Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors

Molecular and Cellular Endocrinology 314 (2010) 101–109

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Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors P. Kempná a , M. Körner b , B. Waser b , G. Hofer a , J.-M. Nuoffer c , J.C. Reubi b , C.E. Flück a,∗ a b c

Department of Pediatrics, Division of Pediatric Endocrinology and Diabetology, University Children’s Hospital Bern, Switzerland Institute of Pathology, Division of Cell Biology and Experimental Cancer Research, University of Bern, Switzerland Institute of Clinical Chemistry, University Hospital Inselspital Bern, 3010 Bern, Switzerland

a r t i c l e

i n f o

Article history: Received 17 April 2009 Received in revised form 13 August 2009 Accepted 13 August 2009 Keywords: NPY Y1 receptor Adrenal Steroidogenesis Tumorigenesis

a b s t r a c t Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Neuropeptide Y (NPY) is a peptidic neurotransmitter abundantly expressed in the central and peripheral nervous system where it regulates many diverse functions, such as eating behavior, mood, and stress (Balasubramaniam, 2002). It is structurally related to the two gut peptide hormones peptide YY (PYY) and pancreatic polypeptide (PP), which are involved in digestion. NPY, PYY, and PP exert their actions through binding to the NPY receptors which are members of the G protein-coupled receptor (GPCR) superfamily. In humans, four NPY receptor subtypes have been identified, namely Y1, Y2, Y4, and Y5 (Michel et al., 1998). Adrenal steroid hormone metabolism is one of the many systems under the control of NPY. It is regulated by NPY not only centrally via the hypothalamo–pituitary–adrenal (HPA) axis, but also peripherally at the level of the adrenal gland. Evidence for the latter derives primarily from rat studies where NPY was found to stimulate mainly aldosterone secretion in vivo (Nussdorfer et al., 1998; Renshaw and Hinson, 2001). Experiments using intact rat adrenal gland preparations suggested that this effect must be indirect as it was mediated by catecholamine release from medullary

∗ Corresponding author at: Pediatric Endocrinology and Diabetology, University Children’s Hospital Bern, Freiburgstrasse 15, G3 812, CH-3010 Bern, Switzerland. Tel.: +41 31 632 0499; fax: +41 31 632 8424. E-mail address: christa.fl[email protected] (C.E. Flück). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.08.010

chromaffine cells expressing NPY receptors (Renshaw et al., 2000). Conversely, direct effects of NPY on dispersed rat adrenal cortical cells were small and inconsistent (Renshaw and Hinson, 2001; Renshaw et al., 2000). In humans, however, the role of NPY and related peptides in adrenal steroid hormone biosynthesis is largely unexplored, and effects may considerably differ from those in the rat. In fact, preliminary studies on the expression of NPY receptor binding sites in the human adrenal gland revealed large amounts of Y1 receptors throughout the cortex, but no NPY receptors in the medulla (Korner et al., 2004a). This finding suggests rather a direct action of NPY, which may be released from intracortical nerve fibers (Li et al., 1999), on adrenal cortical cells, than an indirect action of NPY targeting only medullary chromaffine cells. Furthermore, the relevance of observed Y4 receptor-mediated PP effects on cortisol secretion in dispersed human adrenal cortical cells is unclear, as Y4 receptors were not present in detectable amounts in human adrenal cortical tissues (Korner et al., 2004a). A role of NPY on steroid hormone secretion may not be restricted to physiologic processes, but also occur in neoplasia. This is indeed strongly suggested by the high expression of NPY receptors in different kinds of steroid hormone producing tumors such as adrenal cortical tumors and ovarian sex cord-stromal tumors (Korner et al., 2004a,b). Similarly the presence of NPY peptide in nerve fibers within such tumors suggests a role of NPY in tumor steroidogenesis (Li et al., 1999). Theoretically, NPY peptide released from tumoral nerve fibers could activate tumoral NPY receptors in a paracrine

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P. Kempná et al. / Molecular and Cellular Endocrinology 314 (2010) 101–109 Table 1 Sequences of primers used for RT-PCR.

fashion. However, to date, this has never been explored at the functional level. Therefore, the aims of the present study were to assess the NPY receptor profile of human adrenal H295 cell lines and to find a cell model to study the effect of NPY on human adrenal steroid hormone production and secretion in these cells. We used the well-characterized human adrenal cortical carcinoma cell lines H295A and H295R, which serve as established models for studies of adrenal cortical neoplasia as well as human adrenal steroidogenesis (Gazdar et al., 1990; Rainey et al., 2004; Rodriguez et al., 1997). These cells express all genes for mineralocorticoid, glucocorticoid and adrenal androgen biosynthesis. Furthermore, the NPY receptor expression of the human adrenal gland was comprehensively investigated with in vitro NPY receptor autoradiography.

Leandro, CA, USA). Intensities of specific bands were compared to GAPDH as internal control.

2. Materials and methods

2.4. Protein extraction and Western blot analysis

2.1. Cell cultures and treatments

Cell protein extractions and Western blot analysis of phosphorylated ERK1/2 and MEK were performed according to the protocol provided by the manufacturer (Cell Signaling Technology, Inc., Danvers, MA, USA). Briefly, cells were treated in 5 cm dishes, washed with ice-cold PBS and collected in 300 ␮l of SDS loading buffer (62.5 mM Tris–HCl, pH 6.8; 2% sodium dodecylsulfate, 10% glycerol, 100 mM dithiotreitol, 0.01% bromophenol blue). Lysates were passed few times through a 25G syringe, centrifuged at 12,000 × g for 15 min and then heated for 10 min at 95◦ . Aliquots of protein extracts were resolved by 10% SDS PAGE and blotted on Immobilon P transfer membrane (Milipore, Bedford, MA, USA) using semi-dry transfer method. Staining with antibodies was performed according to manufacturer’s recommendation. Protein bands were visualized by ECL substrate reagent and exposed on ECL Plus films (Amersham Pharmacia Biotech, Dübendorf, Switzerland). Films were scanned by AlphaImager and intensities of protein bands quantified. As control for equal loading total ERK (antibody purchased from Santa Cruz) was used after stripping membranes with 0.2 M NaOH.

