Functional Characterization of the Odorant Receptor 51E2 in Human Melanocytes*

Olfactory receptors, which belong to the family of G-protein-coupled receptors, are found to be ectopically expressed in non-sensory tissues mediating a variety of cellular functions. In this study we detected the olfactory receptor OR51E2 at the transcript and the protein level in human epidermal melanocytes. Stimulation of primary melanocytes with the OR51E2 ligand β-ionone significantly inhibited melanocyte proliferation. Our results further showed that β-ionone stimulates melanogenesis and dendritogenesis. Using RNA silencing and receptor antagonists, we demonstrated that OR51E2 activation elevated cytosolic Ca2+ and cAMP, which could mediate the observed increase in melanin synthesis. Co-immunocytochemical stainings using a specific OR51E2 antibody revealed subcellular localization of the receptor in early endosomes associated with EEA-1 (early endosome antigen 1). Plasma membrane preparations showed that OR51E2 protein is present at the melanocyte cell surface. Our findings thus suggest that activation of olfactory receptor signaling by external compounds can influence melanocyte homeostasis.

Olfactory receptor (OR) 5 genes constitute the basis for the sense of smell and were first described as being expressed exclusively in the olfactory epithelium (1). Later on, a subset of human OR genes have been found to be expressed in various non-olfactory tissues (2)(3)(4). This ectopic expression of OR genes raised the possibility that a subset of ORs may have physiological functions in non-olfactory tissues in addition to their canonical role in odor detection in the sensory neurons. Functionality of olfactory receptors in non-olfactory cell types is becoming better understood. Initially, a lack of correlation in expression levels in different tissues between human-mouse orthologous pairs and between intact and pseudogenized ORs was taken as an indication that functional interpretation of ectopic OR expression cannot be based on transcriptional information (5). Analysis of non-olfactory tissues in human and chimpanzee later revealed that OR orthologous genes are expressed in the same tissues more often than expected by chance alone. Orthologous OR genes with conserved ectopic expression have evolved under stronger evolutionary constraints than OR genes expressed exclusively in the olfactory epithelium (6), indicating that ectopically expressed OR genes have conserved functions in non-olfactory tissues. In the studies performed so far, functional analysis of ectopically expressed ORs revealed a possible role in sperm chemotaxis (7)(8)(9). Human ORs were also shown to mediate serotonin release in enterochromaffin cells (10) and to influence proliferation of prostate cancer cells (11,12). The OR2A4 that localizes to the cytokinetic structures in cells was shown to participate in cytokinesis (13). The mouse OR MOR23 exerts a role in skeletal muscle regeneration by regulating cell adhesion and migration (14). In the murine kidney, ORs and olfactory signaling proteins control the glomerular filtration rate, renin secretion, and blood pressure (15,16). A synthetic sandalwood compound was shown to accelerate wound healing processes by activation of OR2AT4 expressed in human keratinocytes (17).
In contrast to olfactory neurons, where activation of the receptors elicits a receptor current, activation of ectopically expressed receptors can have diverse effects. ORs are G-protein-coupled receptors (GPCRs), which can couple to different intracellular signaling cascades depending on the activation of different types of heterotrimeric G-proteins (18), the interaction with other cellular partners such as arrestins (19) and scaffolding proteins (20), (hetero-)dimerization with other receptors (21), lipid-protein interactions (22), and intracellular localization (23). GPCRs, which are activated by chemical ligands such as small amines, peptide hormones, chemokines, and odorants, can elicit a huge variety of cellular responses, among them cell shape changes and altered adhesion (24) as well as changes in cell proliferation (25).
In the present study we functionally characterized the signaling pathway and the implication of activation of OR51E2 in human melanocytes. We found that OR activation in these cells elicits Ca 2ϩ signals and ultimately regulates cellular proliferation and differentiation, similar to that observed previously in prostate epithelial cells (11).

Experimental Procedures
Cell Culture and Transfection-Reagents for cell culture use were purchased from Life Technologies unless stated otherwise. Hana3a cells were maintained under standard conditions in DMEM supplemented with 10% FBS, 100 units/ml penicillin and streptomycin and 2 mM L-glutamine. Hana3a cells were transiently transfected (3 g of OR cDNA per dish) in 35-mm dishes (Falcon, BD Bioscience) using a standard calcium phosphate precipitation technique. Primary neonatal normal human epidermal melanocytes were a generous gift from Dr. G. Neufang (Beiersdorf, Hamburg, Germany). Melanocytes were maintained under standard conditions in melanocyte growth medium (Cell Systems, St. Katharinen, Germany). For siRNA experiments, melanocytes were transiently transfected with either targeted or scrambled siRNAs using Lipofectamine 2000 (Life Technologies) according to manufacturers' instructions. The transfection rates were Ͻ1% with both OR51E2 siRNA and control siRNA as detected by co-expression of GFP. Cells were analyzed in Ca 2ϩ imaging experiments 2 days after transfection.
