol; BIOTREND,Chemikalien GmbH Technologiezentrum Köln Eupener Str. 157 D-50933 Köln) in the presence or absence of an increasing concentrationof non labeled competitors (G-1, nicotinic acid and nicotinamide).Then, cells were incubated for 2 h at 37 °C and washed three timeswith ice-cold PBS; the radioactivity collected by 100% ethanol extractionwas measured by liquid scintillation counting. Competitor binding wasexpressed as a percentage of maximal specific binding. Each point is themean of three observations.

2.7. Western blot analysis

SkBr3, CAFs and HUVECs were grown in 10-cm dishes and exposedto drugs for the appropriate time, then washed twice with ice-cold PBSand solubilizedwith 50mMHepes buffered solution, pH 7.5, containing150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, and a mixture of protease inhibitors (Aprotinin, PMSF and Na-orthovanadate). Protein concentration in the supernatant was deter-mined according to the Bradford method. Equal amounts (10–50 μg)of the whole cell lysate that was electrophoresed through a reducingSDS/10% (w/v) polyacrylamide gel, were electroblotted onto a nitro-cellulose membrane probed with primary antibodies against CTGF(L-20), c-fos (H-125), phosphorylated ERK1/2 (E-4), ERK2 (C-14),GPER (N-15), ICAM-1 (G-5) and β-actin (C2), all purchased fromSanta Cruz Biotechnology, DBA (Milan, Italy). The levels of proteinsand phosphoproteins were detected with horseradish peroxidase-linked secondary antibodies and revealed using the ECL® System(GE Healthcare, Milan, Italy).

2.8. Proliferation assay

For quantitative proliferation assay, cells (1 × 105) were seeded in24-well plates in regular growth medium. Cells were washed oncethey had attached, transfected for 24 h with shRNA or shGPER (500 ngDNA/well transfectedwith X-tremeGENE9 reagent inmedium contain-ing 2.5% charcoal stripped FBS) and then treated. Every two days, cellswere transfected with shRNA or shGPER and medium was renewed(with treatments) before counting using the Countess Automated CellCounter, as recommended by the manufacturer's protocol (Life Tech-nologies, Milan, Italy).

2.9. Migration assay

Migration assays were performed using Boyden chambers (CostarTranswell, 8 mm polycarbonate membrane). For knockdown experi-ments, cells were transfected with shRNA or shGPER (3 μg DNA/welltransfected with X-treme GENE9 reagent in a medium withoutserum). After 24 h, cells were seeded in the upper chambers. Nicotinicacid and nicotinamide were added to the medium without serum inthe bottom wells. After 6 h, cells on the bottom side of the membranewere fixed and counted.

2.10. Tube formation assay

The day before the experiment, confluent HUVECs were transfectedwith shRNA constructs directed against GPER, CTGF or with an unrelat-ed shRNAconstruct in a regular growthmediumand after 8 h, cellswerestarved overnight at 37 °C in a serum freemediumand treatedwith nic-otinic acid (EBM, Lonza, Milan, Italy). Growth factor-reducedMatrigel®(Cultrex, Trevigen Inc, USA) was thawed overnight at 4 °C on ice and,the day of the assay, plated on the bottom of prechilled 96 well-plates,and left at 37 °C for 1 h for gelification. HUVECs starved and treated

were collected by enzymatic detachment (0.25% trypsin-EDTA solution(Life Technologies, Milan, Italy), counted and resuspended in EBM. Then10,000 cells/well were seeded onMatrigel and incubated at 37 °C. Tubeformation was observed starting from 2 h after cell seeding and quanti-fied by using the software NIH ImageJ.

2.11. Statistical analysis

Statistical analysis was performed using one-way analysis ofvariance (ANOVA) followed by Newman–Keuls' testing to determinedifferences inmeans. P b 0.05was considered as statistically significant.