Human adrenal NCI-H295R cells were purchased from ATCC (ATCC number CRL-2128TM ). Human adrenal NCI-H295A cells were kindly provided by Prof. W.L. Miller (University of California, San Francisco, USA). Cells were cultured under standard conditions in Dulbecco’s modified Eagle’s/Ham’s F12 (DMEM/F12) medium (Gibco BRL Invitrogen, Basel, Switzerland) supplemented with 5% NuI serum, 0.1% ITS (insulin transferrin selenium) Plus and antibiotics (all from Gibco BRL). Starvation medium consisted of DMEM/F12 supplemented with antibiotics only. Human NPY was purchased from Bachem (Bubendorf, Switzerland) and dissolved in either 10 mM acetic acid or PBS at stock concentrations of 500 ␮M. BIBP2336, a specific antagonist of the NPY-Y1 receptor (Y1R) was also purchased from Bachem and dissolved at 5 mM in PBS. Angiotensin II (AngII), PD98059 and forskolin were all purchased from Sigma (Sigma–Aldrich, Basel, Switzerland). Forskolin was dissolved in DMSO at stock concentrations of 10 mM (final concentration 10 ␮M); Ang II was dissolved in PBS and used at final concentrations of 100 nM with 0.001% Brij-35 (Sigma); MEK inhibitor PD98059 was dissolved in DMSO and used at final concentration of 25 ␮M. For steroid labeling and measurements, cells were grown and treated in 6-well plates (Becton Dickinson Labware, Le Pont de Claix, France). Generally, cells were grown in starvation medium for 24 h before starting any treatment. Peptides were either diluted in 10 mM acetic acid/1% BSA (bovine serum albumin (Sigma–Aldrich, Basel, Switzerland)) or PBS/1% BSA and added to the cells repetitively every 24 h. When indicated, cells were treated with antagonist BIBP2336 in 100-fold excess of NPY treatments. 2.2. In vitro NPY receptor autoradiography NPY receptor autoradiography was performed to assess NPY receptor binding sites in the human adrenal cortical carcinoma cell lines H295A and H295R and in human adrenal gland tissues. Eleven fresh-frozen samples of normal adrenal glands were obtained from patients undergoing nephrectomy for renal cancer with informed consent and approval of the Review Board of the Institute of Pathology of the University of Berne. For two of these samples, the NPY receptor expression has been published previously (Korner et al., 2004a). The procedure was performed as described before (Korner et al., 2004b). Briefly, cell pellets or tissue sections mounted on glass slides were incubated with 10,000 cpm/100 ␮l 125 I-PYY (2000 Ci/mmol; Anawa, Wangen, Switzerland) either alone or together with increasing concentrations of cold PYY, the Y1-selective analogs [Leu31 ,Pro34 ]-PYY (Bachem) or BIBP 3226 (Böhringer, Ingelheim, Biberach, Germany), the Y2-selective analog BIIE0246 (Böhringer), the Y4-preferring analog PP (Bachem) or the Y5-selective analog [cPP(1–17) ,NPY(19–23) ,Ala31 ,Aib32 ,Pro34 ]hPP (Dr. Jean Rivier, La Jolla, CA, USA). Slides were exposed to photosensitive films for 7 days. The signal density on the films was quantitatively assessed using tissue standards for iodinated compounds (Amersham, Ailesbury, UK) and a computer-assisted image processing system (Analysis Imaging System, Interfocus, Mering, Germany). Curve fitting analysis was performed with the Graph Pad PRISM program (GraphPad software Inc., San Diego, CA, USA). 2.3. RNA extraction and RT-PCR analysis Total RNA of NCI-H295R cells was extracted using the Nucleospin RNA kit by Macherey Nagel (Oensingen, Switzerland). Total RNA of human adrenal tissues was extracted using the Trizol method as described by the manufacturer (Gibco BRL). RNA was reverse transcribed to cDNA using the Improm RNA Transcriptase kit (Promega, Madison, WI, USA) with either 0.5 ␮g oligo-dT or random hexamers (both from Promega) per 1 ␮g of RNA at 42◦ for 1 h. Aliquots of cDNA obtained from 50 ng RNA were taken for PCR reactions in a final volume of 25 ␮l. For PCR GoTaq Polymerase (Promega) and specific primers were used (see Table 1). PCR conditions for Y1R, NPY and GAPDH were 45 s at 95◦ , 45 s at 57◦ , 45 s at 72◦ for 30 cycles. Aliquots of PCR products were electrophoresed on a 1.5% agarose gel, visualized with ethidium bromide, scanned and quantified using an Alpha Imager 3400 (Alpha Innotech, San

NPY (gi:31542152)

Sense Antisense

5 -CGAGGACGCACCAGCGGAG 5 -CACATTGCAGGGTCTTCAAGC

Y1R (gi:41350310)

Sense Antisense

5 -TCCATCGGACTCTCATAGGTTG 5 -GGAGCCAGCAGACTGCAAATG

GAPDH (gi:83641890)