Western Blotting-80% confluent cells were harvested by scraping, and proteins were prepared by standard methods. Sample aliquots of fractionated melanocytes were mixed with Laemmli buffer (30% glycerol, 3% SDS, 125 mM Tris/Cl, pH 6.8), resolved by 10% SDS-PAGE, and transferred to nitrocellulose membrane (Protan, Schleicher and Schuell, Dassel, Germany). The nitrocellulose membranes were stained with Ponceau S (Sigma), blocked with TBST (150 mM NaCl, 50 mM Tris-Cl, Tween 20, pH 7.4) containing 5% nonfat dried milk (Bio-Rad), and incubated with the primary antibody diluted in 3% dry milk in TBST. After washing and incubation with horseradish peroxidase-coupled secondary antibodies, detection was performed with ECL plus (Amersham Biosciences) and the Fusion-SL image acquisition system (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany). Signal intensities were quantified using ImageJ (v1.4.3.67, Broken Symmetry Software). and isolation of  cell surface proteins for Western blotting analysis were performed using the Thermo Scientific TM Pierce TM Cell Surface  Protein Isolation kit according to the manufacturer's instructions (Thermo Fisher Scientific, Pierce, Rockford, IL).

Cell Surface Protein Isolation-Biotinylation
Single Cell Ca 2ϩ Imaging-Melanocytes were incubated for 45 min, and Hana3a cells were incubated for 30 min in loading buffer, pH 7.4, containing Ringers' solution and 7.5 M Fura-2-AM (Life Technologies). After removal of extracellular Fura-2 by washing, ratiofluorometric Ca 2ϩ imaging was performed using an inverted microscope equipped for ratiometric live cell imaging (IX71, Olympus, Hamburg, Germany) with a 150-watt xenon arc lamp, a motorized fast change filter wheel illumination system for multiwavelength excitation (MT20, Olympus), and a 12-bit 1376 ϫ 1032-pixel charge-coupled device (CCD) camera (F-View II, Olympus). WinNT-based cellR TM imaging software (Olympus) served to collect and quantify spatiotemporal Ca 2ϩ -dependent fluorescence signals (excited at 340 and 380 nm, measured at 510 nm, and calculated as a f340/f380 intensity ratio) of the cells. Compounds were diluted in Ringer's solution or for Ca 2ϩ free experiments in Ringer's solution containing 50 M EGTA and applied using a specialized pressure-driven microcapillary perfusion system designed for instantaneous solution change and focal application. Images were acquired in randomly selected fields of view. Ultrapure ␣-ionone and ␤-ionone were a generous gift of Dr. J. Panten (Symrise, Holzminden, Germany). Steroids were purchased from Steraloids (Steraloids Inc., Newport, RI), aminoethoxydiphenyl borate (2-APB), SKF 96365, GdCl 3 , and BTP2 were from Tocris (Tocris Bioscience, Bristol, UK), and endothelin-1, thapsigargin, and ATP were from Sigma. Odorants were prediluted in DMSO (Sigma) so that the DMSO concentration did not exceed 1‰ (v/v), which was well tolerated by melanocytes. All odorants assayed for potential inhibition of OR51E2 were tested in at least three different series of transfection experiments.
Immunocytochemistry-Cells were seeded on coverslips and maintained as described above. The cells were fixed by incubation with 4% paraformaldehyde at 4°C for 30 min. After blocking with 1% gelatin for 1 h, cells were incubated overnight with the primary antibody. For visualization, secondary fluorescent IgGs (Life Technologies) (1:1000) were used. Micrographs were taken by using a LSM510 Meta confocal microscope (Zeiss, Jena, Germany).
Apoptosis Detection-50 -70% confluent melanocytes were stimulated for 72 h with different concentrations of ␤-ionone or solvent only. Apoptotic activity was detected by Caspase-Glo 3/7 Assay (Promega, Madison, WI). Apoptotic cells were quantified using the terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) based In Situ Cell Death Detection kit (Hoffmann-La Roche) according to the manufacturer's instructions.
Cell Proliferation-Growing melanocytes were plated in 96-well plates at a density of 5 ϫ 10 3 cells/well. After 24 h at 37°C with 5% CO 2 , cells were treated with different concentrations of ␤-ionone. Cell proliferation was assessed after 6 days using CyQUANT cell proliferation assay kit (Life Technologies). For the visualization of proliferating cells via PCNA staining, cells were stimulated for 6 days with ␤-ionone (50 M) or solvent only. After-ward, cells were stained with anti-PCNA antibody (1:500) as described under "Immunocytochemistry" and with Alexa Fluor 546 phalloidin (Life Technologies; 1:200).
cAMP Assay-cAMP concentration was analyzed using a cAMP kit from R&D systems (Minneapolis, MI). In brief, 7 ϫ 10 4 melanocytes were stimulated with growth medium containing odorant or forskolin and 100 M 3-isobutyl-1-methylxanthine (IBMX) as cAMP-phosphodiesterase inhibitor. Afterward, the cells were lysed in 0.1 M HCl in ice-cold ethanol. After 30 min of incubation on ice, cells lysates were desiccated for 5 h at 37°C. Samples were reconstituted in 400 l of 0.1 M HCl. Sample volumes of 100 l per well were transferred to a microtiter plate, and cAMP concentrations per well were measured according to the manufacturers' instructions with the following minor changes. To exclude differences in anti-cAMP antibody binding efficiency, all samples and standards were adjusted to identical ionic conditions; i.e. standards provided by the manufacturer in 10 mM HCl/ethanol were desiccated and reconstituted in 0.1 M HCl. For each tested condition and standard, three replicate experiments were performed, and results were averaged.
Melanin Content Assay-Melanocytes were cultured for 72 h in basal medium containing ␤-ionone (50 M), forskolin (20 M), ␣-ionone (200 M), H89 (10 M), or the solvent only (0.1% DMSO). Melanin contents of stimulated melanocytes were measured according to the method of Oka et al. (26) with a slight modification. After stimulation, cells were harvested by scraping, and cell numbers were counted using a counting chamber (Blaubrand Neubauer improved, Sigma). To take the anti-proliferative effect of ␤-ionone into account, cell numbers were adjusted to 1 ϫ 10 5 before determination of the melanin content. Cell pellets were solubilized in boiling 1 M NaOH for 10 min. Spectrophotometric analysis of melanin content was performed at 400 nm absorbance.