3. Results

3.1. Nicotinic acid and nicotinamide are ligands of GPER

Our [5,29–31] and other [35,36] studies have provided evidence re-garding the ability of diverse compounds to bind to GPER, then acting asagonist or antagonist ligands. In order to provide new insights into theGPER-mediated signalling, we used a fragment-based drug discovery(FBDD) approach which would serve as part of the drug discovery pro-cess. In particular, FBDD is based on the identification of small “buildingblocks” (small chemical fragments, generally with a Mw b 300 Da)which are able to bind, even if weakly, to the selected target. Oncethese blocks are docked to the receptor, they are combined in order toproduce a lead with a higher affinity for the biological target. Hence,we initially built a “in silico” homemade library of small functional moi-eties and thereafter we screened it using as a target the molecularmodel of GPER. Surprisingly, nicotinic acid and the similar moiety nico-tinamide showed good docking scores; we therefore evaluated thestructural/functional similarities between GPER and the nicotinic acidreceptor GPR109A. Although sequence alignment suggests no commonfeature in nicotinic acid binding sites within the two proteins, the com-parison of the three dimensional structural complexes GPER: nicotinicacid and GPR109A: nicotinic acid highlighted some similarity in the li-gand binding mode. Nicotinic acid is placed with a different orientationwithin the two clefts: in GPER (Fig. 1A) the carboxyl tail of the acidpoints towards helix TM III and the aromatic ring towards helix TMVII, while in GPR109A the binding site is displaced towards helix TM Vof about 8.5 Å and rotated of ca. 78°, with the acidic tail of the ligandpointing towards helix TM VI and the pyridyl moiety towards helixTM V (data not shown). Particularly, GPER employs Ser134 OH and Natoms in hydrogen bonds with the carboxyl groups of nicotinic acid,Leu137 in a hydrophobic contact with the pyridyl moiety and Met141to close the binding pocket together with Met309. Moreover, the polarresidue Gln138 is forming a hydrogen bondwith the pyridin-3-yl nitro-gen atom and Phe208 is in π-stacking with the aromatic ring of the nic-otinic acid. On the other hand, in the case of GPR109A, the nicotinic acidform hydrogen bonds with its carboxyl group with Arg89, Arg229 andSer225 and with the nitrogen atom of the aromatic ring with Gln89.Three phenylalanines, Phe171, Phe175 and Phe222 are in hydrophobiccontact with the ligand, Phe175 being in π-stacking with the pyridylmoiety. Met170 close the nicotinic acid binding pocket. Interestingly,many of the residues adopted to bind nicotinic acid in the two pocketsare identical, although they are not conserved when aligning the twoprimary structures as other residues (i.e. Leu137 and Met309 in GPER)are replaced with aminoacids with similar characteristics (Phe171 andPhe222 in GPR109A). The only striking difference in the two bindingpockets is the presence of two basic residues in GPR109A, Arg89 andArg229, that form hydrogen bonds with the carboxyl of the nicotinicacid. This confers to GPR109A a substrate specificity. The bindingmode of nicotinamide to GPER is almost identical to that shown by nic-otinic acid (Fig. 1B). However, a slight decrease in the calculated affinityof nicotinamide for GPERwas foundwith respect to nicotinic acid, prob-ably due to the loss of a hydrogen bond between the carboxyl group ofthe ligand and the Ser134 peptidic nitrogen. Superposing themolecular

Fig. 1. GPER docking simulations. Representation of the GPER binding site with different ligands. The protein is drawn as tan ribbons; residues interacting with the ligands are reported insticks. Nicotinic acid is drawn as purple sticks (panel A) and nicotinamide is represented in green (panel B). The bindingmodes of two known ligands, G-1 (panel C) andMIBE (panel D) arereported for reference and drawn in yellow and orange sticks, respectively.

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structure of the binding sites of GPER for nicotinic acid andG-1 (Fig. 1C),which is a GPER ligand [35], it was evident that the aromatic ring of nic-otinic acid is fully overlying the dioxolic moiety of G-1, being this lastmolecule extended towards a cleft formed by TM I, TM II and TM VII.On the other side, superposition of the complex GPER: nicotinic acidto the one formed by GPER with the full-antagonist MIBE [29] showsas the pyridine of the nicotinic acid occupies the position adopted bythe methyl-dihydropyrrole of MIBE (Fig. 1D). In this case, thepropenyl-propanoate tail of the antagonist, differently from the confor-mation adopted by G-1, is directed towards helix TM I withoutcontacting helices TM I and TM II.