Sense Antisense

5 -GTATCGTGGAAGGACTCAT 5 -TACTCCTTGGAGGCCATGT

18S SYBR (gi:89058284)

Sense Antisense

5 -CTCAACACGGGAAACCTCAC 5 -AGACAAATCGCTCCACCAAC

2.5. Cell proliferation studies Cell viability and proliferation was measured by Cell Titer 96 aqueous nonradioactive cell proliferation assay (Promega). Briefly, cells were plated into 96-well plates at a density of 15,000 cells/well; 24 h later medium was changed to starvation medium and cells were allowed to grow for 24 h. Cells were then treated with 10–100 nM NPY in PBS/0.1% BSA up to 96 h, replacing NPY every 24 h. After 0, 48, 72, 96 h cell proliferation was assessed. For that 20 ␮l of MTS/PMS solution (provided by the assay kit) was added to the cell culture medium and cells were incubated for 3 h before reading the absorbance at 490 nm (microplate reader SLT Rainbow, Tecan, Männedorf, Switzerland) which corresponds directly to the number of viable cells in this assay. 2.6. Steroid labeling NCI-H295R cells were grown in starvation medium on 6-well plates and treated for 48 h with neuropeptides as described above. Steroid metabolism was labeled with 220,000 cpm [3 H] pregnenolone (11.5 Ci/mmol) dissolved in ethanol (max 0.5%, v/v concentration) for 90 min as previously described (Auchus et al., 1998). Steroids were extracted from media as described (Kempna et al., 2007), separated on thin layer chromatography (TLC) plates (Whatman GmbH, Bottmingen, Switzerland) using 3:1 chloroform:ethylacetate as solvent system to resolve 17-hydroxysteroids (Dardis and Miller, 2003; Lin et al., 1991), and then exposed and visualized on a Storm PhosphoImager (Molecular Dynamics, Sunnyvale, CA, USA). Spots corresponding to specific steroids, identified by co-chromatography with a panel of labeled and unlabeled steroid standards, were densitometrically quantified using ImageQuant Software (Molecular Dynamics). Steroid conversion was assessed by calculating the percentage of radioactivity incorporated in a specific steroid hot spot compared to total radioactivity added to the reaction. For comparison, NPY/PYY treated samples were expressed as percentage of untreated control. 2.7. Measurements of cortisol, testosterone, dehydroepiandrosterone and aldosterone Cells were grown and treated in starvation medium for various durations as described. Medium was collected for measurements of specific steroid concentrations. Cells were lysed in 250 ␮l PBS/0.5% NP-40 (Sigma), homogenized by passing through a 25 Gauge syringe and centrifuged at 13,000 × g for 10 min at 4◦ . Supernatants were collected for colorimetric protein measurements (Biorad Protein Assay, Biorad, München, Germany). Cortisol was measured by the competitive immunoassay Modular Analytics E170 (Roche Diagnostics, Basel, Switzerland). Measurement of free testosterone was carried out by the Coat-A-Count solid phase 125 I radioimmunoassay (RIA) as described by the manufacturer (Diagnostic Products Corporation, Los Angeles, CA, USA). Dehydroepiandrosterone (DHEA) was measured

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Fig. 1. Expression of NPY and Y1R in human adrenal NCI-H295 cell lines and human adrenal tissues. (A) In vitro NPY receptor autoradiography on H295R (top row) and H295A (bottom row) cell pellets. Autoradiograms show (a and d) total 125 I-PYY binding, (b and c) 125 I-PYY binding in the presence of 25 nM of the cold Y1-selective analog BIBP3226, and (c and f) 125 I-PYY binding in the presence of 25 nM of the cold Y2-selective analog BIIE 0246. In H295R cells, there is moderate total 125 I-PYY binding (a), which is completely displaced by the Y1-selective ligand (b), but not the Y2-selective ligand (c). This provides evidence of specific Y1 expression in H295R cells. Conversely, in H295A cells, there is virtually no 125 I-PYY binding (d) above background labeling, which, as it is not displaced by either the Y1- or the Y2-selective ligand (e and f), represents non-specific binding; H295A cells display therefore no detectable NPY receptor binding. (B and C) Pharmacological displacement experiments in Y1-expressing H295R cells (B) and Y1-expressing human adrenal cortical tissue (C). In both cases, there is high affinity displacement of 125 I-PYY binding by PYY and the Y1-selective analogs [Leu31 ,Pro34 ]-PYY and BIBP 3226, but low affinity or now displacement by the Y2-selective analog BIIE 0246 and Y4-preferring PP. The Y5-selective analog [cPP(1–17) ,NPY(19–23) ,Ala31 ,Aib32 ,Pro34 ]hPP was inactive at the receptors (data not shown). This rank order of potencies provides pharmacological evidence of specific Y1 expression in H295R cells and human adrenal cortical tissue. (D) Semiquantitative RT-PCR analysis for NPY and Y1R expression in H295R and H295A cells as well as in two samples of human adrenal tissue (adrenal 1, 2). GAPDH was used as an internal control. (E) Quantitative Real Time PCR of Y1R expression in H295R versus H295A cells using 18S as internal control. (F) Representative picture of a human adrenal tissue section illustrating the distribution of Y1 receptors by receptor autoradiography. (a) Hematoxylin–eosin stained section showing the 3 layers of the cortex (C) and the medulla (M). (b) Autoradiagram showing total binding of 125 I-PYY. A homogeneous receptor distribution is seen in all cortical layers, with very little binding in the medulla. (c) Non-specific binding in presence of 25 nM of Y1-selective [Leu31 ,Pro34 ]-PYY.