Differentiation Assay-Melanocytes were cultured for 6 days in basal medium containing ␤-ionone (50 M), forskolin (20 M), or the solvent only (0.1% DMSO). Cell morphology was checked by bright field microscopy using a Zeiss Axioskop2 microscope with 20ϫ magnification. Undifferentiated (bipolar morphology, small cell bodies, less pigmentation) and differentiated melanocytes (multiple dendrite, large cell body, high pigmentation) were quantified.
Synthesis of Fluoresceine-5-isothiocyanate (FITC)-labeled Steroids-FITC-labeled steroids were obtained by chemical synthesis from dihydrotestosterone and dehydrotestosterone. In brief, the 17-␤-OH group of the respective steroid was activated by acylation with carbonyldiimidazole (27)(28)(29) in tetrahydrofuran. The resulting monoimidazolide was treated with excess 4,7,10-trioxa-1,13-tridecanediamine in acetonitrile followed by evaporation and washing with aqueous NaHCO 3 . Treatment of the resulting primary amine intermediate with fluorescein-5-isothiocyanate in DMF in the presence of Hünig's base (iPr 2 NEt) provided the fluorescently labeled steroid ligands. The final products were purified to homogeneity by using preparative HPLC and obtained as orange powders after lyophilization in 12-50% overall yield. Details of synthesis procedures and characterization data for all new compounds ␣ (m.p., high resolution mass spectrometry, 1 H and 13 C NMR, IR) was described in the following section.
All solvents, when not purchased in suitable purity or dryness, were distilled using standard methods. Alternatively, solvents (HPLC grade) were passed through activated alumina columns under dry argon atmosphere (solvent purification system, M. Braun Inertgas-Systeme GmbH, Garching, Germany). Deionized water was used for all experiments. All reagents were purchased from commercial suppliers (Acros, Novabiochem, Sigma) and used without purification. Analytical thin layer chromatography (TLC) was carried out on Merck precoated silica gel plates (60F-254) using ultraviolet light irradiation at 254 nm or phosphomolybdic acid solution as staining reagent (1 wt% in EtOH). Flash column chromatography was performed using silica gel (J. T. Baker, particle size 40 m; pore size 60 Å) under a pressure of 0.3-0.5 bar. Analytical HPLC was performed on an Agilent 1100 system using a C18 gravity 3-m reverse phase column (Macherey & Nagel, Düren, Germany). The separations were started at 10% MeCN (with 0.1% HCOOH) in H 2 O (with 0.1% trifluoroacetic acid) with a flow of 1 ml/min, and the MeCN proportion was linearly increased after 1 min to 100% over a period of 10 min and then kept at constant ratio for a period of 5 min. Preparative HPLC was performed on a Varian system using a C18 gravity 5-m reversed phase column (Macherey & Nagel) using a MeCN in H 2 O gradient. 1 H and 13 C NMR spectra were recorded on a Varian Mercury VX 400 (400.1 MHz ( 1 H) and 100.6 MHz ( 13 C)) spectrometer. Chemical shifts are expressed in parts per million (ppm), and the spectra were calibrated to residual solvent signals of CDCl 3 (7.26 ppm ( 1 H) and 77.0 ppm ( 13 C)), CD 3 OD (3.31 ppm ( 1 H), and 49.0 ppm ( 13 C)), respectively. Coupling constants are given in Hertz (Hz), and the following notations indicate the multiplicity of the signals: s (singlet), d (doublet), t (triplet), q (quartet), qui (quintet), sext (sextet), sept (septet), m (multiplet), app (apparent), br (broad signal). Unless otherwise stated, NMR spectra were recorded at 27°C. Low resolution mass spectra were recorded on a Thermo Finnigan LCQ ESI spectrometer (source voltage 70 keV). High resolution FAB (fast atom bombardment) spectra were recorded on a Jeol SX 102 A (matrix: meta-nitrobenzyl alcohol). High resolution electrospray ionization spectra were recorded on a Thermo Electron LTQ Orbitrap (source voltage 3.8 kV, resolution: 60000) spectrometer.

An Olfactory Receptor Influences Melanocyte Pigmentation
Compound 9 (50 mg crude, 0.13 mmol) was dissolved in DMF (5 ml), and FITC (162 mg, 0.39 mmol) and EtNiPr 2 (116 l, 0.65 mmol) were added. The mixture was stirred, and turnover was monitored by HPLC. After 4 h, the solvent was evaporated, and the residue was purified using preparative HPLC. Pure fractions were combined and lyophilized to give the title compound 11 as an orange solid (14 mg, 12% yield after 2 steps), m.

Results
Olfactory Receptors Are Expressed in Human Melanocytes-Frog melanophores have previously been shown to disperse their melanosomes in response to odorants, although the concurrent increase in intracellular cAMP levels on pigment dispersion was discussed controversially (30,31). Moreover, screening for naturally occurring compounds with antiproliferative activity on melanoma cells has led to the identification of the odorant 4-allyl-2-methoxyphenol (eugenol) (32,33).
