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Melanocortin-3 receptor activates MAP kinase via PI3 kinase
Regulatory Peptides 139 (2007) 115 – 121 www.elsevier.com/locate/regpep
Melanocortin-3 receptor activates MAP kinase via PI3 kinase Biaoxin Chai, Ji-Yao Li, Weizhen Zhang, John B. Ammori, Michael W. Mulholland ⁎ Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA Received 22 September 2006; received in revised form 6 November 2006; accepted 7 November 2006 Available online 22 December 2006
Abstract HEK 293 cells stably expressing human melanocortin-3 receptor (MC3R) were exposed to melanocortin receptor agonist, NDP-MSH (10− 10– 10 M). ERK1/2 was phosphorylated in a dose-dependent manner with an EC50 of 3.3 ± 1.5 × 10− 9 M, similar to the IC50 of NDP-MSH binding to the MC3R. ERK1/2 phosphorylation was blocked by the melanocortin receptor antagonists SHU9119. NDP-MSH-induced ERK1/2 phosphorylation was sensitive to pertussis toxin and the PI3K inhibitor, wortmannin. Rp-cAMPS, BAPTA-AM and Myr-PKC did not inhibit the NDP-MSH-induced ERK1/2 phosphorylation. NDP-MSH stimulated cellular proliferation in a dose-dependent manner with a similar EC50 to ERK1/2 phosphorylation, 2.1 ± 0.6 × 10− 9 M. Cellular proliferation was blocked by AGRP (86–132) and by the MEK inhibitor, PD98059. The NDP-MSH did not inhibit serum deprivation-induced apoptosis. MC3R activation induces ERK1/2 phosphorylation via PI3K and this pathway is involved in cellular proliferation in HEK cells expressing MC3R. © 2006 Elsevier B.V. All rights reserved. −6
Keywords: Melanocortin-3 receptor; NDP-MSH; Mitogen-activated protein kinase
1. Introduction The melanocortin-3 receptor (MC3R) is a member of a family of seven transmembrane receptors, consisting of five subtypes, MC1-MC5R [1]. Both the MC3R and MC4R have pivotal roles in central control of energy homeostasis. In the brain, MC3R is expressed in the hypothalamus, thalamus, septum, hippocampus, olfactory cortex, and amygdala [2]. Hypothalamic pro-opiomelanocortin (POMC) neurons, crucial in regulation of nutrient intake, express MC3R [3]. The role of MC3R in control of food intake remains unsettled. MC3R-deficient mice demonstrate increased adiposity, reduced lean mass, and a higher ratio of weight gain to food intake [4]. A recent study has shown that peripheral injection of a selective melanocortin receptor agonist stimulates feeding, acting via MC3R to reduce POMC neuronal Abbreviations: α-MSH, α-melanocyte stimulating hormone; NDP-MSH, [Nle4, D-Phe7]-α-melanocyte stimulating hormone; MAPK, mitogen-activatedprotein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; GPCR, Gprotein coupled receptor; MCR, melanocortin receptor; SHU9119, Ac-Nle-c [Asp-His-D-Nal-Arg-Trp-Lys]-NH2. ⁎ Corresponding author. 2101 Taubman Center, 1500 E. Medical Center Dr. Ann Arbor, MI 48109-0346, USA. Tel.: +1 734 936 3236; fax: +1 734 763 5625. E-mail address: [email protected] (M.W. Mulholland). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.003
activity while stimulating the activity of AgRP/NPY neurons [5]. Investigators have recently reported hyperphagia in MC3R knockout mice [6], coupled with reduced muscle oxidative metabolism [7]. In contrast, Marsh et al. have reported a modest stimulatory effect of AgRP, an antagonist of melanocortin receptors, in MC4R knockout animals [8]. In addition to the central nervous system, MC3R is also expressed in peripheral tissues. MC3R expression has been localized to placenta, gut [9], heart [10] and monocytes [11]. The diverse localization of MC3R suggests activities beyond energy homeostasis, and a variety of biological functions have been reported. MC3R activation mediates the protective influences of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias in rats [12]. In mouse models, activation of MC3R exhibits anti-inflammatory effects, and the receptor has been suggested as a potential therapeutic target for inflammatory conditions [13,14]. The conventional understanding of MC3R activity involves both adenylyl cyclase and inositol phospholipid–Ca2+-mediated signaling systems [15,16]. Recent studies have demonstrated that MC4R activation may also induce MAP kinase activation in vitro and in vivo. In vitro, melanocortin peptides activated MAPK in COS-1 cells, in CHO-K1 cells transfected with MC4R [17,18]
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and in GT1-1 cells with endogenous MC4R receptor expression [19]. In vivo, central administration of the melanocortin agonist MTII increased the number of phospho-MAPK-immunoreactive cells in the paraventricular nucleus of the rat [17]. In rats, an extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in the solitary nucleus has been reported to mediate melanocortin suppression of food intake [20,21]. We have previously reported that MAP kinase activation by MC4R is involved in anti-apoptotic processes in GT1-1 cells [19]. The current study sought to investigate the role of MAP kinase in MC3R signaling and the effects of that pathway on cellular proliferation. 2. Materials and methods 2.1. Chemicals and antibodies NDP-MSH (a long-acting potent α-MSH analogue, [Nle4 , D-Phe 7 ] α-MSH) and SHU9119 (synthetic melanocortin antagonist) were purchased from Bachem (King of Prussia, PA). The ERK1/2-specific inhibitor, PD98059, was purchased from Calbiochem (San Diego, CA). Myr-PKC (Myr-PKC 19– 27), wortmannin, Rp-adenosine 3′,5′-cyclic monophorothioate triethylammonium salt (Rp-cAMPS), pertussis toxin (PTX), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-phospho-p44/42 antibody and anti-P44/42 antibody were obtained from Cell Signaling Technologies (Beverly, MA). Secondary anti-IgG HRP antibody was purchased from Santa Cruz Biotechnology. 2.2. Cell culture HEK 293 cells, stably transfected with the coding region of the human MC3R gene expressed in pcDNA3.1 (Invitrogen, Carlsbad, CA), were used for this study. This cell line has previously been characterized [22,23]. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) with 100 U/ml penicillin and 100 U/ml streptomycin. Cells were plated on 100 mm dishes and maintained at 37 °C in a water-saturated atmosphere of 95% O2 and 5% CO2. 2.3. MAP kinase assay Cells were seeded on 6 well plates and cultured in DMEM media supplemented with 10% FBS for 18 h. The media were replaced with serum-free DMEM, incubated for 14–18 h, and then treated with various materials. For studies with inhibitors of signaling pathways, cells were treated with pathway inhibitors or vehicle for 1 h before the addition of NDP-MSH. After treatment, the media were removed and cells were washed twice with ice-cold phosphate buffered saline solution and then lysed with a buffer (9803, Cell Signaling Technologies) that contained phosphatase inhibitors and protease inhibitors supplemented with 1 mM PMSF. Lysates were centrifuged at 16,000 g at 4 °C for 10 min, and supernatants were retained. The