by the DSL-8900 radioimmunoassay kit (Diagnostic Systems Laboratories Inc., Webster, TX, USA). Aldosterone measurement was done by competitive RIA on a gamma counter (Diagnostic Products Corporation, Los Angeles, CA, USA). Concentrations of cortisol, testosterone, DHEA and aldosterone were calculated per mg of total protein in each sample. 2.8. Real Time PCR The mRNA levels of CYP17 and HSD3B2 were quantitatively analysed by Real Time PCR using an ABI7500 Sequence Detection System (Applied Biosystems, Foster

City, CA, USA) (Kempna et al., 2007; Samandari et al., 2007). Briefly, PCR reactions were performed in 96-well plates (Abgene, Epsom, UK) using cDNA prepared as described above (50 ng/20 ␮l), TaqMan Universal PCR mixture and 1 ␮l of specific primers and probes obtained as assay-on-demandTM gene expression products (Applied Biosystems, www.appliedbiosystems.com). For the analysis of CYP21A2 and SULT2A1 ABsolute QPCR SYBR Green Mix (Abgene) and specific primers were used (Samandari et al., 2007). 18S rRNA was employed as the reference gene for all qPCR analyses. Relative expression values were determined by the 2−Ct method (Livak and Schmittgen, 2001). Results were expressed as percentage of untreated control.

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Table 2 Expression of NPY-Y1 receptors in the human adrenal. Location Cortex

Zona glomerulosa Zona fasciculata Zona reticularis

Medulla Blood vessels a

# of cases tested

Incidence

Densitya

11 11 11 8 10

11/11 11/11 11/11 4/8 10/10

2468 2384 2493 738 806

± ± ± ± ±

320 228 338 131 163

Density represents dpm/mg tissue (mean ± SEM).

2.9. Data analysis Data represent the mean of 3–5 independent experiments ± SD (with the exception of Fig. 2B, which shows the SEM of two independent experiments). Statistical significance (p < 0.05** and p < 0.01*) was tested using one- or two-way analyses of variance (ANOVA) with post hoc testing.

3. Results 3.1. Human adrenal H295R cells and human adrenal tissues specifically express the NPY-Y1 receptor subtype Adrenal cortical tumors, both hormonally active and inactive, express high levels of NPY receptors, specifically Y1 (Korner et al., 2004a). These findings raise the question whether adrenal cortical cell proliferation or steroidogenesis might be regulated by NPY through Y1 receptors. To study the role of NPY and Y1 receptors, we used human adrenal carcinoma H295 cell lines as an established model for such studies (Gazdar et al., 1990; Rainey et al., 2004; Rodriguez et al., 1997). Two subclones, H295A and H295R cells, exist with different steroidogenic characteristics (Gazdar et al., 1990; Rainey et al., 2004; Rodriguez et al., 1997; Samandari et al., 2007). Recently, we compared both cell lines directly and found that H295R cells produce more androgens than H295A cells and differ in 3␤HSDII and 17,20-lyase activity (Samandari et al., 2007). We now performed binding experiments on both cell lines using 125 I-labeled PYY. H295R cells, but not H295A cells were found to specifically express NPY receptors (Fig. 1A). Pharmacological displacement experiments showed that the NPY receptors in H295R cells displayed a high affinity for the Y1selective ligands [Leu31 ,Pro34 ]-PYY and BIBP 3226, but low or no affinity for the Y2-selective ligand BIIE 0246, Y4-preferring PP, or the Y5-selective ligand [cPP(1–17) ,NPY(19–23) ,Ala31 ,Aib32 ,Pro34 ]hPP (Fig. 1B). This provides pharmacological evidence of specific expression of Y1 receptors, but not Y2, Y4, or Y5 receptors in H295R cells (Fig. 1A and B). Of note, this is in accordance with the exclusive Y1 receptor expression found in original human adrenal cortical tumor tissues (Korner et al., 2004a). Performing RT-PCR analysis on RNA extracted from both cell lines, we found that H295R cells expressed the Y1 receptor at higher levels than H295A cells (Fig. 1D). Quantitative PCR analysis confirmed this result; H295A cells were found to express significantly less Y1 receptors compared to H295R cells (Fig. 1E). Thus H295R and H295A cells differed in Y1 expression as well as ligand binding markedly. Expression of the NPY peptide was not found in either H295R or H295A cell lines (Fig. 1D). By contrast, expression of both Y1 receptor and NPY peptide was found by RT-PCR from total RNA of normal human adrenals consisting of cortex and medulla (Fig. 1D). This is in agreement with the reported NPY expression in the adrenal medulla of various mammals (Li et al., 1999). Autoradiography studies of normal human adrenal tissue sections revealed that NPY receptors were highly expressed in the cortex in all samples (Table 2). NPY receptors were homogeneously distributed throughout the zona glomerulosa, zona fasciculata, and zona reticularis (Fig. 1F). In the medulla, only a weak and focal NPY receptor expression was discerned in 50% of the cases (Table 2). The

Fig. 2. NPY activates the MAPK pathway in H295R cells. Cells were treated with NPY for various timings and activation of ERK and its kinase MEK was assessed by Western blot using phospho-specific antibodies. (A) Representative Western blot showing phosphorylation of MEK and ERK after 5–30 min stimulation with 100 nM NPY. To prove specificity of NPY on ERK phosphorylation, cells were pre-treated with 25 ␮M PD98059, an inhibitor of MEK, for 30 min prior to addition of NPY. (B) Quantification of effect of NPY treatment on P-ERK/ERK and P-MEK/ERK; mean ± SEM of two independent experiments. *Unspecific band.