In order to further evaluate the ability of nicotinic acid and nicotin-amide to bind to GPER, we then performed whole cell binding assays

Fig. 2. Nicotinic acid and nicotinamide are GPER ligands. Competition curves of increasing concmaximum specific [5,6-3H] nicotinic acid binding in SkBr3 cells transfectedwith shRNA or shGP(B) Efficacy of GPER silencing obtained using shGPER. (D) Efficacy of GPER over-expression obtathree separate experiments performed in triplicate. Note that the amount of tracer bound in th

by using [5,6-3H] nicotinic acid in GPER-positive SkBr3 cells that donot express both isoforms (GPR109A and GPR109B) of the nicotinicacid receptor (Supplemental Fig. 1). Nicotinic acid, nicotinamide andG-1 displaced the radiolabeled nicotinic acid in a dose-dependentmanner; however silencing GPER expression by transfecting cells witha shGPER, the radiolabeled nicotinic acid was not displaced by all com-pounds used (Fig. 2A). Additional evidence regarding the binding prop-erties of nicotinic acid and nicotinamide to GPER was also provided byperforming whole cell binding assays in GPER-negative Hek293 cellsthat do not express both isoforms (GPR109A and GPR109B) of the nico-tinic acid receptor (Supplemental Fig. 1). Of note, nicotinic acid, nicotin-amide and G-1 showed the capability to displace [5,6-3H] nicotinic acidonly in Hek293 cells engineered to express GPER (Fig. 2C). Collectively,

entration of nicotinic acid (NA), nicotinamide (NM), and G-1 expressed as a percentage ofER (A), and in Hek293 cells transfectedwith a vector or an expression vector of GPER (C).ined using GPER expression vector (pGPER). Each data point represents themean± SD ofe absence of competitor was arbitrarily set to 100%.

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the abovementioned findings demonstrate that both compounds areligands of GPER.

3.2. Nicotinic acid and nicotinamide induce GPER-mediated ERK activationand gene expression

To verify whether the binding of nicotinic acid and nicotinamide toGPER triggers ERK phosphorylation which characterizes the ligand acti-vation of this receptor [37], we used the GPER-positive SkBr3 cells andCAFs, which are key players of the tumor microenvironment [38], asthese cells lack both isoforms (GPR109A and GPR109B) of the nicotinicacid receptor (Supplemental Fig. 1). Interestingly, the rapid ERK activa-tion induced by both compounds (Fig. 3A–D) occurred through GPER asdetermined by knocking-downGPER expression (Fig. 4A,D). In addition,using the EGFR inhibitor AG1478 (AG) and the MEK inhibitor PD98059(PD), we determined that the EGFR/ERK transduction signalling is in-volved in the GPER-mediated response to both compounds in SkBr3cells and CAFs (Fig. 4C,F). As the activation of the GPER/EGFR/ERKpathway has been shown to regulate the transcription of GPER-targetgenes [3,7], we then ascertained that nicotinic acid and nicotinamidestimulate the mRNA and protein expression of c-fos and CTGF(Fig. 5A–D). Accordingly, in SkBr3 cells and CAFs the induction of c-fosand CTGF by both compoundswas prevented silencingGPER expression(Fig. 6A,D) aswell as using AG and PD that are EGFR andMEK inhibitors,respectively (Fig. 6C,F). Taken together, these data suggest that nicotinicacid and nicotinamide activate the GPER/EGFR/ERK signalling in bothSkBr3 cells and CAFs.

3.3. Nicotinic acid and nicotinamide stimulate cell proliferation andmigration through GPER

As a biological counterpart of the aforementioned results, we evalu-ated the potential of the nicotinic acid and nicotinamide to trigger cellproliferation and migration. Notably, both compounds induced prolifera-tive and migratory effects in SkBr3 cells and CAFs, whereas the silencingof GPER expression abrogated these responses (Fig. 7A–G). Overall,these findings demonstrate that nicotinic acid and nicotinamide inducerelevant biological effects acting as GPER agonists in SkBr3 cells and CAFs.

Fig. 3. Nicotinic acid and nicotinamide activate ERK1/2. ERK1/2 phosphorylation (p-ERK1/2) in10 μMnicotinamide (NM), as indicated. Side panels show densitometric analysis of the blots noiments. (●) indicates p b 0.05 for cells receiving vehicle (-) versus treatments.

3.4. Nicotinic acid inhibits ICAM-1 expression through GPER

In endothelial cells the exposure to tumor necrosis factor (TNF-α)induces the up-regulation of the intercellular cell adhesion molecule-1(ICAM-1),which promotes the recruitment of leukocytes and thereafterthe progression of the inflammatory process [39]. As nicotinic acid wasshown to inhibit the up-regulation of ICAM-1 induced by TNF-α inHUVECs [40], we evaluated whether GPER is involved in this anti-inflammatory response triggered by nicotinic acid in HUVECs that donot express both isoforms (GPR109A and GPR109B) of the nicotinicacid receptor (Supplemental Fig. 1). Remarkably, the inhibitory effectselicited by nicotinic acid from 10 μM (data not shown) to 1 mM onthemRNA and protein induction of ICAM-1 upon exposure to TNF-α oc-curred through GPER, as determined by transfecting HUVECs withshGPER (Fig. 8A-B).