protein content of supernatants was determined using the BioRad protein assay reagent (Richmond, CA). 2.4. Western blotting Protein samples from cell lysates (15–20 μg) were subjected to electrophoresis separation on a 10% polyacrylamide gel (BioRad) and then transblotted onto Immobilon™-P PVDF membrane (Millipore Corp., Bedford, MA). Blots were blocked at room temperature for 1 h in 5% milk in TBS–Tween 20 (0.05%) and then incubated overnight in primary antibody diluted 1/1000. Membranes were washed three times in TBS–Tween (0.05%) and then incubated for 1 h with secondary antibody, diluted 1/5000 in 5% milk TBS–Tween (0.05%). Detection was performed using SuperSignal West Pico chemiluminescent detection reagents (Pierce, Rockford, IL). 2.5. Cell proliferation assay The effect of the NDP-MSH on cell proliferation was investigated using a commercially available proliferation kit (XTT II, Roche Diagnostics, Indianapolis, IN, USA). Briefly, the cells were plated in 96-well culture plates at a density of 5000 cells per well in 0.1 ml 10% FBS DMEM medium and allowed to grow for 24 h. The media were removed and replaced with 0.1% BSA DMEM medium. Cells were preincubated in with vehicle (DMSO) or PD98059 for 1 h, then NDP-MSH was added to various final concentrations. After 24 h of culture, 50 μl of XTT reaction solution ((sodium 3′-(1-phenylaminocarbonyl)-3,4-tetrazolium)-bis (4-metoxy-6-nitro) benzene sulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate; mixed in proportion 50:1) was added to the wells. Optical density was read at 450 nm in an enzyme-linked immunosorbent assay plate reader after 4 h incubation of the plates with XTT. 2.6. Apoptosis detection DNA fragmentation was quantitatively evaluated by Cell Death Detection ELISAPLUS (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer's instructions [19,24]. Cells were plated at a density of 10,000 cells per well in 96-well plates. The following day, cells were treated and incubated at 37 °C for 2 or 4 h, followed by the addition of 150 μl of lysis buffer. Samples were incubated for 30 min at room temperature. Lysates were centrifuged at 200 g for 10 min; 20 μl of the supernatant was removed for assessment of DNA fragmentation. 2.7. Data analysis Experiments were performed at least three times. Data are expressed as mean ± SEM of experiments. Curves were fitted and EC50 were calculated using Graphpad Prism 4.0 (Graphpad Software, San Diego, CA). For Western blots, analysis of densitometry was performed using Kodak 1D 3.6 software (Eastman Kodak, New Haven, CT). Differences were analyzed by unpaired two-tailed Student's t test. The values of p < 0.05 were taken as significant.
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3. Results 3.1. NDP-MSH activates ERK1/2 by MC3R HEK 293 cells expressing human MC3R were treated with 100 nM NDP-MSH for various times and cell lysates were used to detect the phosphorylated form of ERK1/2. Western blotting with a specific ERK1/2 antibody revealed increased phosphorylation of ERK1/2 in response to NDP-MSH, maximal at 5 min, returning to control values by 45 min (Fig. 1A). Both ERK1 and ERK2 were phosphorylated following NDP-MSH treatment, with the amount of phosphorylated ERK2 greater than that of ERK1. To determine if ERK1/2 activation occurred in a doseresponsive manner, cells were treated with increasing concentrations of NDP-MSH, ranging from 10− 10 to 10− 6 M, and phosphorylated ERK1/2 was determined. NDP-MSH activated ERK1/2 phosphorylation in a dose-dependent manner with an EC50 of 3.3 ± 1.5 × 10− 9 M (Fig. 1B). The EC50 of ERK1/2 phosphorylation is similar to the IC50 of NDP-MSH binding to MC3R [25,26]. To examine whether NDP-MSH-induced ERK1/2 activation is mediated by the MC3R receptor, the melanocortin receptor antagonist SHU9119 was used. HEK 293 cells expressing MC3R were pretreated with 10− 7 M or 10− 6 M SHU9119 for 30 min. NDP-MSH-mediated ERK1/2 activity was significantly inhibited by SHU9119 at 10− 7 M and was completely blocked at 10− 6 M (Fig. 1C).
Fig. 1. NDP-MSH induces ERK1/2 phosphorylation in HEK 293 cells expressing human MC3R. (A) Time course of NDP-MSH-stimulated ERK1/2 phosphorylation. HEK 293 cells expressing MC3R were incubated in serumfree medium overnight before treatment with 100 nM NDP-MSH for 0, 2, 5, 10, 20, or 45 min. The cells were lysed and lysates were used for Western blot analysis. (B) Dose response of NDP-MSH stimulated ERK1/2 phosphorylation. Cells were incubated in serum-free medium overnight and treated with various concentrations of NDP-MSH ranging from 10− 10 to 10− 6 M for 5 min. Cell lysates were used for Western blot analysis. Western blots were scanned and the optical density of each band was measured and the band of highest density in a Western blot was defined as 100%. (C) Melanocortin antagonist SHU9119 blocked NDP-MSH-stimulated ERK1/2 phosphorylation. Cells were incubated in serum-free medium overnight and treated with SHU9119 (10− 7 M and 10− 6 M) for 30 min before treatment with 100 nM NDP-MSH for 5 min. Lysates were used for Western blot analysis. Phosphorylated ERK1/2 (p-ERK1/2) was determined using anti-phospho-ERK1/2 antibody and the blots were then stripped and reblotted with anti-ERK1/2 antibody to determine total protein loading.
Fig. 2. Pertussis toxin sensitivity. (A) HEK 293 cells expressing human MC3R were seeded on 6 well plates and incubated overnight, then PTX (50 ng/ml) was added and incubated for 18 h. The cells were treated with 100 nM NDP-MSH for 5 min. The cells were lysed and lysates were used for Western blot analysis using anti-phospho-ERK1/2 antibody. The optical density of each band was measured and the band of highest density in a Western blot was defined as 100%. The blot is typical of an experiment that was replicated three times.
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3.2.2. Involvement of PI3 kinase Vongs et al. have reported that Phosphatidylinositol 3-Kinase (PI3K) is involved in MC4R-induced ERK1/2 activation in CHO-K1 cells expressing human MC4R [18]. The PI3K inhibitor wortmannin inhibited ERK1/2 phosphorylation by 91% at concentrations of 50 nM [28] (Fig. 3). 3.2.3. Independent of PKA pathway The cyclic AMP-protein kinase A (PKA) pathway is a conventional signaling pathway of melanocortin receptors. To determine if the PKA pathway is involved in MC3R-induced ERK1/2 activation, Rp-cAMPS was used to block PKA. RpcAMPS did not affect ERK activation at concentrations up to 100 μM (Fig. 4).
Fig. 3. PI3K pathway is involved NDP-MSH-induced ERK1/2 phosphorylation. HEK 293 cells expressing human MC3R were incubated in serum-free medium overnight and then incubated with PI3K inhibitor wortmannin at concentration of 2, 10 and 50 nM for 1 h. Cells were incubated with 100 nM NDP-MSH for 5 min. The cells were lysed and lysates were used for Western blot analysis using anti-phospho-ERK1/2 antibody. The blots were stripped and reblotted with antiERK1/2 antibody. The blot is typical of an experiment that was replicated three times.