NPY receptors in both adrenal cortex and medulla corresponded exclusively to Y1 receptors, based on pharmacological competition experiments (Fig. 1C). 3.2. NPY modulates Y1R dependent MAPK signaling in H295R cells NPY receptors belong to the G protein-coupled receptors (GPCRs). Stimulation of such receptors activates or represses primarily the cAMP pathway; however cross-talking to other signaling pathways is well known. The activation of Y1 receptors is generally associated with a reduction of cAMP accumulation, an increase of intracellular free calcium, and a modulation of the mitogenactivated protein kinase (MAPK) pathway via several signaling molecules, including protein kinase C (PKC) (Crespo et al., 1994; Gudermann et al., 2000; Mannon and Mele, 2000; Pedrazzini, 2004; Pedrazzini et al., 2003). The involvement of MAPK signaling in the modulation of cell proliferation by NPY via Y1 receptors has been described for several cell systems including prostate cancer cells (Ruscica et al., 2006). Furthermore, human adrenal steroidogenesis is regulated by cAMP-dependent, protein kinase A (PKA) and MAPK-PKC mediated pathways (Stocco et al., 2005). Therefore, in order to analyse the functional effect of NPY on specific Y1 receptors in H295R cells, cells were treated with NPY for various durations and stimulation of the MAPK pathway was analysed by assessing the phosphorylation status of MEK and ERK kinases by Western blot (Fig. 2). Short time treatment with NPY increased phosphorylation of both kinases significantly (Fig. 2A and B). Strongest activation was found after treatment for 5–15 min. As in previous work, we found that only one of the two ERK isoforms, ERK2/p42, was phosphorylated in H295R cells (Kempna et al., 2007). To confirm specificity of this effect, we treated our cells with the MEK inhibitor PD98059 before stimulation with NPY. Addition of the inhibitor completely abolished NPY induced phosphorylation of ERK, but did not affect phosphorylation of MEK (Fig. 2A). By contrast, phosphorylation of both kinases remained unchanged after long-term treatment (48 h) with NPY (data not shown).

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Fig. 3. Effect of NPY on proliferation of H295R cells. Cells were split into 96-well plates at a density of 15,000 cells/well and cultured in starvation medium in the presence or absence of different concentrations of NPY up to 96 h. Cell proliferation and viability were measured colorimetrically as described in Section 2. The amount of viable cells corresponds to OD 490 nm. Results are given as mean ± SD of three independent experiments.

3.3. Effect of NPY on proliferation of NCI-H295R cells NPY is reported to either stimulate or inhibit cell proliferation in various cell lines (Korner and Reubi, 2007). In previous studies, we found that NPY receptors are highly expressed in adrenal cortical tumors, pheochromocytomas, paragangliomas, neuroblastomas, glioblastomas, Ewing sarcomas, breast cancers, as well as in all layers of the normal human adrenal cortex (Korner and Reubi, 2008; Korner et al., 2004a, 2008). However no correlation was found between the expression of NPY receptors and the type of carcinoma, with respect to proliferative activity (Korner et al., 2004a). To test whether NPY regulates the proliferation of adrenal H295R cells, we measured the amount of viable cells under standard growth conditions with and without repetitive NPY treatment every 24 h for up to 96 h. We used a commercially available proliferation assay for which the absorbance at 490 nm corresponds directly to the number of viable and metabolically active cells. Treatment of H295R cells with 10 or 100 nM NPY for up to 96 h did not change their proliferation (Fig. 3). 3.4. Effect of NPY on steroidogenesis in NCI-H295R cells To assess the effect of NPY on steroid hormone production of H295R cells, cells were treated with 100 nM NPY or PYY for 24 or 48 h. PYY is closely related to NPY and shows a similar specific affinity to the NPY receptors. Steroid hormone production was assessed by labeling steroidogenesis of cells with [3 H] pregnenolone, which is the precursor of all steroid hormones, followed by steroid extraction from medium and thin layer chromatogra-

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phy (TLC). This method allows identification of different steroid hormones at the same time. Both NPY and PYY inhibited the production of 11-deoxycortisol (11-DOC), an intermediate product of cortisol synthesis, in H295R cells by about 30%, while other steroid hormone metabolites were not changed significantly (Fig. 4). Because this result suggested that NPY might inhibit cortisol production, we measured concentrations of cortisol in the cell culture medium after treatment with NPY or PYY by a specific immunoassay. We found that both peptides inhibited cortisol production (Fig. 5A). To assess the specificity of the effect of NPY on cortisol production, cells were then treated with increasing concentrations of NPY in the presence or absence of the specific Y1 receptor antagonist BIBP2336 in 100-fold excess. NPY dose-dependently and significantly inhibited cortisol production. Presence of BIBP2336 blocked the effect of NPY, confirming that NPY inhibits cortisol production through Y1 receptors specifically (Fig. 5B). Adrenocorticotropic hormone (ACTH) is the strongest stimulator for cortisol production in adrenocortical cells. However, H295R cells express the ACTH receptor only weakly and its activation does not result in any detectable effect on steroidogenesis (Samandari et al., 2007), but it has been shown to stimulate cell proliferation (Janes et al., 2008). Thus, to assess whether NPY inhibits cortisol production also in stimulated H295R cells, the effect of NPY on cortisol production was measured in cells treated with forskolin. Forskolin is a known activator of the adenylate cyclase and increases intracellular cAMP levels similar to GPCRs such as the ACTH receptor. Similar to basal conditions, NPY treatment inhibited cortisol production of forskolin stimulated H295R cells by about 15–20%, while co-treatment with BIBP2336 clearly blocked this effect of NPY on forskolin-induced cortisol production (Fig. 5C).