We, therefore, analyzed whether ORs are expressed in melanocytes. We started by analyzing receptor expression in cultured human melanocytes by RT-PCR (Fig. 1A) and found different ORs to be expressed, among them OR51E2, an olfactory receptor that was previously also detected in prostate cells and in the olfactory epithelium (11). Because the OR51E2 ligand, the isoprenoid ␤-ionone (with an odor of violet), was included in the earlier studies on the effect of odorants on pigment dispersion (30,31), we specifically studied the expression of this receptor in detail. RT-PCR with intron-spanning primers to exclude amplification from genomic DNA contamination revealed that OR51E2 transcripts are expressed in human epidermal melanocytes (NHEM) from donors of ethnic origins with differing pigmentation levels (Fig. 1B). We also detected OR51E2 protein in human epidermal melanocytes by Western blotting and immunocytochemical analysis using an OR51E2specific antibody (Fig. 1, C and D). Specificity of the antibody was demonstrated by co-immunocytochemical staining of Hana3a cells heterologously expressing rho-tagged OR51E2 (Fig. 1E).
Effect of ␤-Ionone on Proliferation and Apoptosis-Because OR51E2 has been shown to be involved in the regulation of prostate cancer cell growth (11), we investigated the effect of the OR51E2 ligand ␤-ionone on melanocyte proliferation.

An Olfactory Receptor Influences Melanocyte Pigmentation
Melanocytes were treated for 6 days in basal medium containing different concentrations of ␤-ionone, and the DNA content was determined to reveal the number of cells. ␤-Ionone stimulation decreased the cell number in a significant manner, even at submicromolar concentrations ( Fig. 2A). The maximal decrease in proliferation (ϳ30%) was observed after 6 days of treatment with 50 M ␤-ionone. This result was verified via immunocytochemical staining with an antibody against the PCNA as a marker for cell proliferation (Fig. 2B).
As we observed decreased cell numbers upon exposure to ␤-ionone, we investigated the influence of ␤-ionone on cell apoptosis rates in primary human melanocytes. Primary melanocytes were stimulated for 72 h before detection of apoptosis by assessment of Caspase 3/7 activity, as indicative of an activated apoptotic signaling pathway, and TUNEL staining to quantify apoptotic cells (Fig. 2C). The Caspase 3/7 assay showed a nominal increase of the caspase activity after stimulation with 50 M ␤-ionone, which was not significant. However, TUNEL staining implicated that long term ␤-ionone stimulation does not induce apoptosis in melanocytes. We, therefore, conclude that the observed decrease in cell number after stimulation with 50 M ␤-ionone is a result of a declining proliferation rate.

␤-Ionone Induces Melanogenesis and Differentiation-We
next investigated the effect of ␤-ionone on melanogenesis and dendritogenesis as indicators of pigment cell differentiation (34). To observe whether ␤-ionone stimulation induces melanogenesis in normal melanocytes, the melanin content of normal human melanocytes was determined. Forskolin, a potent stimulator of adenylate cyclases, which cause melanogenesis and dendritogenesis of mammalian pigment cells (35,36), served as the positive control. The ␤-ionone-induced increase of the melanin content was similar to that of forskolin treatment after 72 h cultivation in basal medium containing the respective stimuli (Fig. 2D). Co-stimulation with the OR51E2 competitive antagonist ␣-ionone (11) demonstrates that the observed increase in melanogenesis depends on activation of OR51E2. We furthermore identified PKA as a key mediator of OR51E2-triggered melanogenesis by using the specific PKA inhibitor H89 (37), which inhibited the ␤-ionone-induced melanogenesis when the cells were pre-stimulated. Concordantly, qPCR analysis revealed that ␤-ionone stimulation for 72 h induced tyrosinase expression (Fig. 2E), a promoter of melanocyte melanogenesis. This was confirmed with Western blotting analysis that showed an increase in tyrosinase protein levels (Fig. 2F). Moreover, we examined the effect of ␤-ionone on melanocyte morphology as an indicator for cell differentiation. Melanocyte differentiation involves branching and extension of the dendrites. Primary melanocytes cultured in basal medium were treated for 6 days with 50 M ␤-ionone, and the number of dendrites was quantified. The majority of control cells exhibited a typical bipolar morphology, whereas most of the ␤-ionone-treated cells had multiple dendrites. Quantification of this effect showed that treatment with ␤ionone induced a morphological change in dendrites and significantly increased the percentage of cells with more than two dendrites (Fig. 2G).
As melanocyte cellular responses to external growth and differentiation signals can be mediated by MAP kinases (38 -40), we studied activation (phosphorylation) of prominent mem-bers of the MAP kinase family in ␤-ionone-treated melanocytes. Western blotting analysis using antibodies specific for phosphorylated and non-phosphorylated extracellular stressregulated kinase (p44/42 MAPK) and p38 MAPK revealed that both kinases are activated in ␤-ionone-treated melanocytes (Fig. 2H). ␤-Ionone stimulation did not result in a marked increase in phosphorylation of SAPK/JNK (data not shown). In summary, we could show that activation of OR51E2 affects pigment cell melanogenesis and differentiation by activation of a cAMP-PKA-mediated pathway.