3.2.4. Effect of Ca2+ and PKC in NDP-MSH-induced ERK1/2 phosphorylation It has been reported that MC3R activation may initiate intracellular calcium signaling. To investigate whether the Ca2+protein kinase C (PKC) pathway is involved in MC3R-induced ERK activation in HEK 293 cells expressing MC3R, the cellpermeable calcium chelator, BAPTA-AM, and a cell-permeable PKC specific inhibitor, Myr-PKC, were used. Neither BAPTAAM (10 μM) nor Myr-PKC (10 μM) affected MC3R-induced ERK phosphorylation (Fig. 5). 3.3. MC3R activation stimulates cellular proliferation via MAP kinase pathway
3.2. Signal pathway involved in ERK activation by NDP-MSH 3.2.1. Pertussis toxin sensitivity Melanocortin receptors are seven transmembrane receptors which signal by coupling to G-proteins. Stimulation of ERK activity by G-protein coupled receptors is mediated by Gi protein in some cells [27]. Involvement of Gi in biological processes is implied by sensitivity of responses to pertussis toxin (PTX) pretreatment. PTX catalyses the ADP ribosylation of Gi protein heterotrimers and inhibits Gi signaling function. Pretreatment of the MC3R cells for 18 h with PTX at concentrations of 50 ng/ml blocked ERK1/2 phosphorylation by approximately 75% [19] (Fig. 2).
Fig. 4. Effect of PKA inhibitor Rp-cAMPs on NDP-MSH-induced ERK1/2 phosphorylation. HEK 293 cells expressing human MC3R were incubated in serum-free DMEM overnight and preincubated for 1 h with the PKA inhibitor Rp-cAMPs at concentrations of 50, 100 μM, and then cells were incubated with 100 nM NDP-MSH for 5 min. Cells were lysed and lysates were used for Western blotting analysis by using anti-phospho-ERK1/2 antibody. The blots were then stripped and reblotted with anti-ERK1/2 antibody. The blot is typical of an experiment that was replicated three times.
Because activation of ERK is commonly associated with cell growth, we next determined whether NDP-MSH could stimulate
Fig. 5. Involvement of calcium and PKC in NDP-MSH-induced ERK1/2 phosphorylation. (A) HEK 293 cells expressing human MC3R were incubated in serum-free medium overnight and then preincubated with the intracellular calcium chelator BAPTA-AM at concentrations of 5 and 10 nM for 40 min. The cells were incubated with 100 nM NDP-MSH for 5 min. Cells were lysed and lysates were used for Western blot analysis. (B) Cells were incubated in serum-free medium overnight and then preincubated with the PKC inhibitor, Myr-PKC, at concentrations from 1 to 10 μM for 1 h before being treated with 100 nM NDP-MSH for 5 min. Cell lysates were used for Western blot analysis. Phosphorylated ERK1/2 was immunoblotted by using anti-phospho-ERK1/2 antibody and the blots were then stripped and reblotted with anti-ERK1/2 antibody to determine total protein loading. The blots are typical of an experiment that was replicated three times.
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Fig. 6. NDP-MSH stimulates HEK 293 cell proliferation via MC3R. HEK 293 cells expressing human MC3R were seeded on 96-well plates and cultured with DMEM containing 10% FBS for 18–24 h. Cells were then incubated with NDPMSH ranging from 10− 10 to 10− 6 M in 0.1% BSA media without (■) or with 0.4 μM AGRP (Δ) for 24 h. Cell proliferation was determined using XTT method.
cellular proliferation. HEK cells expressing MC3R were incubated with NDP-MSH at concentrations ranging from 10− 10 to 10− 6 M in 0.1% BSA DMEM media. NDP-MSH stimulated cell proliferation in a dose-dependent manner with an EC50 of 2.1 ± 0.6 × 10− 9 M, similar to the EC50 of NDP-MSH stimulated ERK1/2 activation (Fig. 6). To examine whether NDP-MSH stimulated cellular proliferation is mediated by MC3R, the melanocortin receptor antagonist AGRP (86–132) was used. Cellular proliferation was assayed in the presence of 0.4 μM AGRP (86–132) with a range of NDPMSH concentrations from 10− 10 to 10− 6 M. As showed in Fig. 6, cellular proliferation was shifted to the right with an EC50 of 3.0 ± 0.7 × 10− 8 M, fourteen times higher than control values. Because NDP-MSH activates the ERK1/2 signaling pathway, we further investigated the involvement of ERK1/2 activation in NDP-MSH-induced cellular proliferation. The MEK specific inhibitor PD98059 was used to block the ERK1/2 signal pathway. Cells were incubated with vehicle, NDP-MSH (0.1 μM), or NDP-
Fig. 7. MAP kinase inhibitor PD98059 blocks NDP-MSH-induced cell growth. HEK 293 cells expressing human MC3R were seeded on 96-well plates and cultured with DMEM containing 10% FBS for 18–24 h. Cells were then preincubated in 0.1% BSA DMEM media with vehicle or with PD98059 for 1 h, and then exposed to vehicle or NDP-MSH (0.1 μM) for 24 h. Cell proliferation was determined using XTT method.
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Fig. 8. NDP-MSH has no effect on serum deprivation-induced apoptosis. Cells were seeded on 96-well plates and grown for 28 h (>80% confluency). Cells were incubated with 10% FBS; 0.1% BSA or with 0.1% BSA plus 10− 7 M NDP-MSH. Nuclei-free supernatants were prepared for assay of DNA fragmentation.
MSH plus 1 μM or 5 μM PD98059, respectively. Cellular proliferation was determined using the XTT method. PD98059 1 μM and 5 μM significantly inhibited NDP-MSH-induced cellular proliferation (Fig. 7). 3.4. Effects of NDP-MSH on serum deprivation-induced apoptosis Serum deprivation induced increased apoptosis in HEK 293 cells expressing MC3R as determined by DNA fragmentation. NDP-MSH (0.1 μM) did not inhibit serum deprivation-induced apoptosis (Fig. 8). 4. Discussion The principal finding of the current study is that melanocortin-3 receptor occupation stimulates ERK1/2 activity. When HEK 293 cells expressing MC3R were exposed to NDP-MSH, ERK1/2 was phosphorylated in a dose-dependent manner with an EC50 similar to the IC50 of NDP-MSH binding to the MC3R receptor. ERK1/2 phosphorylation was blocked by the melanocortin receptor antagonists SHU9119, indicating a receptormediated event. MC3R mediated ERK1/2 phosphorylation via the Gi protein–PI3K signaling system. The actions of NDPMSH were pertussis toxin sensitive and were inhibited by the PI3K inhibitor wortmannin. In this study, NDP-MSH stimulated cell proliferation in a dose-responsive manner; the EC50 of NDP-MSH-induced cell growth closely matched that of NDPMSH-induced ERK1/2 phosphorylation. Cellular proliferation could be blocked by a melanocortin receptor antagonist, AGRP (86–132), and by a specific MEK inhibitor. The conventional understanding of melanocortin receptor signaling has centered around cyclic AMP-protein kinase A and alterations in intracellular Ca2+([Ca2+]i). In this study which used HEK 293 cells expressing MC3R, neither PKA nor Ca2+ signaling was required for NDP-MSH-stimulated ERK1/2 phosphorylation. While numerous studies have demonstrated