3.5. Effect of NPY on testosterone, DHEA and aldosterone production in H295R cells Treatment of H295R cells with both NPY and PYY inhibited the production of testosterone slightly, however this effect did not reach statistical significance (Fig. 6A). However, treatment with 100 nM NPY decreased DHEA production of H295R cells significantly by about 20% (Fig. 6B). Under basal conditions our assay is not able to detect aldosterone in cell culture medium of H295R cells (Samandari et al., 2007). Therefore, to measure aldosterone, cells were stimulated by AngII. Accordingly, we assessed the effect of NPY on AngII stimulated aldosterone production. Repetitive treatment with AngII for up to 72 h increased aldosterone concentrations in the cell medium to detectable levels, however co-treatment with NPY did not alter aldosterone levels compared to AngII treatment alone (Fig. 6C).

Fig. 4. Effect of NPY and PYY on steroidogenesis in H295R cells. Cells were treated with 100 nM NPY or PYY for 48 h. Steroid production was labeled by incubating cells with [3 H] pregnenolone for 90 min prior to steroid extraction and separation by thin layer chromatography (TLC). (A) Representative TLC plate visualized by phosphoimaging. (B) Quantification of three independent experiments as described in Section 2, mean ± SD (*p < 0.01). 4A: androstenedione, Preg: pregnenolone, DHEA: dehydroepiandrosterone, 17OHProg: 17␣-hydroxyprogesterone, 17OHPreg: 17␣-hydroxypregnenolone, 11DOC: 11-deoxycortisol.

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Fig. 5. Effect of NPY and PYY on cortisol production in H295R cells. (A) Cells were treated with 100 nM NPY or PYY for 6–48 h and cortisol production was measured in the cell culture medium by RIA. (B) H295R cells were treated with 1–100 nM NPY in the presence or absence of 100-fold excess of BIBP2336, a specific antagonist of Y1R. Cortisol production was measured by RIA 48 h after these treatments. (C) H295R cells were treated with 100 nM NPY in presence or absence of 10 ␮M BIBP2336 for 72 h and cortisol production was stimulated by treatment with 10 ␮M forskolin (FSK) during the last 24 h. All data shown in A–C represent the mean ± SD of 4–5 independent experiments. a: significance tested against untreated controls, *p < 0.01, **p < 0.05.

3.6. Effect of NPY on gene expression of steroidogenic enzymes Steroidogenesis may be regulated acutely (within minutes to hours) and/or chronically. Long-term treatment with small peptides such as NPY may cause a chronic response that involves changes in the expression of steroidogenic genes. Therefore, we analysed in H295R cells the expression of genes encoding steroidogenic enzymes after treatment with NPY for 48 h using quantitative Real Time PCR (Fig. 7). Chronic NPY treatment resulted in a marked decrease in the expression of the HSD3B2 gene (Fig. 7A) and the CYP21A2 gene (Fig. 7B). By contrast, chronic NPY treatment did not affect the expression of the CYP17 (Fig. 7C) and the SULT2A1 gene (Fig. 7D). Activity of 3␤HSDII (encoded by the HSD3B2 gene) is crucial for mineralocorticoid, glucocorticoid and sex steroid production. Thus inhibition of this enzyme by NPY may explain the observed decrease in cortisol and testosterone production in part. In addition, cortisol synthesis requires 21-hydroxylase activity (encoded by CYP21A2), which, when inhibited, will decrease corti-

Fig. 6. Effect of NPY on testosterone, DHEA and aldosterone production. H295R cells were treated with 10–100 nM NPY or 100 nM PYY for 24–72 h. (A) Testosterone concentrations were measured by RIA in the culture medium after 24 and 48 h. (B) DHEA concentrations were measured by RIA after 48 h. (C) Similarly, AngII stimulated aldosterone production was measured 72 h after repetitive treatment with 10 and 100 nM NPY. Data represent the mean ± SD of 4 independent experiments.

sol production further. Although aldosterone synthesis requires the activities of both 3␤HSDII and 21-hydroxylase, NPY treatment did not change AngII stimulated aldosterone secretion in H295R cells. As we were only able to detect aldosterone in H295R cell culture medium after AngII stimulation and as AngII is reported to stimulate gene expression of CYP21A2 and HSD3B2 (Ye et al., 2009), we tested the hypothesis that the inhibitory effect of NPY on aldosterone might have been covered by the stimulatory effect of AngII. For that, we performed quantitative RT-PCR looking at HSD3B2 and CYP21A2 gene expression levels in H295R cells after NPY and AngII treatment. We found that AngII stimulated the expression of both genes markedly but co-treatment with NPY did not attenuate this effect (data not shown). 4. Discussion In previous studies, we have shown that NPY receptors are highly expressed in both human adrenal tumors and normal adrenal tissues suggesting a functional role for NPY targeting these receptors in the adrenals (Korner et al., 2004a). The present study

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Fig. 7. NPY modulates the expression of specific steroidogenic genes. H295R cells were treated with 10 or 100 nM NPY for 48 h. Total RNA was isolated and gene expression was analysed using Real Time PCR. (A and C) Expression of HSD3B2 and CYP17A1 was assessed by Taqman Real Time PCR. (B and D). Expression of CYP21A2 and SULT2A1 were assessed by SYBR Green Real Time PCR. 18S rRNA served as internal control for all. Note that NPY inhibited HSD3B2 and CYP21A2 expression. Data represent the mean ± SD of three independent experiments. *p < 0.01, **p < 0.05.

