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Activation of MAP kinase by MC4-R through PI3 kinase
Regulatory Peptides 120 (2004) 113 – 118 www.elsevier.com/locate/regpep
Activation of MAP kinase by MC4-R through PI3 kinase Aurawan Vongs, Nicole M. Lynn, Charles I. Rosenblum * Department of Metabolic Research-Obesity, Merck Research Laboratories, Merck and Co., P.O. Box 2000, RY80M-213 Rahway, NJ 07065, USA Received 7 January 2004; received in revised form 18 February 2004; accepted 25 February 2004 Available online 2 April 2004
Abstract The melanocortin 4 receptor (MC4-R) is a Gas-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP-a-MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate. D 2004 Elsevier B.V. All rights reserved. Keywords: MC4-R; Melanocortin; p42/p44 MAPK; ERK1; ERK2; PI3K
1. Introduction 1.1. Melanocortin 4 receptor (MC4-R) introduction The MC4-R is a member of a five-member family of Gprotein-coupled receptors. Each of these receptors is modulated by one or more melanocortin peptides. Melanocortin peptides are produced by proteolytic processing of proopiomelanocortin (POMC) (reviewed in Refs. [1,2]). MC4R is widely expressed throughout the central nervous system though the highest levels expression are observed in the hypothalamus, including the paraventricular nucleus, and in the dorsal motor nucleus of the brainstem [3], implying a role in the regulation of feeding behavior. Indeed, MC4-R null mice become obese [4,5] as do humans with inactive MC4-R or diminished MC4-R function [6 –8]. Recent studies determined that 4% of severe childhood obesity is due to mutations in MC4-R [9,10]. Both null mice and humans expressing MC4-R variants show increased linear growth. While decreased MC4-R activity increases food intake, stimulation of MC4-R has the opposite effect. Treatment of normal rodents with MC4-R selective compounds lowers feed intake [11].
MC4-R is a Gas-coupled receptor, and as such, upon agonist binding, activates adenylyl cyclase; the subsequent increase in cAMP activating protein kinase A (PKA) [12 – 14]. Mountjoy et al. [15] showed that MC4-R stimulation causes cholera toxin-sensitive increases in cytosolic calcium due to release of intracellular calcium stores by an undefined mechanism. Other GPCRs are known to activate multiple intracellular signaling pathways (reviewed by Gutkind [16] and Liebmann [17]). Here, we have examined if MC4-R modulated pathways other than Gas stimulation of adenylyl cyclase. Corroborating the recent findings of Daniels et al. [18], we observed increased phosphorylation and enzymatic activity of the mitogen-activated kinases (MAPK) p42 and p44 following treatment with melanocortin agonist NDP-aMSH. Furthermore, this activation was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors but not PKA inhibitors, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.
2. Materials and methods 2.1. Chemicals
* Corresponding author. Tel.: +1-732-594-5958; fax: +1-732-5943337. E-mail address: [email protected] (C.I. Rosenblum). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.02.018
NDP-a-MSH and SHU-9119 were purchased from Bachem (King of Prussia, PA). PMSF, Tris – HCl, Earle’s
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A. Vongs et al. / Regulatory Peptides 120 (2004) 113–118
buffered salt solution and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin was purchased from American Bioanalytical (Natick, MA). PVDF membranes were purchased from Schleicher and Schuell (Keene, NH). Wortmannin and Rp-cAMPS were purchased from Sigma-Aldrich. Sp-cAMPS was purchased from Biolog (Bremen, Germany). HEPES was purchased from Invitrogen (Carlsbad, CA). Anti-phosphorylated MAP kinase antibody (9106), LY294002 and an in vitro MAP kinase phosphorylation assay were purchased from Cell Signaling Technologies (Beverly, MA). Anti-p42/p44 MAP kinase antibody (06-182) was purchased from Upstate Biotechnology (Lake Placid, NY). A cAMP SPA assay and ECL plus chemiluminescent protein detection reagent were purchased from Amersham Biosciences (Piscataway, NJ).
jC. After thorough washing the blot was incubated with anti-mouse-HRP conjugate (1:5000) for 1 h at room temperature. Following thorough washing, the bound antibody was detected using ECL plus reagent (Amersham Bioscience) and exposed to Bio-Max film (Eastman Kodak, Rochester, NY). Antibodies were then stripped using the Amersham ECL plus protocol. Following blocking in TBST, 5% non-fat dried milk, it was incubated overnight in antiMAP kinase antibody, 1:5000 at 4 jC. After thorough washing, the blot was incubated with anti-rabbit – HRP conjugate (1:3000) for 1 h at room temperature. Following thorough washing, the bound antibody was detected using ECL plus reagent and exposed to Bio-Max film.