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an increase in cAMP upon MC3R activation, the role of intracellular Ca2+ as a signaling mechanism is less settled [29]. Mountjoy et al. have reported that HEK 293 cells with mouse MC3R transfection responded to α-MSH or desacetyl α-MSH with increased [Ca2+]i and cAMP [16]. In contrast, Kim et al. have reported that HEK 293 cells transfected with MC3R respond to human agouti protein, an antagonist of melanocortin receptors, with a slow, sustained increase in [Ca2+]i [30]. γ3MSH, a MC3R agonist, evoked oscillatory [Ca2+]i increases in approximately 15% of cultured neonatal rat pituitary cells [31]. The MC3/MC4 receptor antagonist SHU9119 blocked these calcium transients in 50% of the responding cells. Langouche et al. have reported that γ3MSH, α-MSH and NDP-MSH produce oscillating Ca2+ signaling in GH3 cells, but surprisingly, that mRNA for MC3R could not be detected by PCR [32]. Studies performed in vivo support a role for nitric oxide in MC3R signaling. The role of nitric oxide in MC3R signaling has been studied with synthetic peptides with preferential binding to MC3R [33]. In a mouse model of forebrain injury, peptides showing binding activity for MC3R dose-dependently inhibited lipopolysaccharide-induced NO overproduction [33]. Nitric oxide has also been implicated in MC3-mediated reproductive behavior. In rats, reproductive posturing is stimulated by administration of α-MSH into the medial preoptic area of the brain [34]. Both MC3 and MC4 receptors are expressed in this area, but lordosis could not be blocked by a specific MC4 receptor antagonist. The α-MSH-stimulated behavior was abolished by the NO synthase inhibitor L-NAME. The MC3R has not previously been reported to stimulate ERK1/2 phosphorylation. The ability of NDP-MSH to activate ERK1/2 signaling is consistent with prior observations relating to the MC4R. Daniels et al. have reported that the melanocortin agonist MTII activated MAPK phosphorylation in COS-1 cells transfected with MC4R [35]. Vongs et al. also reported that NDP-MSH activated the ERK1/2 in CHO-K1 cells transfected with MC4R [18]. In GT1-1 cells with endogenous MC4R receptor expression, we have previously demonstrated that NDP-MSH exposure increased MAPK phosphorylation [19]. In vivo, central administration of the melanocortin agonist MTII increased the number of phospho-MAPK-immunoreactive cells in the paraventricular nucleus of the rat [35]. It has been reported that phosphorylation of ERK1/2 in the solitary nucleus is required for food intake suppression by exogenous cholecystokinin [20] and that activation of the ERK pathway is required for inhibition of nutrient intake by peripheral CCK or central MT II [21]. ERK signaling cascades play a crucial role in transduction of extracellular signals. These cascades affect proliferation, differentiation and development in a variety of cell types. In the current study, in HEK 293 cells expressing MC3R, NDP-MSHstimulated ERK activation was associated with increased cellular proliferation. Lorsignol et al. have reported that γ3MSH, in nanomolar concentrations, stimulated DNA replication in thyrotrophs and somatotrophs derived from immature rat pituitary [31] with similar effects, though at higher concentrations, for lactotrophs. In cultured pituicytes, γ3MSH increased mitosis but did not affect differentiation. In the current study,
MC3R activation was not shown to affect serum deprivationinduced apoptosis. In contrast, we have previously demonstrated that MC4R activation inhibits apoptosis in GT1-1 cells with endogenous MC4R expression [19]. Potential functional correlates of MC3R activation of ERK1/2 signaling are currently unknown. Hypothalamic pro-opiomelanocortin (POMC) neurons regulate food intake through release of αMSH [36]. While the MC4R has a crucial role in maintaining metabolic homeostasis, the role of the MC3R is less well-defined [6]. MC3R agonists can stimulate feeding via MC3R, even in satiated animals. MC3R-deficient mice demonstrate increased fat mass, hyperleptinemia and hyperinsulinemia [37]. Disordered nutrient intake is exacerbated by a diet high in fat in MC3Rdeficient animals [37]. Expression of MC3R in areas of the brain beyond the hypothalamus and in the periphery suggests other potential actions as well. Melanocortins have been reported to be protective in animal models of ischemic brain injury [38]. Peripheral melanocortin administration reduced biochemical and histological markers of ischemia-induced brain damage. In addition to protective actions, neurotrophic properties have been suggested [38]. MC3 receptors have been reported to be protective in myocardial ischemia/reperfusion-induced arrhythmias [12,39]. Acknowledgement This work was supported by NIH grant DK054032. References [1] Gantz I, Fong TM. The melanocortin system. Am J Physiol Endocrinol Metab 2003;284:E468–74. [2] Roselli-Rehfuss L, Mountjoy KG, Robbins LS, Mortrud MT, Low MJ, Tatro JB, et al. Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system. Proc Natl Acad Sci U S A 1993;90:8856–60. [3] Jegou S, Boutelet I, Vaudry H. Melanocortin-3 receptor mRNA expression in pro-opiomelanocortin neurones of the rat arcuate nucleus. J Neuroendocrinol 2000;12:501–5. [4] Chen AS, Marsh DJ, Trumbauer ME, Frazier EG, Guan XM, Yu H, et al. Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nat Genet 2000;26:97–102. [5] Marks DL, Hruby V, Brookhart G, Cone RD. The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R). Peptides 2006;27:259–64. [6] Butler AA. The melanocortin system and energy balance. Peptides 2006;27:281–90. [7] Sutton GM, Trevaskis JL, Hulver MW, McMillan RP, Markward NJ, Babin MJ, et al. Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors. Endocrinology 2006;147:2183–96. [8] Marsh DJ, Miura GI, Yagaloff KA, Schwartz MW, Barsh GS, Palmiter RD. Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues. Brain Res 1999;848:66–77. [9] Gantz I, Konda Y, Tashiro T, Shimoto Y, Miwa H, Munzert G, et al. Molecular cloning of a novel melanocortin receptor. J Biol Chem 1993;268:8246–50. [10] Chhajlani V. Distribution of cDNA for melanocortin receptor subtypes in human tissues. Biochem Mol Biol Int 1996;38:73–80. [11] Taherzadeh S, Sharma S, Chhajlani V, Gantz I, Rajora N, Demitri MT, et al. alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages. Am J Physiol 1999;276: R1289–94.