indicates that human adrenocortical H295R cells express Y1 receptors. Assessing receptor signaling, cell proliferation and steroid hormone production, we found that NPY activates Y1 receptor signaling and modulates steroidogenesis of H295R cells specifically via Y1; by contrast, we found no effect of NPY on cell proliferation. NPY receptors belong to the GPCRs which typically involve adenylate cyclase for their signaling. Downstream signaling can involve activation of the MAPK pathway which can result in alterations of cell proliferation as shown for some prostate cancer cell lines (Ruscica et al., 2006). In addition, the MAPK pathway is implicated in the regulation of a broad range of cellular processes in endocrine cells including steroid hormone biosynthesis (Stocco et al., 2005). So far, little has been reported on Y1 receptor signaling on human adrenal cells. We show that NPY is able to enhance ERK1/2 phosphorylation in H295R cells. Thus, H295R cells do not only express Y1 receptors which specifically bind NPY peptide, but these receptors are truly functional. Our studies show that NPY inhibits both basal and forskolin stimulated cortisol production of adrenal H295R cells within 24 h by 45% and 20%, respectively. NPY also inhibited basal DHEA production by 20%. Similarly, testosterone production seemed slightly decreased whereas AngII stimulated aldosterone production remained unchanged. To date the direct effect of NPY on adrenal steroid hormone production has been mainly investigated in rats (Spinazzi et al., 2005). In contrast to our findings in human adrenal cells, both in vivo and in vitro studies in the rat revealed no effect on corticosterone production except for one study that suggested an inhibitory effect of NPY on corticosterone secretion in cultured rat adrenocortical cells (Malendowicz et al., 1990). Likewise, NPY is reported to increase plasma aldosterone concentrations in rats in vivo in a dose-dependent manner, however in vitro studies reveal controversial results (Spinazzi et al., 2005). NPY was first found to inhibit aldosterone production in dispersed mouse zona glomerulosa cells (Neri et al., 1990, 1991). Subsequent studies did not confirm this anti-secretagogue action of NPY for mineralocorticoids, but suggested that NPY might modulate the secretory response of the zona glomerulosa cells to their main stimulators, namely AngII and ACTH (Hinson et al., 1995; Hinson and Kapas, 1995), or even exert its action indirectly via catecholamines

(Renshaw et al., 2000). Overall these effects were only observed at high concentrations of NPY and were therefore thought unlikely to be of any physiological relevance (Nussdorfer and Gottardo, 1998). Thus, the absence of any change in aldosterone secretion in H295R cells upon combined NPY/AngII stimulation within the picomolar concentration range, adds another inconclusive result to the published literature. Whether it is the complexity of the NPY action on aldosterone production or the different in vitro and in vivo models used, remains unknown. In our opinion, the H295R cell model may not qualify best for studying the effect of NPY on aldosterone production for the following reasons: First, basal aldosterone levels in H295R cell cultures are undetectable. Second, stimulation with AngII – although making aldosterone measurable – rises levels only to pM compared to nM when measuring cortisol; and assessment of an inhibition is extremely difficult when dealing with small amounts. Third, AngII has been shown to enhance CYP21A2 and HSD3B2 gene expression (Bird et al., 1998; Ye et al., 2009). We show in this study that NPY represses basal CYP21A2 and HSD3B2 expression but is not able to attenuate the effect of AngII stimulation. Thus stimulation with AngII may cover a much smaller inhibitory effect of NPY, possibly explaining why we did not find an alteration in aldosterone concentrations. Finally, the in vitro system might not reflect the in vivo system and H295R tumor cells may not reflect normal adrenal zona glomerulosa cells. The inhibitory effect of NPY on DHEA production of H295R cells remains unexplained. Compared to the inhibitory effect of NPY on cortisol, the effect on DHEA is weaker. The fact that CYP21A2 and HSD3B2 expressions are decreased by NPY would rather suggest an increase in DHEA. A decrease in DHEA could result from an inhibition of CYP17A1 activity or an increase in SULT2A1 activity. However, in our studies we only analysed expression of these genes. Thus we cannot exclude an effect of NPY on the proteins of these genes at the posttranslational level. For that CYP17A1 would be a good candidate as the 17,20 lyase activity of this enzyme which is essential for DHEA production, is regulated through co-factors (P450 oxidoreductase, cytochrome b5) and phosphorylation (Tee et al., 2008). As reported studies on the effect of NPY on adrenal steroidogenesis are almost exclusively performed in rodents, and as the adrenal cortex of rodents does not produce androgens, our