3.5. Nicotinic acid promotes tube formation through GPER

As nicotinic acid has been previously reported to promote angiogen-esis [41], we evaluatedwhether this ability of nicotinic acidmay involveGPER. Hence, we performed endothelial tube formation assay thatrepresents a useful model system towards the evaluation of theneoangiogenesis process [42]. Nicotinic acid promoted from 10 μM(data not shown) to 1 mM the formation of capillary-like structures inHUVECs through GPER and its target gene CTGF, which has been dem-onstrated to be involved in this biological response [43] (Fig. 9A,B,D).The aforementioned results were quantified and recapitulated in panelsF–H of Fig. 9. Taken together, our findings demonstrated that GPERme-diates relevant physiological effects induced by nicotinic acid in cellslacking both isoforms (GPR109A and GPR109B) of the nicotinic acidreceptor (Supplemental Fig. 1).

4. Discussion

In the present study, we have demonstrated that nicotinic acid andnicotinamide acting as agonists for GPER induce stimulatory effectsin breast cancer cells and CAFs. In particular, we have ascertainedthat both molecules trigger the up-regulation of the GPER target genesc-fos and CTGF through the activation of the EGFR/ERK transduction

SkBr3 cells (A-B) and CAFs (C-D) treated with vehicle (-) or 10 μM nicotinic acid (NA) orrmalized to ERK2. Each data point represents the mean± SD of three independent exper-

Fig. 4. Nicotinic acid and nicotinamide activate ERK1/2 through GPER. The ERK1/2 activation observed in SkBr3 cells and CAFs treated for 5 minwith 10 μMnicotinic acid (NA) and 10 μMnicotinamide (NM)was abolished transfecting cells with shGPER (A, D). (B, E) Efficacy of GPER silencing obtained using shGPER. Moreover, the ERK1/2 phosphorylation in SkBr3 cells andCAFs treated for 5minwith 10 μMnicotinic acid (NA) or 10 μMnicotinamide (NM)was prevented using 10 μMEGFR inhibitor AG1478 (AG) or 10 μMMEK inhibitor PD98089 (PD) (C, F).Side panels show densitometric analysis of the blots normalized to ERK2. Each data point represents the mean ± SD of three independent experiments. (●) indicates p b 0.05 for cellsreceiving vehicle (-) versus treatments.

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pathway and stimulate cell proliferation and migration in a GPER-dependent manner. Interestingly, in HUVECs nicotinic acid preventedthrough GPER the up-regulation of ICAM-1 upon exposure to the pro-inflammatory cytokine TNF-α and induced the endothelial tube forma-tion through both GPER and CTGF.

Increasing evidence has recently highlighted the potential roleexerted by GPER in diverse pathophysiological conditions [1]. As itconcerns the malignant diseases, GPER expression has been associatedwith negative clinical features and poor survival rates in diversetypes of tumors [11]. GPER may act as an estrogen receptor as it bindsto estrogens, antagonists of the classical estrogen receptors (ERs),phytoestrogens and xenoestrogens [3–6]. Considering the promiscuous

Fig. 5.Nicotinic acid and nicotinamide induce the expression of GPER target genes. In SkBr3 cellCTGF expression at bothmRNA (1 h of treatments, panels A–B) and protein level (4 h of treatmeexperiments, gene expressionwas normalized to 18S expression and results are shown as fold cexperiments, side panels show densitometric analyses of the blots normalized to β-actin. Eachb 0.05 for cells receiving vehicle (-) versus treatments.

activity exhibited by many of these compounds through GPER and theclassical ERs, the identification of ligands targeting these receptors in aselective fashion does represent a challenge towards the evaluation ofthe differential properties displayed by ligands through each type of es-trogen receptors. In this regard, our and other studies contributed to thediscovery of diverse compounds acting as GPER agonists or antagonists,hence allowing the assessment of the exclusive biological responsesmediated by this GPCR family member [30,35,36]. In addition, we iden-tified the first ligand of both GPER and ERα, named MIBE, which ex-hibits the exclusive capacity to inhibit both the GPER and ERα-mediated signalling, hence opening new perspectives towards a morecomprehensive treatment in breast cancer [29]. Further searching for

s and CAFs, 10 μMnicotinic acid (NA) and 10 μMnicotinamide (NM) up-regulate c-fos andnts, panels C–D), as evaluated by real-time PCR and immunoblotting, respectively. In RNAhanges of mRNA expression compared to cells treatedwith vehicle (-). In immunoblottingdata point represents the mean ± SD of three independent experiments. (●) indicates p