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[26] Jackson PJ, McNulty JC, Yang YK, Thompson DA, Chai B, Gantz I, et al. Design, pharmacology, and NMR structure of a minimized cystine knot with agouti-related protein activity. Biochemistry 2002;41:7565–72. [27] Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity. Cell Signal 2001;13:777–85. [28] de Obanos MP, Zabalza MJ, Prieto J, Herraiz MT, Iraburu MJ. Leucine stimulates procollagen alpha1(I) translation on hepatic stellate cells through ERK and PI3K/Akt/mTOR activation. J Cell Physiol 2006;209:580–6. [29] Stanley SA, Davies S, Small CJ, Gardiner JV, Ghatei MA, Smith DM, et al. gamma-MSH increases intracellular cAMP accumulation and GnRH release in vitro and LH release in vivo. FEBS Lett 2003;543:66–70. [30] Kim JH, Kiefer LL, Woychik RP, Wilkison WO, Truesdale A, Ittoop O, et al. Agouti regulation of intracellular calcium: role of melanocortin receptors. Am J Physiol 1997;272:E379–84. [31] Lorsignol A, Vande Vijver V, Ramaekers D, Vankelecom H, Denef C. Detection of melanocortin-3 receptor mRNA in immature rat pituitary: functional relation to gamma3-MSH-induced changes in intracellular Ca2+ concentration? J Neuroendocrinol 1999;11:171–9. [32] Langouche L, Roudbaraki M, Pals K, Denef C. Stimulation of intracellular free calcium in GH3 cells by gamma3-melanocyte-stimulating hormone. Involvement of a novel melanocortin receptor? Endocrinology 2001;142:257–66. [33] Muceniece R, Zvejniece L, Liepinsh E, Kirjanova O, Baumane L, Petrovska R, et al. The MC3 receptor binding affinity of melanocortins correlates with the nitric oxide production inhibition in mice brain inflammation model. Peptides 2006;27:1443–50. [34] Nocetto C, Cragnolini AB, Schioth HB, Scimonelli TN. Evidence that the effect of melanocortins on female sexual behavior in preoptic area is mediated by the MC3 receptor; participation of nitric oxide. Behav Brain Res 2004;153:537–41. [35] Daniels D, Patten CS, Roth JD, Yee DK, Fluharty SJ. Melanocortin receptor signaling through mitogen-activated protein kinase in vitro and in rat hypothalamus. Brain Res 2003;986:1–11. [36] Mounien L, Bizet P, Boutelet I, Vaudry H, Jegou S. Expression of melanocortin MC3 and MC4 receptor mRNAs by neuropeptide Y neurons in the rat arcuate nucleus. Neuroendocrinology 2005;82:164–70. [37] Zhang Y, Kilroy GE, Henagan TM, Prpic-Uhing V, Richards WG, Bannon AW, et al. Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin. FASEB J 2005;19:1482–91. [38] Tatro JB. Melanocortins defend their territory: multifaceted neuroprotection in cerebral ischemia. Endocrinology 2006;147:1122–5. [39] Mioni C, Giuliani D, Cainazzo MM, Leone S, Bazzani C, Grieco P, et al. Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors. Eur J Pharmacol 2003;477:227–34.
Melanocortin-3 receptor activates MAP kinase via PI3 kinase Biaoxin Chai, Ji-Yao Li, Weizhen Zhang, John B. Ammori, Michael W. Mulholland ⁎ Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA Received 22 September 2006; received in revised form 6 November 2006; accepted 7 November 2006 Available online 22 December 2006
Abstract HEK 293 cells stably expressing human melanocortin-3 receptor (MC3R) were exposed to melanocortin receptor agonist, NDP-MSH (10− 10– 10 M). ERK1/2 was phosphorylated in a dose-dependent manner with an EC50 of 3.3 ± 1.5 × 10− 9 M, similar to the IC50 of NDP-MSH binding to the MC3R. ERK1/2 phosphorylation was blocked by the melanocortin receptor antagonists SHU9119. NDP-MSH-induced ERK1/2 phosphorylation was sensitive to pertussis toxin and the PI3K inhibitor, wortmannin. Rp-cAMPS, BAPTA-AM and Myr-PKC did not inhibit the NDP-MSH-induced ERK1/2 phosphorylation. NDP-MSH stimulated cellular proliferation in a dose-dependent manner with a similar EC50 to ERK1/2 phosphorylation, 2.1 ± 0.6 × 10− 9 M. Cellular proliferation was blocked by AGRP (86–132) and by the MEK inhibitor, PD98059. The NDP-MSH did not inhibit serum deprivation-induced apoptosis. MC3R activation induces ERK1/2 phosphorylation via PI3K and this pathway is involved in cellular proliferation in HEK cells expressing MC3R. © 2006 Elsevier B.V. All rights reserved. −6
Keywords: Melanocortin-3 receptor; NDP-MSH; Mitogen-activated protein kinase
1. Introduction The melanocortin-3 receptor (MC3R) is a member of a family of seven transmembrane receptors, consisting of five subtypes, MC1-MC5R [1]. Both the MC3R and MC4R have pivotal roles in central control of energy homeostasis. In the brain, MC3R is expressed in the hypothalamus, thalamus, septum, hippocampus, olfactory cortex, and amygdala [2]. Hypothalamic pro-opiomelanocortin (POMC) neurons, crucial in regulation of nutrient intake, express MC3R [3]. The role of MC3R in control of food intake remains unsettled. MC3R-deficient mice demonstrate increased adiposity, reduced lean mass, and a higher ratio of weight gain to food intake [4]. A recent study has shown that peripheral injection of a selective melanocortin receptor agonist stimulates feeding, acting via MC3R to reduce POMC neuronal Abbreviations: α-MSH, α-melanocyte stimulating hormone; NDP-MSH, [Nle4, D-Phe7]-α-melanocyte stimulating hormone; MAPK, mitogen-activatedprotein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; GPCR, Gprotein coupled receptor; MCR, melanocortin receptor; SHU9119, Ac-Nle-c [Asp-His-D-Nal-Arg-Trp-Lys]-NH2. ⁎ Corresponding author. 2101 Taubman Center, 1500 E. Medical Center Dr. Ann Arbor, MI 48109-0346, USA. Tel.: +1 734 936 3236; fax: +1 734 763 5625. E-mail address: [email protected] (M.W. Mulholland). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.003
activity while stimulating the activity of AgRP/NPY neurons [5]. Investigators have recently reported hyperphagia in MC3R knockout mice [6], coupled with reduced muscle oxidative metabolism [7]. In contrast, Marsh et al. have reported a modest stimulatory effect of AgRP, an antagonist of melanocortin receptors, in MC4R knockout animals [8]. In addition to the central nervous system, MC3R is also expressed in peripheral tissues. MC3R expression has been localized to placenta, gut [9], heart [10] and monocytes [11]. The diverse localization of MC3R suggests activities beyond energy homeostasis, and a variety of biological functions have been reported. MC3R activation mediates the protective influences of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias in rats [12]. In mouse models, activation of MC3R exhibits anti-inflammatory effects, and the receptor has been suggested as a potential therapeutic target for inflammatory conditions [13,14]. The conventional understanding of MC3R activity involves both adenylyl cyclase and inositol phospholipid–Ca2+-mediated signaling systems [15,16]. Recent studies have demonstrated that MC4R activation may also induce MAP kinase activation in vitro and in vivo. In vitro, melanocortin peptides activated MAPK in COS-1 cells, in CHO-K1 cells transfected with MC4R [17,18]