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study is the first to report an effect of NPY on DHEA production. However, the mechanism behind this effect awaits further exploration. Human adrenal cortical tumors are either hormonally active or inactive. If they are hormonally active, they produce large amounts of either mineralocorticoids or glucocorticoids or adrenal androgens, or a combination of these steroid hormones resulting in Conn’s or Cushing’s syndrome. H295R cells originate from a human adrenocortical carcinoma that predominantly produced androgens and glucocorticoids (Gazdar et al., 1990). Unexpectedly, NPY does not stimulate these secretory activities of H295R cells, but inhibits them, particularly cortisol production and secretion. Indeed, our findings suggest that NPY rather counteracts secretagogue functions mediated by other receptors in these tumors. Aberrant expression of GPCRs has been observed in the adrenals of some patients presenting with ACTH-independent Cushing’s syndrome (Lacroix et al., 2004; Mazzuco et al., 2007). These receptors were regulated by various peptides including gastric inhibitory polypeptide, catecholamines, vasopressin, serotonin and luteinizing hormone/human chorionic gonadotropin. Characteristically, aberrant GPCRs drive excessive cortisol production uncontrolled by the physiological feedback loop of the HPA axis (Lacroix et al., 2004). Our results indicate that Y1 receptors do not play such a stimulatory role in adrenal tumorigenesis. NPY has been associated with adrenal steroidogenesis for long. So far, it was mainly thought to exert its effect on the adrenal cortex indirectly via modulating the HPA axis centrally or via second messengers (e.g. leptin) originating from the adipose tissue (Roberge et al., 2007); in addition, a local indirect effect of NPY via the release of catecholamines from postganglionic sympathetic fibers or medullary chromaffin cells in the adrenals was implicated (Spinazzi et al., 2005). However, to date there has been some debate in the literature whether NPY acts directly on adrenocortical cells (Whitworth et al., 2003). In the present study, we now show several lines of evidence that NPY is able to directly target adrenocortical cells: First, NPY binds highly specifically to Y1 receptors that are abundantly expressed in H295R cells and in all layers of the normal human adrenal cortex. Second, NPY is able to activate the MAPK pathway which is a typical downstream signaling pathway of Y1 receptor—adenylate cyclase and can modulate steroidogenesis. Third, NPY stimulation of H295R cells leads to a decrease in cortisol production and altered gene expression of specific steroidogenic enzymes involved in cortisol biosynthesis specifically via Y1 receptors. Therefore, we believe that beside ACTH, NPY is a regulator of adrenal steroidogenesis, at least in human adrenal H295R tumor cells. However, as NPY receptors were also found in normal adrenal cortex tissues and as detailed binding studies revealed high specificity of these receptors for normal human tissues similar to H295R cells, we suggest that NPY also plays a functional role in normal adrenal steroidogenesis. Unfortunately, to our knowledge so far no laboratory was able to establish primary cultures of normal adrenal cortex cells to confirm study results obtained from H295 cells. In contrast to H295R cells, we found that H295A cells which originate from same adrenocortical tumor, did not express NPY receptors and differ in other characteristics; but this might be due to different selection and culture conditions over time (see this study and (Samandari et al., 2007)). Only recently, another human adrenocortical carcinoma HAC15 cell line has been characterized (Parmar et al., 2008). HAC15 cells may be used for future studies as they are responsive to AngII and ACTH and, therefore, resemble more the normal human adrenal cortex. Presently, the exact interplay between NPY and ACTH as well as the nature of the feedback of cortisol on peripheral and central NPY production remain elusive. From our data, we suggest that the inhibitory NPY–Y1 receptor signaling system is a counterbalance to the stimulatory ACTH–MC2R signaling on steroid hormone

biosynthesis of the normal adrenal cortex and thus plays a role in acute stress response. But further studies are needed to confirm this hypothesis. Modulation of cell proliferation by NPY is reported for several cancers (Kitlinska et al., 2005; Korner and Reubi, 2007). It can be stimulatory or inhibitory, even within the same cancer type (Ruscica et al., 2006). This appears, however, not to be the case for adrenal corticocarcinoma H295R cells. Although these cells express the Y1 receptor abundantly, specific binding of NPY to these receptors is not able to modulate cell growth. High expression of Y1 and Y2 receptors has been described for several endocrine and non-endocrine tumors such as glioblastomas, Ewing sarcomas, breast carcinomas, ovarian cancers, and adrenal gland and related tumors (Korner and Reubi, 2007, 2008; Korner et al., 2008). It is generally known that activation of various peptide receptors by specific ligands can affect tumor cell proliferation, angiogenesis and hormone release (Reubi, 2003). These peptide receptors may also be of potential use for diagnostic and therapeutic tumor management. Somatostatin analogues, for instance, are successfully used for diagnostic imaging studies of gut neuroendocrine tumors, as well as for therapeutic inhibition of hormone release and growth of some hormonally active tumors (Korner and Reubi, 2007; Reubi, 2003). However, prerequisite for such in vivo targeting is not only a high peptide hormone receptor expression in the tumor, but also a low background expression in normal tissues (Korner and Reubi, 2007; Reubi, 2003). In addition to high expression in tumors, our study showed high Y1 receptor expression in all layers of the normal human adrenal cortex and moderate expression in blood vessels and in some samples of the adrenal medulla. Thus high Y1 receptor expression is not specific for adrenal tumors suggesting that NPY analogues may not be ideal candidates for targeting tumors specifically. Overall, our study shows that NPY peptide is a direct, negative regulator of adrenal cortisol biosynthesis in H295R cells. So far, ACTH is known as the main regulator of adrenal cortisol production. The relationship between the two peptides in modulating adrenal steroidogenesis is unknown. Further studies are needed to find out whether NPY and related peptides may be of potential therapeutic use as specific inhibitors of adrenal cortisol production. Acknowledgments We thank Prof. Walter L. Miller, University of California San Francisco for providing the NCI-H295A cells for this study. We also thank Dr. Jean Rivier, The Salk Institute, La Jolla, CA, for the ligand [cPP(1–17) ,NPY(19–23) ,Ala31 ,Aib32 ,Pro34 ]hPP, and Böhringer Ingelheim (Biberach, Germany) for the ligand BIIE 0246. This work was supported by the Swiss National Science Foundation grant 320000-116299 to CEF. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2009.08.010. References Auchus, R.J., Lee, T.C., Miller, W.L., 1998. Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer. J. Biol. Chem. 273, 3158–3165. Balasubramaniam, A., 2002. Clinical potentials of neuropeptide Y family of hormones. Am. J. Surg. 183, 430–434. Bird, I., Mason, J., Rainey, W.E., 1998. Protein kinase A, protein kinase C and Ca2+ regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells. J. Clin. Endocrinol. Metab. 83, 1592–1597. Crespo, P., Xu, N., Simonds, W.F., Gutkind, J.S., 1994. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature 369, 418–420.

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