OR51E2 Activation in Melanocytes-To investigate if ␤ionone exerts its effects on melanocytes by activation of OR51E2, we first examined the effects of short term ␤-ionone stimulation on the level of intracellular Ca 2ϩ in melanocytes via the Ca 2ϩ imaging method. Activation of an OR expressed in olfactory sensory neurons as well as in heterologous cell systems, induces intracellular Ca 2ϩ signals as a result of OR-initiated signaling. Stimulation of Fura-2-loaded melanocytes with ␤-ionone also caused a rise in cytosolic Ca 2ϩ (Fig. 3A). However, this ␤-ionone-induced Ca 2ϩ signal was different in response kinetic compared with olfactory sensory neurons and Hana3a cells expressing OR51E2. Olfactory sensory neurons and OR-expressing Hana3a cells show a fast transient Ca 2ϩ signal upon odorant-ligand stimulation, whereas application of ␤-ionone in melanocytes caused a slow but robust increase in cytosolic Ca 2ϩ that started rising after 1 min from the begin of the application (Fig. 3B). When the ligand was supplied continuously, the maximal amplitude was reached after 5 min application of ␤-ionone. The signal decreased afterward in the presence of the ligand, indicating a desensitization of the signaling cascade (Fig. 3C). The ␤-ionone-induced rise in intracellular Ca 2ϩ was found to be dose-dependent, and the threshold concentration to trigger a cellular response to ␤-ionone was ϳ10 M (Fig. 3D).
We next aimed to confirm that the ␤-ionone induced increase in cytosolic Ca 2ϩ is dependent on activation of OR51E2. We, therefore, performed Ca 2ϩ imaging experiments with reduced OR51E2 expression rate due to RNA silencing (Fig. 3E). ␤-Ionone-induced Ca 2ϩ signal amplitudes were quantified and compared with those in non-transfected control cells within the same experiment. The average of the ␤-ionone-mediated Ca 2ϩ signal amplitude was found to be significantly reduced (ϳ85%) in siRNA-expressing melanocytes compared with control cells (Fig. 3, F and G). Expression of scrambled control siRNA did not show an effect on the ␤-ionone-induced Ca 2ϩ responses in transfected cells; neither did the transfection procedure alter Ca 2ϩ responses of cells to endothelin-1 (Fig.  3G), which was reported to induce a Ca 2ϩ rise in melanocytes through action on the endothelin-1 B receptor (41). We further showed that the ␤-ionone-evoked Ca 2ϩ rise could be prevented by co-application of the OR51E2 competitive antagonist ␣-ionone (11) (Fig. 3I), whereas ␣-ionone alone did not produce Ca 2ϩ signals (Fig. 3H). Together, these data show that ␤-ionone produces an increase in the cytosolic Ca 2ϩ concentration in melanocytes and that the ␤-ionone induced Ca 2ϩ rise is mediated by the activation of OR51E2.
OR51E2 Signaling in Melanocytes-We next aimed to elucidate the signaling mechanism of OR51E2 in primary human melanocytes. To determine the origin of the agonist-evoked Ca 2ϩ rise, we used Ringer's solution with varying Ca 2ϩ concentrations. The ␤-ionone-induced Ca 2ϩ increase still occurred after removal of the extracellular Ca 2ϩ , suggesting that release from intracellular stores contributes to the ␤-ionone-induced Ca 2ϩ signal (Fig. 4, A and G). The signal amplitude was significantly reduced (by ϳ30%), the signal onset was delayed, and the rise in intracellular Ca 2ϩ was slower under Ca 2ϩ -free conditions. Extracellular Ca 2ϩ is, therefore, considered to significantly account for the ␤-ionone-induced Ca 2ϩ response in melanocytes. The observation that thapsigargin prestimulation suppresses the ␤-ionone-induced Ca 2ϩ response (Fig. 4, B and G) might reflect a reciprocal regulation between the OR51E2initiated pathway and store-operated calcium entry (SOCE). To identify potential Ca 2ϩ channels mediating the Ca 2ϩ influx, we tested pharmacological inhibitors of SOCE and transient receptor potential (TRP) channels. Whereas ORAI and TRP channel blocker 2-APB significantly diminished the ␤-ionone-induced Ca 2ϩ signal (Fig. 4, C and G), TRPC and SOCE channel blocker SKF 96365 (Fig. 4, D and G) and SOCE inhibitor BTP2 (Fig. 4, E and G) as well as stretch-activated calcium channel, TRPA1, and TRPC blocker GdCl 3 (Fig. 4, E and G) did not influence the ␤-ionone induced Ca 2ϩ rise in melanocytes when co-applied with ␤-ionone.