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and in GT1-1 cells with endogenous MC4R receptor expression [19]. In vivo, central administration of the melanocortin agonist MTII increased the number of phospho-MAPK-immunoreactive cells in the paraventricular nucleus of the rat [17]. In rats, an extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in the solitary nucleus has been reported to mediate melanocortin suppression of food intake [20,21]. We have previously reported that MAP kinase activation by MC4R is involved in anti-apoptotic processes in GT1-1 cells [19]. The current study sought to investigate the role of MAP kinase in MC3R signaling and the effects of that pathway on cellular proliferation. 2. Materials and methods 2.1. Chemicals and antibodies NDP-MSH (a long-acting potent α-MSH analogue, [Nle4 , D-Phe 7 ] α-MSH) and SHU9119 (synthetic melanocortin antagonist) were purchased from Bachem (King of Prussia, PA). The ERK1/2-specific inhibitor, PD98059, was purchased from Calbiochem (San Diego, CA). Myr-PKC (Myr-PKC 19– 27), wortmannin, Rp-adenosine 3′,5′-cyclic monophorothioate triethylammonium salt (Rp-cAMPS), pertussis toxin (PTX), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-phospho-p44/42 antibody and anti-P44/42 antibody were obtained from Cell Signaling Technologies (Beverly, MA). Secondary anti-IgG HRP antibody was purchased from Santa Cruz Biotechnology. 2.2. Cell culture HEK 293 cells, stably transfected with the coding region of the human MC3R gene expressed in pcDNA3.1 (Invitrogen, Carlsbad, CA), were used for this study. This cell line has previously been characterized [22,23]. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) with 100 U/ml penicillin and 100 U/ml streptomycin. Cells were plated on 100 mm dishes and maintained at 37 °C in a water-saturated atmosphere of 95% O2 and 5% CO2. 2.3. MAP kinase assay Cells were seeded on 6 well plates and cultured in DMEM media supplemented with 10% FBS for 18 h. The media were replaced with serum-free DMEM, incubated for 14–18 h, and then treated with various materials. For studies with inhibitors of signaling pathways, cells were treated with pathway inhibitors or vehicle for 1 h before the addition of NDP-MSH. After treatment, the media were removed and cells were washed twice with ice-cold phosphate buffered saline solution and then lysed with a buffer (9803, Cell Signaling Technologies) that contained phosphatase inhibitors and protease inhibitors supplemented with 1 mM PMSF. Lysates were centrifuged at 16,000 g at 4 °C for 10 min, and supernatants were retained. The
protein content of supernatants was determined using the BioRad protein assay reagent (Richmond, CA). 2.4. Western blotting Protein samples from cell lysates (15–20 μg) were subjected to electrophoresis separation on a 10% polyacrylamide gel (BioRad) and then transblotted onto Immobilon™-P PVDF membrane (Millipore Corp., Bedford, MA). Blots were blocked at room temperature for 1 h in 5% milk in TBS–Tween 20 (0.05%) and then incubated overnight in primary antibody diluted 1/1000. Membranes were washed three times in TBS–Tween (0.05%) and then incubated for 1 h with secondary antibody, diluted 1/5000 in 5% milk TBS–Tween (0.05%). Detection was performed using SuperSignal West Pico chemiluminescent detection reagents (Pierce, Rockford, IL). 2.5. Cell proliferation assay The effect of the NDP-MSH on cell proliferation was investigated using a commercially available proliferation kit (XTT II, Roche Diagnostics, Indianapolis, IN, USA). Briefly, the cells were plated in 96-well culture plates at a density of 5000 cells per well in 0.1 ml 10% FBS DMEM medium and allowed to grow for 24 h. The media were removed and replaced with 0.1% BSA DMEM medium. Cells were preincubated in with vehicle (DMSO) or PD98059 for 1 h, then NDP-MSH was added to various final concentrations. After 24 h of culture, 50 μl of XTT reaction solution ((sodium 3′-(1-phenylaminocarbonyl)-3,4-tetrazolium)-bis (4-metoxy-6-nitro) benzene sulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate; mixed in proportion 50:1) was added to the wells. Optical density was read at 450 nm in an enzyme-linked immunosorbent assay plate reader after 4 h incubation of the plates with XTT. 2.6. Apoptosis detection DNA fragmentation was quantitatively evaluated by Cell Death Detection ELISAPLUS (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer's instructions [19,24]. Cells were plated at a density of 10,000 cells per well in 96-well plates. The following day, cells were treated and incubated at 37 °C for 2 or 4 h, followed by the addition of 150 μl of lysis buffer. Samples were incubated for 30 min at room temperature. Lysates were centrifuged at 200 g for 10 min; 20 μl of the supernatant was removed for assessment of DNA fragmentation. 2.7. Data analysis Experiments were performed at least three times. Data are expressed as mean ± SEM of experiments. Curves were fitted and EC50 were calculated using Graphpad Prism 4.0 (Graphpad Software, San Diego, CA). For Western blots, analysis of densitometry was performed using Kodak 1D 3.6 software (Eastman Kodak, New Haven, CT). Differences were analyzed by unpaired two-tailed Student's t test. The values of p < 0.05 were taken as significant.
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3. Results 3.1. NDP-MSH activates ERK1/2 by MC3R HEK 293 cells expressing human MC3R were treated with 100 nM NDP-MSH for various times and cell lysates were used to detect the phosphorylated form of ERK1/2. Western blotting with a specific ERK1/2 antibody revealed increased phosphorylation of ERK1/2 in response to NDP-MSH, maximal at 5 min, returning to control values by 45 min (Fig. 1A). Both ERK1 and ERK2 were phosphorylated following NDP-MSH treatment, with the amount of phosphorylated ERK2 greater than that of ERK1. To determine if ERK1/2 activation occurred in a doseresponsive manner, cells were treated with increasing concentrations of NDP-MSH, ranging from 10− 10 to 10− 6 M, and phosphorylated ERK1/2 was determined. NDP-MSH activated ERK1/2 phosphorylation in a dose-dependent manner with an EC50 of 3.3 ± 1.5 × 10− 9 M (Fig. 1B). The EC50 of ERK1/2 phosphorylation is similar to the IC50 of NDP-MSH binding to MC3R [25,26]. To examine whether NDP-MSH-induced ERK1/2 activation is mediated by the MC3R receptor, the melanocortin receptor antagonist SHU9119 was used. HEK 293 cells expressing MC3R were pretreated with 10− 7 M or 10− 6 M SHU9119 for 30 min. NDP-MSH-mediated ERK1/2 activity was significantly inhibited by SHU9119 at 10− 7 M and was completely blocked at 10− 6 M (Fig. 1C).
Fig. 1. NDP-MSH induces ERK1/2 phosphorylation in HEK 293 cells expressing human MC3R. (A) Time course of NDP-MSH-stimulated ERK1/2 phosphorylation. HEK 293 cells expressing MC3R were incubated in serumfree medium overnight before treatment with 100 nM NDP-MSH for 0, 2, 5, 10, 20, or 45 min. The cells were lysed and lysates were used for Western blot analysis. (B) Dose response of NDP-MSH stimulated ERK1/2 phosphorylation. Cells were incubated in serum-free medium overnight and treated with various concentrations of NDP-MSH ranging from 10− 10 to 10− 6 M for 5 min. Cell lysates were used for Western blot analysis. Western blots were scanned and the optical density of each band was measured and the band of highest density in a Western blot was defined as 100%. (C) Melanocortin antagonist SHU9119 blocked NDP-MSH-stimulated ERK1/2 phosphorylation. Cells were incubated in serum-free medium overnight and treated with SHU9119 (10− 7 M and 10− 6 M) for 30 min before treatment with 100 nM NDP-MSH for 5 min. Lysates were used for Western blot analysis. Phosphorylated ERK1/2 (p-ERK1/2) was determined using anti-phospho-ERK1/2 antibody and the blots were then stripped and reblotted with anti-ERK1/2 antibody to determine total protein loading.
Fig. 2. Pertussis toxin sensitivity. (A) HEK 293 cells expressing human MC3R were seeded on 6 well plates and incubated overnight, then PTX (50 ng/ml) was added and incubated for 18 h. The cells were treated with 100 nM NDP-MSH for 5 min. The cells were lysed and lysates were used for Western blot analysis using anti-phospho-ERK1/2 antibody. The optical density of each band was measured and the band of highest density in a Western blot was defined as 100%. The blot is typical of an experiment that was replicated three times.