2.2. Cell culture
Growth medium was removed from confluent cultures of CHO-hMC4-R or CHO-K1 cells. Cells were placed in assay solution (EBSS, 0.1% BSA, 25 mM HEPES, 5 mM MgCl2) and treated with various concentrations of NDP-a-MSH at 37jC for the length of time indicated in the Results. For studies with inhibitors of signaling pathways, cells were transferred into assay solution and treated with pathway inhibitors or vehicle for 30 min before addition of 100 nM NDP-a-MSH for 10 min. Cells were washed with ice-cold phosphate buffered saline solution and lysed with a cell lysis buffer (no. 9803, Cell Signaling Technologies) that contains phosphatase inhibitors and protease inhibitors and that was supplemented with 1 mM PMSF. Lysates were passed through a 25-gauge needle three times and centrifuged at 16,000 g at 4 jC for 15 min, and supernatants were retained. The protein concentration of lysates were determined using the Bio-Rad protein assay (Richmond, CA).
In vitro MAP kinase enzymatic activity was determined using a kit in which an Elk-1 fusion protein serves as substrate for activated MAP kinase. However, the protocol was modified from the manufacture’s to create a moderate throughput protein dot-blot assay rather than a western blot assay. 200 Ag of cell lysate was immunoprecipitated with 7 Al of anti-phospho p42/p44 MAP kinase antibody-conjugated agarose beads for 16 h at 4 jC. This material was then loaded into a Handee spin cup column (Pierce Biotechnology, Rockford, IL) and was centrifuged at 300 g for 10 s to remove supernatant. The agarose conjugate was washed once with 400 Al of cell lysis buffer and two times with 400 Al kinase reaction buffer (25 mM Tris (pH 7.5), 5 mM hglycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). Fifty microliters of kinase reaction buffer, 200 AM ATP and 2 Ag GST – Elk-1 fusion protein were added to the agarose conjugate and were incubated at 30 jC for 30 min. The reaction was terminated by centrifugation at 15,000 g for 30 s and incubation at 100 jC for 4 min. Twenty microliters of reaction product was spotted onto PVDF membrane (Schleicher and Schuell) using a 96-well vacuum manifold (Schleicher and Schuell). Immunochemistry was used to detected the phosphorylated Elk-1 using reagents and following the protocol provided with the assay kit. The amount of phosphorylation was quantitated using an Eastman Kodak Image Station 440C (Rochester, NY). Statistical analyses were performed using GraphPad Prism for Windows version 4.0 (San Diego, CA).
2.4. Western blot analysis
2.6. cAMP assays
Twenty micrograms of cell lysate was prepared for SDS –PAGE using Laemmli sample buffer (Bio-Rad) and proteins were separated on a 10% polyacrylamide gel (Invitrogen). The proteins were electrophoretically transferred onto PVDF membrane (Invitrogen) and the blot was blocked in 50 mM Tris –HCl pH 8, 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T) and 5% non-fat dried milk. The blot was incubated with anti-phospho-p42/p44 MAP kinase antibody (1:2000) in TBS-T, 5% BSA overnight at 4
CHO-MC4R cells were treated as described in the Results. cAMP accumulation was determined using an RIA [19].
The creation and characterization of CHO-K1 cells expressing human MC4-R (CHO-hMC4-R) was previously described [19]. Cells were cultured in Iscove’s modified Dulbecco medium supplemented with 10% fetal bovine serum, 1 mg/ml G418, (Invitrogen) and 100 AM sodium hypoxanthine and 160 AM thymidine. 2.3. Cell treatment
2.5. In vitro MAP kinase assays
3. Results To assess whether stimulation of the MC4-R receptor modulates p42/p44 MAPK, CHO-hMC4-R cells were
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treated with the MC4-R agonist NDP-a-MSH for 10 min. p42 (ERK2) and p44 (ERK1) MAPK phosphorylation was assessed by western blot (Fig. 1A). Both p42 and p44 were found to be phosphorylated following NDP-aMSH treatment. Though the absolute amount of phosphorylated p42 was greater than that of p44, the relative increases in phosphorylated p42 and p44 were found to be equal as measured by image quantitation of the blot itself. We used an in vitro substrate phosphorylation assay to measure enzymatically activated p42/p44 MAPK. In this assay, phosphorylated p42/p44 MAPK is immunoprecipitated and phosphorylation of Elk-1, a substrate of p42/p44, is determined by immunoblot analysis. We modified a commercial kit to create a moderate throughput assay utilizing spin columns as reaction vessels and dot blotting, eliminating PAGE and a protein transfer step. Utilizing a dot-blot manifold permitted spotting of up to 96 samples per membrane. In order to determine if p42/p44 MAPK activation was occurring via MC4-R activation, wild-type CHO-K1 cells and CHO-hMC4-R cells were treated with NDP-a-MSH. p42/p44 MAPK activation, as measured by Elk-1 phosphorylation, did not occur in the CHO-K1 cells lacking hMC4-R (Fig. 1B). In another experiment, pre-treatment of the CHOhMC4-R cells with SHU-9119, a potent MC4-R antagonist, inhibited NDP-a-MSH-mediated p42/p44 MAPK activity (Fig. 1C). Together, these observations conclusively demonstrate that stimulation of MC4-R causes phosphorylation and activation of p42 and p44. 3.1. Time course of activation We studied p42/p44 MAPK activation by treating hMC4-R CHO cells with 100 nM NDP-a-MSH for various periods of time. Substantial phosphorylation of the Elk-1 substrate was observed by 5 min of stimulation. Activity was maximal by 10 min of treatment (Fig. 2). Interestingly,
Fig. 1. (A) NDP-a-MSH treatment causes phosphorylation of p42 and p44 in CHO-K1 cells expressing human MC4-R. Cells were treated with 0, 100 nM or 1 AM NDP-a-MSH for 10 min. Phosphorylated p42/p44 MAPK was determined by western blot analysis using anti-phospho-p42/p44 MAPK. Blots were stripped and blotted with anti-p42/p44 MAPK to determine total p42/p44 MAPK. (B) p42/p44 MAPK activation is mediated through MC4R. Wild-type and CHO-hMC4-R were treated with 100 nM NDP-a-MSH for 10 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Open bars: wild-type CHO-K1 cells; solid bars: CHO-hMC4-R. Results represent the mean F S.E.M. of three independent measurements. An asterisk indicates a statistically significant difference from unmarked bars ( p < 0.01) by one-way ANOVA. (C) p42/p44 MAPK activation is blocked by inhibitors of MC4-R. Prior to treatment with 100 nM NDP-a-MSH for 10 min, CHO-hMC4-R were incubated 5 AM SHU9119 for 30 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent measurements. An asterisk indicates a statistically significant difference from unmarked bars ( p < 0.05) by one-way ANOVA.