In olfactory sensory neurons, ORs couple to G␣ olf , a protein homologous to G␣ s , resulting in activation of an adenylate cyclase and generation of cAMP. We here investigated whether OR51E2 activation in melanocytes also modulates cellular cAMP levels. We determined cAMP levels in primary melano-cytes and found that ␤-ionone stimulation increased the intracellular cAMP concentration 4-fold. This effect was suppressed by co-stimulation with the OR51E2 inhibitor ␣-ionone, indicating that the elevation of cAMP levels upon ␤-ionone treatment is caused by activation of OR51E2 (Fig. 4H). Usage of pharmacological tools that specifically inhibit key enzymes typically activated by GPCR stimulation (adenylate cyclase, phos- The gray Ca 2ϩ imaging trace is representative for the vehicle controls (0.1% DMSO). Application of the solvent did not result in any changes in cytosolic Ca 2ϩ . Cytosolic Ca 2ϩ levels were monitored as integrated f340/f380 fluorescence ratio expressed as a function of time. C, in prolonged stimulation the maximal signal amplitude was reached after a 5-min application of ␤-ionone. After reaching the maximum peak, the cytosolic Ca 2ϩ concentration decreases until a plateau phase was sustained. ␤-Ionone was applied for 9 min. D, ␤-ionone induced Ca 2ϩ increase is dose-dependent. ␤-ionone was applied for 9 min at different concentrations to ensure maximal signal amplitude generation. Signal amplitude was quantified, normalized to the maximal peak height, and is displayed as a function of the applied ␤-ionone concentration (n ϭ 49 -129 cells). E, Ca 2ϩ imaging on primary melanocytes transfected with a plasmid encoding for siRNA directed against OR51E2. As the plasmid encodes for GFP under the same promoter, siRNA-expressing cells can be identified via GFP fluorescence (left panel); pseudocolor images were captured of Fura-2 loaded cells during Ca 2ϩ imaging experiment (right panel). OR51E2-siRNA/GFP-expressing melanocytes (turquoise labeling) showed no significant changes in the Fura-2 ratio upon application of ␤-ionone, whereas non-transfected cells (blue labeling) did. F, ␤-ionone-induced Ca 2ϩ signals in siRNA transfected (turquoise labeling) and control melanocytes (blue labeling) displayed as a function of time. 50 M ␤-ionone was applied for 5 min. G, quantification of the relative signal amplitudes of the ␤-ionone evoked Ca 2ϩ signals, normalized to the ␤-ionone response of control cells (n ϭ 109 cells). n ϭ 4 independent transfections were analyzed. Shown are OR51E2-siRNA-expressing melanocytes (n ϭ 42 siRNA-expressing cells) and melanocytes transfected with a plasmid encoding for scrambled OR51E2-siRNA (control siRNA, n ϭ 27). OR51E2-siRNA-transfected (n ϭ 23) and control cells (n ϭ 64) were stimulated with 40 nM endothelin-1 (ET-1) to control that siRNA expression did not affect cell viability. Error bars represent the S.E. Significance was calculated by Student's t test for each sample group referring to the signal amplitude in control melanocytes (***, p Ͻ 0.001). H, representative Ca 2ϩ imaging traces of Fura-2-loaded melanocytes. ␣-Ionone (250 M) was applied for 5 min. Cytosolic Ca 2ϩ levels were monitored as integrated f340/f380 fluorescence ratio expressed as a function of time. I, co-application of the OR51E2-specific inhibitor ␣-ionone reduced the ␤-ionone-induced Ca 2ϩ signals (n ϭ 48). ␤-Ionone (50 M) and the ␣-ionone and ␤-ionone 2:1 mix (100 M and 50 M, respectively) were applied for 4 min.
pholipase C) did not allow for straightforward interpretation of the Ca 2ϩ data, probably due to the observed cytotoxic side effects of the tested inhibitors MDL-12,330A, edelfosine and U73122 in melanocytes. However, the pharmacological characterization using inhibitors of Ca 2ϩ signaling (thapsigargin, 2-APB, SKF, BTP2, GdCl 3 ) indicate that the OR51E2-triggered Ca 2ϩ signal is constituted by Ca 2ϩ release from intracellular stores and an additive Ca 2ϩ influx from the extracellular space. Ca 2ϩ influx may be mediated by members of the TRP ion channel family that are expressed in melanocytes such as TRPM family members (42), TRPA1 (43), or TRPV1 (44) or calcium release-activated channels, such as ORAI1/2 (45). However, an involvement of ORAI channels, TRPC channels, and TRPA1 could be excluded because SKF, BTP2, and GdCl 3 did not block the ␤-ionone-induced Ca 2ϩ signal at tested concentrations. Therefore, we suggest that members of the TRPM family mediate the observed Ca 2ϩ influx. This assumption is supported by the finding that 2-APB, which blocks inter alia TRPM3, TRPM7, and TRPM8, significantly reduced the ␤-ionone-induced Ca 2ϩ signal.
Subcellular Localization of OR51E2-A well characterized GPCR that is involved in biogenesis of the pigment producing organelles, the melanosomes, is the ocular albinism type 1 (46 -51). Ocular albinism type 1, an atypical GPCR, primarily localizes to intracellular endolysosomes and the melanosomes of retinal pigment epithelial cells rather than the cell surface (52,53).
We, therefore, investigated where OR51E2 resides in human epidermal melanocytes. To determine the subcellular localization of the receptor, we performed immunofluorescence studies to visualize OR51E2 protein. Melanocytes were labeled with an antibody against OR51E2. In epidermal melanocytes, immunofluorescence staining revealed that OR51E2 is localized in a vesicular pattern throughout the cytosol (Fig. 5A). OR51E2containing vesicles accumulated at the extremity of the dendrites. Localization at the plasma membrane was not apparent in immunocytochemical stainings. Co-staining with antibodies targeting several cellular organelles revealed that OR51E2 did not localize to the endoplasmic reticulum, the Golgi apparatus, melanosomes, and lysosomes (data not shown) but showed colocalization with EEA-1 (Fig. 5B), an early endosomal marker. Analysis of numerous cells revealed that there was clear OR51E2 labeling at the plasma membrane.
Because immunocytochemical methods failed to give clear information about membrane localization of OR51E2, we next aimed to resolve whether receptor activation occurs also at the plasma membrane or only at intracellular membranes. To provide evidence for plasma membrane localization of OR51E2, we detected the protein by Western blotting in surface preparations of primary melanocytes (Fig. 5C).