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3.2.2. Involvement of PI3 kinase Vongs et al. have reported that Phosphatidylinositol 3-Kinase (PI3K) is involved in MC4R-induced ERK1/2 activation in CHO-K1 cells expressing human MC4R [18]. The PI3K inhibitor wortmannin inhibited ERK1/2 phosphorylation by 91% at concentrations of 50 nM [28] (Fig. 3). 3.2.3. Independent of PKA pathway The cyclic AMP-protein kinase A (PKA) pathway is a conventional signaling pathway of melanocortin receptors. To determine if the PKA pathway is involved in MC3R-induced ERK1/2 activation, Rp-cAMPS was used to block PKA. RpcAMPS did not affect ERK activation at concentrations up to 100 μM (Fig. 4).
Fig. 3. PI3K pathway is involved NDP-MSH-induced ERK1/2 phosphorylation. HEK 293 cells expressing human MC3R were incubated in serum-free medium overnight and then incubated with PI3K inhibitor wortmannin at concentration of 2, 10 and 50 nM for 1 h. Cells were incubated with 100 nM NDP-MSH for 5 min. The cells were lysed and lysates were used for Western blot analysis using anti-phospho-ERK1/2 antibody. The blots were stripped and reblotted with antiERK1/2 antibody. The blot is typical of an experiment that was replicated three times.
3.2.4. Effect of Ca2+ and PKC in NDP-MSH-induced ERK1/2 phosphorylation It has been reported that MC3R activation may initiate intracellular calcium signaling. To investigate whether the Ca2+protein kinase C (PKC) pathway is involved in MC3R-induced ERK activation in HEK 293 cells expressing MC3R, the cellpermeable calcium chelator, BAPTA-AM, and a cell-permeable PKC specific inhibitor, Myr-PKC, were used. Neither BAPTAAM (10 μM) nor Myr-PKC (10 μM) affected MC3R-induced ERK phosphorylation (Fig. 5). 3.3. MC3R activation stimulates cellular proliferation via MAP kinase pathway
3.2. Signal pathway involved in ERK activation by NDP-MSH 3.2.1. Pertussis toxin sensitivity Melanocortin receptors are seven transmembrane receptors which signal by coupling to G-proteins. Stimulation of ERK activity by G-protein coupled receptors is mediated by Gi protein in some cells [27]. Involvement of Gi in biological processes is implied by sensitivity of responses to pertussis toxin (PTX) pretreatment. PTX catalyses the ADP ribosylation of Gi protein heterotrimers and inhibits Gi signaling function. Pretreatment of the MC3R cells for 18 h with PTX at concentrations of 50 ng/ml blocked ERK1/2 phosphorylation by approximately 75% [19] (Fig. 2).
Fig. 4. Effect of PKA inhibitor Rp-cAMPs on NDP-MSH-induced ERK1/2 phosphorylation. HEK 293 cells expressing human MC3R were incubated in serum-free DMEM overnight and preincubated for 1 h with the PKA inhibitor Rp-cAMPs at concentrations of 50, 100 μM, and then cells were incubated with 100 nM NDP-MSH for 5 min. Cells were lysed and lysates were used for Western blotting analysis by using anti-phospho-ERK1/2 antibody. The blots were then stripped and reblotted with anti-ERK1/2 antibody. The blot is typical of an experiment that was replicated three times.
Because activation of ERK is commonly associated with cell growth, we next determined whether NDP-MSH could stimulate
Fig. 5. Involvement of calcium and PKC in NDP-MSH-induced ERK1/2 phosphorylation. (A) HEK 293 cells expressing human MC3R were incubated in serum-free medium overnight and then preincubated with the intracellular calcium chelator BAPTA-AM at concentrations of 5 and 10 nM for 40 min. The cells were incubated with 100 nM NDP-MSH for 5 min. Cells were lysed and lysates were used for Western blot analysis. (B) Cells were incubated in serum-free medium overnight and then preincubated with the PKC inhibitor, Myr-PKC, at concentrations from 1 to 10 μM for 1 h before being treated with 100 nM NDP-MSH for 5 min. Cell lysates were used for Western blot analysis. Phosphorylated ERK1/2 was immunoblotted by using anti-phospho-ERK1/2 antibody and the blots were then stripped and reblotted with anti-ERK1/2 antibody to determine total protein loading. The blots are typical of an experiment that was replicated three times.
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Fig. 6. NDP-MSH stimulates HEK 293 cell proliferation via MC3R. HEK 293 cells expressing human MC3R were seeded on 96-well plates and cultured with DMEM containing 10% FBS for 18–24 h. Cells were then incubated with NDPMSH ranging from 10− 10 to 10− 6 M in 0.1% BSA media without (■) or with 0.4 μM AGRP (Δ) for 24 h. Cell proliferation was determined using XTT method.
cellular proliferation. HEK cells expressing MC3R were incubated with NDP-MSH at concentrations ranging from 10− 10 to 10− 6 M in 0.1% BSA DMEM media. NDP-MSH stimulated cell proliferation in a dose-dependent manner with an EC50 of 2.1 ± 0.6 × 10− 9 M, similar to the EC50 of NDP-MSH stimulated ERK1/2 activation (Fig. 6). To examine whether NDP-MSH stimulated cellular proliferation is mediated by MC3R, the melanocortin receptor antagonist AGRP (86–132) was used. Cellular proliferation was assayed in the presence of 0.4 μM AGRP (86–132) with a range of NDPMSH concentrations from 10− 10 to 10− 6 M. As showed in Fig. 6, cellular proliferation was shifted to the right with an EC50 of 3.0 ± 0.7 × 10− 8 M, fourteen times higher than control values. Because NDP-MSH activates the ERK1/2 signaling pathway, we further investigated the involvement of ERK1/2 activation in NDP-MSH-induced cellular proliferation. The MEK specific inhibitor PD98059 was used to block the ERK1/2 signal pathway. Cells were incubated with vehicle, NDP-MSH (0.1 μM), or NDP-
Fig. 7. MAP kinase inhibitor PD98059 blocks NDP-MSH-induced cell growth. HEK 293 cells expressing human MC3R were seeded on 96-well plates and cultured with DMEM containing 10% FBS for 18–24 h. Cells were then preincubated in 0.1% BSA DMEM media with vehicle or with PD98059 for 1 h, and then exposed to vehicle or NDP-MSH (0.1 μM) for 24 h. Cell proliferation was determined using XTT method.
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Fig. 8. NDP-MSH has no effect on serum deprivation-induced apoptosis. Cells were seeded on 96-well plates and grown for 28 h (>80% confluency). Cells were incubated with 10% FBS; 0.1% BSA or with 0.1% BSA plus 10− 7 M NDP-MSH. Nuclei-free supernatants were prepared for assay of DNA fragmentation.