Fig. 6.Nicotinic acid and nicotinamide up-regulate c-fos and CTGF expression through GPER. (A, D) SkBr3 cells and CAFs were transfected with shRNA or shGPER and then treated for 4 hwith vehicle (-), 10 μMnicotinic acid (NA) or 10 μMnicotinamide (NM). (B, E) Efficacy of GPER silencing obtained using shGPER. (C, F) immunoblots showing c-fos and CTGF protein ex-pression in SkBr3 cells and CAFs treated for 4 h with vehicle (-), 10 μMnicotinic acid (NA) or 10 μMnicotinamide (NM) alone or in combination with 10 μMEGFR inhibitor AG1478 (AG),10 μMMEK inhibitor PD98089 (PD). Side panels show densitometric analysis of the blots normalized to β-actin. Each data point represents the mean ± SD of three independent exper-iments. (●) indicates p b 0.05 for cells receiving vehicle (-) versus treatments.

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ligand-activated GPER signalling, the current study has assessed thatnot only nicotinic acid but also nicotinamide can engage GPER in trig-gering relevant biological responses, as demonstrated by using different

Fig. 7. Nicotinic acid and nicotinamide induce proliferative and migratory effects in SkBr3 cellsotinic acid (NA) or 10 μM nicotinamide (NM) in the presence of shRNA or shGPER and then cogration assays, cells were transfectedwith shRNA or shGPER and then treated for 6 hwith vehicsilencing obtained using shGPER. Values shown are mean ± SD of three independent experimtreatments.

model systems. Hence, our results would provide novel insights into themolecularmechanisms bywhichGPERmaymediate pathophysiologicaleffects induced by niacin in diverse cell contexts, in accordance with

and CAFs. In proliferation assays, cells were treated for 5 days with vehicle (-), 10 μM nic-unted on day 6 (A, C). (B, D) Efficacy of GPER silencing obtained using shGPER. In the mi-le (-), 10 μMnicotinic acid (NA) or 10 μMnicotinamide (NM) (E, G). (F, H) efficacy of GPERents performed in triplicate. (●, ○) indicate p b 0.05 for cells receiving vehicle (-) versus

Fig. 8.Nicotinic acid prevents throughGPER the up-regulation of ICAM-1 induced by TNF-α in HUVECs. Evaluation of ICAM-1 expression at bothmRNA (A) and protein levels (B) inHUVECs transfected with shRNA or shGPER for 24 h and then treated for 6 h with 1 mMnicotinic acid (NA) and 50 ng/ml TNF-α. In B, the side panel shows densitometric analysisof the blot normalized to β-actin. (C) Efficacy of GPER silencing obtained using shGPER.Each data point represents the mean ± SD of three independent experiments. (●) indi-cates p b 0.05 for cells receiving vehicle (-) versus treatments.

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previous studies demonstrating the promiscuous activity elicited by oneligand through different GPCRs [44].

Nicotinic acid has been acknowledged as an important modulator ofinflammation given its ability to prevent the TNF-α-induced increase ofthe leukocyte adhesion molecules ICAM-1 and platelet endothelial celladhesion molecule (PECAM-1), which promote leukocyte recruitmentcontributing to the progression of the inflammatory process [45]. In ad-dition, nicotinic acid has been shown to reduce the expression of pro-inflammatory chemokines in a pertussis toxin-sensitivemanner, thus in-dicating that these effects may occur through the involvement of GPCRs[46]. As it concerns nicotinamide, its involvement in the regulation of in-flammation has been argued as it attenuated the endotoxin-induced re-lease of several pro-inflammatory cytokines [47]. Likewise, G-1-inducedendothelial GPER activation has been reported to prevent the up-regulation of the pro-inflammatory leukocyte adhesion molecules uponTNF-α stimulation [48]. In accordancewith the aforementioned findings,herewe have demonstrated that nicotinic acid inhibits throughGPER theup-regulation of ICAM-1 induced by TNF-α in HUVECs. Together, theseobservations suggest that ligand (including niacin)-inducedGPER signal-ling may be considered as a transduction mechanism involved in thecomplex inflammatory processes that characterize several pathophysio-logical conditions. In this regard, further studies are needed to betterevaluate the potential role exerted by GPER in the aforementioned bio-logical contexts. Our current results have also provided evidence thatnicotinic acid triggers through the GPER/CTGF signalling the formationof endothelial tubes, in line with previous studies showing that nicotinicacid may promote angiogenesis [49]. Nevertheless, it remains to be