To distinguish whether the ␤-ionone induced Ca 2ϩ signal results from activation of surface or cytosolic OR51E2, we generated ligands for receptor activation, which are less membrane-permeable than the unmodified ligands. ␤-Ionone exhibits hydrophobic structural properties and may, therefore, be membrane-permeable, making it possible that the receptor is activated by the ligand inside an intracellular compartment. Other OR51E2 activating ligands described previously are the steroids 4,6-androstadien-17␣-ol-3-one, 1,4,6-androstadien-3,17-dione, and 6-dehydrotestosterone (11). For chemical structures, see Scheme 7. Activation of OR51E2 by these steroid ligands (100 M) could be shown in melanocytes (Fig. 5D), whereas stimulation with dihydrotestosterone (DHT), which does not activate recombinant OR51E2, failed to induce Ca 2ϩ signals in melanocytes. Nota- bly, the Ca 2ϩ signal induced by OR51E2-activating steroid ligands was significantly higher compared with the ␤-ionone induced Ca 2ϩ responses (Fig. 5D).
These data suggested that ␤-ionone might be a weaker agonist and that the A/B-ring region of the steroids was important for ligand activity, whereas the D ring should be amenable to modification. To study the cellular site of activation of OR51E2, we synthesized an OR51E2-activating dehydrotestosterone appended with the membrane-impermeable anionic dye molecule FITC by means of a water-soluble PEG linker. As control, we used DHT that was shown to be inactive on OR51E2 (11). The FITC label was attached to the OH-group of dihydrotestosterone and 6-dehydrotestosterone on position 17 (see Scheme 7 and "Experimental Procedures"). Due to the change in physicochemical properties these dye-labeled, hydrophilic steroid ligands are expected to exhibit a reduced cell membrane permeability compared with the parental steroids.
Interestingly, the FITC-tagged dehydrotestosterone derivative was found to activate recombinant OR51E2 as shown by Ca 2ϩ imaging experiments on Hana3a cells transiently expressing OR51E2 (Fig. 5E). Similarly, application of the same FITCtagged steroid ligand to primary human melanocytes triggered Ca 2ϩ responses (Fig. 5, F and G). Although the maximal signal amplitude was smaller compared with untagged 6-dehydrotestosterone, it resembled the Ca 2ϩ response amplitude evoked by ␤-ionone. Neither treatment of melanocytes with the inactive steroid DHT nor with FITC-DHT induced a comparable Ca 2ϩ response in the cells (Fig. 5, F and G), indicating that OR51E2 protein, which is present at the plasma membrane of melanocytes, received the external signal.
Repetitive ␤-ionone stimulation of melanocytes resulted in an increase in the Ca 2ϩ response amplitudes (Fig. 5H). When we exposed melanocytes to ␤-ionone before re-stimulation in Ca 2ϩ imaging experiments, we observed significant increases in the ␤-ionone induced Ca 2ϩ levels that were dependent on the duration of preexposure (Fig. 5, H and I). We assume that these effects could be caused by increased insertion of OR51E2 into the plasma membrane upon activation of the receptor, thereby increasing the low amounts of functional OR51E2 at the cell surface of melanocytes.

Functionality of an Ectopically Expressed Odorant
Receptor in Melanocytes-The first evidence for an influence of odorants on pigment cells was obtained in the 1980s, when the superfamily of odorant receptors was unknown. Frog melanophores were shown to disperse their melanosomes in response to odorants, and odorant-induced pigment dispersion was accompanied by rises in intracellular cAMP levels (30). Corresponding studies discovered cinnamaldehyde and ␤-ionone to have pigment dispersing activity in fish melanophores (31), but neither substances resulted in a measurable rise in cAMP levels. We describe here that ␤-ionone led to an increase in the Ca 2ϩ concentration and elevated cAMP levels. In addition, melanin content increased in primary epidermal melanocytes, possibly due to up-regulated expression of tyrosinase. Moreover, we describe that OR51E2 mediated ␤-ionone-induced effects in melanocytes. By gene-silencing and by using a specific antagonist, we could clearly show that the ␤-ionone-induced Ca 2ϩ signal in melanocytes depends on the expression of OR51E2.
OR51E2 belongs to the family of odorant receptors and is expressed in prostate cancer cells (11). Analysis of the role of OR51E2 in melanocytes revealed that the receptor affects melanocyte proliferation, differentiation, and melanogenesis and indicates that ectopically expressed ORs have important cellular functions. As OR51E2 expression is up-regulated in human prostate cancer cells compared with healthy prostate epithelial cells, the receptor may be used as a potential tumor biomarker (54 -57). Therefore, it would be interesting to investigate OR51E2 expression and function in melanoma cells derived from epidermal melanocytes. OR51E2 acts as a cell surface steroid receptor that mediates rapid, non-genomic, steroidal signaling in prostate cancer cells (11). The previously identified steroid ligands of OR51E2 activate Ca 2ϩ responses in melanocytes in a similar fashion. The exquisite selectivity of this receptor with respect to the molecular structure of the agonist suggests that endogenous ligands should exist to regulate the proliferation and differentiation of melanocytes. It is tempting to speculate that this yet elusive endogenous ligand for OR51E2 may be a keratinocyte-derived factor that is chemically similar to the identified agonists, possibly a steroidal compound.
OR51E2 Localization-Intracellular localization of GPCRs was previously shown in retinal pigment epithelial cells. The atypical GPCR ocular albinism type 1 is localized to membranes of melanosomes and late endosomes/lysosomes, although low amounts of the receptor were detected also at the plasma membrane (58). Ocular albinism type 1 is considered to regulate melanosome biogenesis by transducing signals through activation of heterotrimeric G-proteins on the cytoplasmatic side of the organelle membrane (46,48,59).