MSH plus 1 μM or 5 μM PD98059, respectively. Cellular proliferation was determined using the XTT method. PD98059 1 μM and 5 μM significantly inhibited NDP-MSH-induced cellular proliferation (Fig. 7). 3.4. Effects of NDP-MSH on serum deprivation-induced apoptosis Serum deprivation induced increased apoptosis in HEK 293 cells expressing MC3R as determined by DNA fragmentation. NDP-MSH (0.1 μM) did not inhibit serum deprivation-induced apoptosis (Fig. 8). 4. Discussion The principal finding of the current study is that melanocortin-3 receptor occupation stimulates ERK1/2 activity. When HEK 293 cells expressing MC3R were exposed to NDP-MSH, ERK1/2 was phosphorylated in a dose-dependent manner with an EC50 similar to the IC50 of NDP-MSH binding to the MC3R receptor. ERK1/2 phosphorylation was blocked by the melanocortin receptor antagonists SHU9119, indicating a receptormediated event. MC3R mediated ERK1/2 phosphorylation via the Gi protein–PI3K signaling system. The actions of NDPMSH were pertussis toxin sensitive and were inhibited by the PI3K inhibitor wortmannin. In this study, NDP-MSH stimulated cell proliferation in a dose-responsive manner; the EC50 of NDP-MSH-induced cell growth closely matched that of NDPMSH-induced ERK1/2 phosphorylation. Cellular proliferation could be blocked by a melanocortin receptor antagonist, AGRP (86–132), and by a specific MEK inhibitor. The conventional understanding of melanocortin receptor signaling has centered around cyclic AMP-protein kinase A and alterations in intracellular Ca2+([Ca2+]i). In this study which used HEK 293 cells expressing MC3R, neither PKA nor Ca2+ signaling was required for NDP-MSH-stimulated ERK1/2 phosphorylation. While numerous studies have demonstrated
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an increase in cAMP upon MC3R activation, the role of intracellular Ca2+ as a signaling mechanism is less settled [29]. Mountjoy et al. have reported that HEK 293 cells with mouse MC3R transfection responded to α-MSH or desacetyl α-MSH with increased [Ca2+]i and cAMP [16]. In contrast, Kim et al. have reported that HEK 293 cells transfected with MC3R respond to human agouti protein, an antagonist of melanocortin receptors, with a slow, sustained increase in [Ca2+]i [30]. γ3MSH, a MC3R agonist, evoked oscillatory [Ca2+]i increases in approximately 15% of cultured neonatal rat pituitary cells [31]. The MC3/MC4 receptor antagonist SHU9119 blocked these calcium transients in 50% of the responding cells. Langouche et al. have reported that γ3MSH, α-MSH and NDP-MSH produce oscillating Ca2+ signaling in GH3 cells, but surprisingly, that mRNA for MC3R could not be detected by PCR [32]. Studies performed in vivo support a role for nitric oxide in MC3R signaling. The role of nitric oxide in MC3R signaling has been studied with synthetic peptides with preferential binding to MC3R [33]. In a mouse model of forebrain injury, peptides showing binding activity for MC3R dose-dependently inhibited lipopolysaccharide-induced NO overproduction [33]. Nitric oxide has also been implicated in MC3-mediated reproductive behavior. In rats, reproductive posturing is stimulated by administration of α-MSH into the medial preoptic area of the brain [34]. Both MC3 and MC4 receptors are expressed in this area, but lordosis could not be blocked by a specific MC4 receptor antagonist. The α-MSH-stimulated behavior was abolished by the NO synthase inhibitor L-NAME. The MC3R has not previously been reported to stimulate ERK1/2 phosphorylation. The ability of NDP-MSH to activate ERK1/2 signaling is consistent with prior observations relating to the MC4R. Daniels et al. have reported that the melanocortin agonist MTII activated MAPK phosphorylation in COS-1 cells transfected with MC4R [35]. Vongs et al. also reported that NDP-MSH activated the ERK1/2 in CHO-K1 cells transfected with MC4R [18]. In GT1-1 cells with endogenous MC4R receptor expression, we have previously demonstrated that NDP-MSH exposure increased MAPK phosphorylation [19]. In vivo, central administration of the melanocortin agonist MTII increased the number of phospho-MAPK-immunoreactive cells in the paraventricular nucleus of the rat [35]. It has been reported that phosphorylation of ERK1/2 in the solitary nucleus is required for food intake suppression by exogenous cholecystokinin [20] and that activation of the ERK pathway is required for inhibition of nutrient intake by peripheral CCK or central MT II [21]. ERK signaling cascades play a crucial role in transduction of extracellular signals. These cascades affect proliferation, differentiation and development in a variety of cell types. In the current study, in HEK 293 cells expressing MC3R, NDP-MSHstimulated ERK activation was associated with increased cellular proliferation. Lorsignol et al. have reported that γ3MSH, in nanomolar concentrations, stimulated DNA replication in thyrotrophs and somatotrophs derived from immature rat pituitary [31] with similar effects, though at higher concentrations, for lactotrophs. In cultured pituicytes, γ3MSH increased mitosis but did not affect differentiation. In the current study,
MC3R activation was not shown to affect serum deprivationinduced apoptosis. In contrast, we have previously demonstrated that MC4R activation inhibits apoptosis in GT1-1 cells with endogenous MC4R expression [19]. Potential functional correlates of MC3R activation of ERK1/2 signaling are currently unknown. Hypothalamic pro-opiomelanocortin (POMC) neurons regulate food intake through release of αMSH [36]. While the MC4R has a crucial role in maintaining metabolic homeostasis, the role of the MC3R is less well-defined [6]. MC3R agonists can stimulate feeding via MC3R, even in satiated animals. MC3R-deficient mice demonstrate increased fat mass, hyperleptinemia and hyperinsulinemia [37]. Disordered nutrient intake is exacerbated by a diet high in fat in MC3Rdeficient animals [37]. Expression of MC3R in areas of the brain beyond the hypothalamus and in the periphery suggests other potential actions as well. Melanocortins have been reported to be protective in animal models of ischemic brain injury [38]. Peripheral melanocortin administration reduced biochemical and histological markers of ischemia-induced brain damage. In addition to protective actions, neurotrophic properties have been suggested [38]. MC3 receptors have been reported to be protective in myocardial ischemia/reperfusion-induced arrhythmias [12,39]. Acknowledgement This work was supported by NIH grant DK054032. References [1] Gantz I, Fong TM. The melanocortin system. Am J Physiol Endocrinol Metab 2003;284:E468–74. [2] Roselli-Rehfuss L, Mountjoy KG, Robbins LS, Mortrud MT, Low MJ, Tatro JB, et al. Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system. Proc Natl Acad Sci U S A 1993;90:8856–60. [3] Jegou S, Boutelet I, Vaudry H. Melanocortin-3 receptor mRNA expression in pro-opiomelanocortin neurones of the rat arcuate nucleus. J Neuroendocrinol 2000;12:501–5. [4] Chen AS, Marsh DJ, Trumbauer ME, Frazier EG, Guan XM, Yu H, et al. Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nat Genet 2000;26:97–102. [5] Marks DL, Hruby V, Brookhart G, Cone RD. The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R). Peptides 2006;27:259–64. [6] Butler AA. The melanocortin system and energy balance. Peptides 2006;27:281–90. [7] Sutton GM, Trevaskis JL, Hulver MW, McMillan RP, Markward NJ, Babin MJ, et al. Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors. Endocrinology 2006;147:2183–96. [8] Marsh DJ, Miura GI, Yagaloff KA, Schwartz MW, Barsh GS, Palmiter RD. Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues. Brain Res 1999;848:66–77. [9] Gantz I, Konda Y, Tashiro T, Shimoto Y, Miwa H, Munzert G, et al. Molecular cloning of a novel melanocortin receptor. J Biol Chem 1993;268:8246–50. [10] Chhajlani V. Distribution of cDNA for melanocortin receptor subtypes in human tissues. Biochem Mol Biol Int 1996;38:73–80. [11] Taherzadeh S, Sharma S, Chhajlani V, Gantz I, Rajora N, Demitri MT, et al. alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages. Am J Physiol 1999;276: R1289–94.
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