further investigated the GPER-mediated action of nicotinic acid in stimu-lating new blood vessels formation. Collectively, the present data indi-cate that nicotinic acid may elicit relevant biological actions in a GPER-dependent manner, beyond those exerted via the well-established cog-nate receptor GPR109A [23]. Of note, certain effects induced by nicotinicacid may occur regardless of the GPR109A, suggesting that alternativemechanisms are involved in the various biological functions triggeredby nicotinic acid [50]. For instance, in mice lacking GPR109A nicotinicacid did not induce anti-lipolytic effects although the treatment wasstill able to modulate the serum lipid levels [51]. In the aforementionedstudy, the administration ofGPR109A agonists inwild-typemice showedanti-lipolytic properties without modifying the plasma lipid profile cor-roborating the results obtained using GPR109A knockout mice. In orderto explain these biological responses, it has been suggested that nicotinicacidmay act as an inhibitor of the acyl-CoAdiacylglycerol acyltransferase2 (DGAT2), which is one of the two terminal enzymes regulating the bio-synthesis of TGs [52]. In addition, it has been proposed that nicotinic acidmay suppress the expression of the transcriptional coactivator PGC-1band the inhibitor of lipoprotein lipase named apoC-III, hence reducingfree fatty acid flux toward the liver [53]. These effects would ultimatelypromote the clearance of the VLDL-TGs then lower plasma TGs, althoughprevious studies have also reported the ability of niacin to influence theproduction of VLDL-TGs [54]. Overall, these data including our own sug-gest that multiple factors may play a role together with or regardless ofthe GPR109A in mediating the biological effects induced by niacin.

The initial evaluation of the basic transduction modes shared byGPCRs has suggested a relatively simple system based on a ligand-dependent “linear cascade” through a specific member of this receptorfamily [55]. Conversely, increasing evidence support the potential of asingle compound to activate different GPCRs, which consequently gen-erate an intricate network strengthening the functional responses to aligand.

Biologically, the discovery of multiple signalling partners mediatingthe stimulation of a distinct agent may represent a challenge indeciphering one of the most elaborate cellular transmission systems.Despite the remarkable structural diversity of GPCR agonists and thelow sequence homology among GPCRs, further studies are needed inorder to identify the sequencemotifs involved in the promiscuous activ-ity of GPCR ligands.

5. Conclusions

The present findings provide novel insights on the ability of niacin toelicit diverse responses through GPER signalling in diverse cell contexts.Hence, the promiscuous activity exerted by niacin via both GPR109Aand GPER may open new avenues towards a better understanding ofthe mechanisms involved in its biological action exerted in differentpathophysiological conditions, including malignant diseases.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cellsig.2014.03.011.

Competing interests

The authors declare that they have no competing interests towardsany aspect of the work described in this manuscript.

Author contributions

M.F.S. and R.L. conceived and designed the experiments; M.F.S. andE.M.D.F. performed the experiments; M.F.S., R.L. and E.M.D.F. analysedthe data; C.R. performed the docking simulation; S.A. provided biologi-cal samples; M.M. contributed reagents/materials/analysis tools; M.F.S.and M.M. wrote the paper.

1474 M.F. Santolla et al. / Cellular Signalling 26 (2014) 1466–1475

1475M.F. Santolla et al. / Cellular Signalling 26 (2014) 1466–1475

Acknowledgments

This work was supported by Associazione Italiana per la Ricerca sulCancro (AIRC, project n. 12849/2012), AIRC project Calabria 2011(http://www.airc.it/) and Fondazione Cassa di Risparmio di Calabria eLucania. C.R. wish to thank the “Compagnia di San Paolo di Torino” forthe generous support to his research.

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tion in HUVECs transfected for 24 h with shRNA (A), shGPER (B) or shCTGF (D) and thenhGPER and efficacy of CTGF silencing obtained using shCTGF respectively. Quantification ofesentative of three independent experiments performed in triplicate. (●) indicates p b 0.05