OR51E2 was mainly localized in early endosomes associated with EEA-1 in human epidermal melanocytes. Endosomal organelles are direct precursors to premelanosomes (stage I melanosomes) (60). As in other cells, two distinct early endosomal domains can be distinguished, tubulovesicular structures containing the bulk of EEA-1 and globular structures, which hold enriched concentrations of the melanoma-associated protein melan-A. However, we could exclude localization of OR51E2 to the globular endosomal domains by co-labeling OR51E2 and melan-A. Although immunocytochemical signals were not apparent in immunocytochemical analysis, we clearly demonstrated plasma membrane localization of OR51E2 by cell surface preparations and Western blotting analysis. Activation of OR51E2 in melanocytes still occurred when using a steroid agonist for OR51E2 with reduced membrane permeability, indicating that the observed Ca 2ϩ signals result from activation of OR51E2 protein at the cell surface rather than cytosolic OR51E2.
Signaling Cascade of OR51E2 in Normal Human Epidermal Melanocytes-The present study shows that signaling of OR51E2 in melanocytes involves cAMP, potentially TRPM family members, Ca 2ϩ , and activation of PKA and MAPKs. The OR51E2-induced Ca 2ϩ signal was found to be partially dependent on extracellular Ca 2ϩ , as the amplitude of the response was significantly reduced in the absence of Ca 2ϩ . However, pharmacological characterization revealed that the influx of extracellular Ca 2ϩ is neither mediated via store-operated Ca 2ϩ channels of the ORAI family, nor by TRPC, TRPV, and TRPA1 channels but indicate an involvement of other TRP channels. Based on previous reports and our results (with given limitations of the employed Ca 2ϩ imaging technique), we conclude that the molecular identity of the TRP channels is in the TRPM family as members of this TRP family are expressed in melanocytes and can be blocked by 2-APB while remaining unaffected by the other tested inhibitors.
The proposed OR51E2 initiated signal transduction pathway in melanocytes may involve PLC-mediated signaling as well. Unfortunately, common inhibitors of PLC appeared unsuitable for application on melanocytes. In classical olfactory tissues, olfactory receptors can couple to both adenylyl cyclase-and PLC-mediated pathways (61).
In prostate cancer cells, OR51E2 activates TRPV6 via Srckinase in a G-protein-independent manner (62). However, comparison of the OR51E2 signaling in prostate cancer cells and melanocytes reveals differences, such as an involvement of adenylate cyclase (11). Thus, the signal transduction cascade of ectopically expressed ORs seems to be majorly determined by the cellular background and not by the properties of the receptor protein.
Role of OR51E2 on Melanogenesis and Pigment Cell Differentiation-Pigment production in vertebrates is modulated by a variety of hormones and neurotransmitters acting on transmembrane receptors located on the cell surface (63,64). Activation of the ET-1 receptor increases cytosolic Ca 2ϩ concentrations via ORAI1 channels, thereby stimulating melanogenesis (45,65). The intracellular Ca 2ϩ concentration itself was found not to be essential for melanogenesis but to play an important role in modulating the responses of melanocytes to melanogenic stimuli (67,68). Activation of OR51E2 resulted in a prolonged increase in the intracellular Ca 2ϩ concentration and led to an induction of melanogenesis via the cAMP pathway. Regulation of melanogenesis was shown to involve stimulation of adenylate cyclase followed by an increase in the intracellular cAMP level and activation of cAMP-dependent protein kinases (PKA) (69). Activated PKA is involved in the phosphorylation of cAMP-responsive element-binding protein (CREB) and CREB-binding protein. Phosphorylated CREB leads to an activation of microphthalmia-associated transcription factor (MITF), whereas MITF regulates the expression (but not activity) of melanogenic enzymes (tyrosinase, TYRP1, and TYRP2) (for review, see Ref. 70). Thus, the observed up-regulation of tyrosinase likely results from OR51E2 triggered activation of PKA.
Downstream signaling of OR51E2 in epidermal melanocytes also involves activation of p38 MAPK and p44/42 MAPK (ERK1/2) as possible modulators of melanogenesis. These MAPKs are described to control pigment cellular responses to melanogenetic stimuli by induction of tyrosinase proteasomal degradation, thus antagonizing activated cAMP signaling (71).
Effects of OR51E2 activation on melanocyte morphology and melanogenesis implicate that OR51E2 is also involved in regulation of melanocyte differentiation. Many coat color genes, known for their ability to regulate melanosome formation, control in addition melanoblast migration, proliferation, and differentiation and melanosome distribution. Thus, melanocyte proliferation and differentiation is not only regulated by typical growth factor receptors but also by genes classically known for their role in pigment formation.
Interestingly, repetitive cell stimulation in Ca 2ϩ imaging experiments showed an increased responsiveness of melanocytes pretreated with ␤-ionone. We hypothesize that OR51E2 that is localized in the early endosome may translocate to the plasma membrane upon activation of cell surface receptors. A new paradigm is emerging in some cellular contexts, in which stocks of functional GPCRs retained within intracellular compartments can be rapidly mobilized to the plasma membrane to maintain sustained physiological responsiveness (66). However, further data must be acquired to confirm such a role for OR51E2 in human melanocytes.
The present study constitutes a new example for the functionality of ectopic ORs. It also contributes to the understanding the molecular processes involved in regulation of skin pigmentation by showing that the ectopically expressed olfactory receptor OR51E2 is functionally expressed in melanocytes. Pigment cells respond to the OR51E2 ligand with decreased proliferation rates and increased melanogenesis. As OR51E2-induced signaling mechanisms could influence melanocyte homeostasis, receptor-activating steroids or terpenoids might provide novel compounds for the treatment of pigmentation disorders and proliferative pigment cell disorders such as melanoma.