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though activity declined, at 45 min, Elk-1 phosphorylation remained threefold the basal level. In subsequent experiments, cells were treated with agonist for 10 min. 3.2. Dose response To determine if p42/p44 MAPK activation occurred in a dose responsive manner, cells were treated with increasing amounts of NDP-a-MSH (Fig. 3). The EC50 of NDP-aMSH was determined to be approximately 1 nM, very similar to that observed for hMC4-R stimulated cAMP production (Fig. 4B).
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Fig. 2. Time course of NDP-a-MSH-induced p42/p44 MAPK activity. CHOhMC4-R cells were treated with 100 nM NDP-a-MSH for the indicated period of time. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent measurements.
3.3. Signaling pathway Some Gas-coupled GPCRs have been shown to activate p42/p44 MAPK via Gas through PKA, while others couple via Ghg activation of PI3K. PI3K, in turn, modulates a Src-mediated activation of Ras [17]. Therefore, cells were pre-treated with compounds that inhibit either PKA or PI3K. Cells were pre-incubated with Rp-cAMPS, a PKA antagonist, or the PI3K inhibitors wortmannin and LY294002. Pre-treatment of cells with 100 AM Rp-cAMPS did not inhibit NDP-a-MSH stimulated Elk-1 phosphorylation (Fig. 4A), demonstrating that p42/p44 MAPK activation is not a PKA-dependent event. Treatment of cells with the cAMP analog Sp-cAMPS (100 AM) did stimulate
Fig. 3. NDP-a-MSH treatment of CHO-hMC4-R cells causes dosedependent stimulation of p42/p44 MAPK activity. Cells were treated with various concentrations of NDP-a-MSH for 10 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent measurements.
Fig. 4. (A) Inhibitors of PI3K but not PKA block p42/p44 MAPK activity. Thirty minutes prior to treatment with NDP-a-MSH, CHO-hMC4-R cells were incubated with vehicle, 100 AM Rp-cAMPS, 50 nM wortmannin or 25 AM LY294002. Cells were then treated with assay solution with or without 100 nM NDP-a-MSH for 10 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent experiments. (B) PI3K inhibitors do not alter MC4-R mediated cAMP accumulation. Thirty minutes prior to assay, CHO-hMC4-R cells were incubated with vehicle, 100 AM RpcAMPS, 50 nM wortmannin or 25 AM LY294002. Cells were then treated with various amounts of NDP-a-MSH for 45 min and cAMP was measured by SPA assay. Results represent the mean F S.E.M. of three independent measurements. An asterisk indicates a statistically significant difference from unmarked bars ( p < 0.01) by one-way ANOVA.
p42/p44 MAPK indicating that PKA activation of p42/p44 MAPK is functional in CHO-K1 cells (not shown). Pretreatment of cells with either 50 nM wortmannin or 25 AM LY294002 inhibited p42/p44 MAPK activity, demonstrating that MC4-R-mediated p42/p44 MAPK activation is mediated through PI3K. It is of interest to note that the amount of Elk-1 phosphorylation appears to be diminished to below basal levels by treatment with the PI3K inhibitors. This is likely due to inhibition of constitutive MC4-R activity [20, 21]. In order to be certain that these concen-
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trations PI3K inhibitors were not altering Gas-mediated adenylyl cyclase activity, indirectly altering p42/p44 MAPK activity, cells were assayed for cAMP accumulation following MC4-R agonist stimulation following pre-treatment with the PI3K antagonists. No alteration in the efficacy or potency of NDP-a-MSH was observed following PI3K inhibitor pre-treatment (Fig. 4B).
4. Discussion We demonstrate that human MC4-R, stably expressed in CHO-K1 cells, activates p42/p44 MAPK through a PI3Kmediated pathway. We did this by modifying a commercial assay that measures p42/p44 MAPK enzymatic activity using western blotting and converted it to moderate throughput method. p42/p44 MAPK was activated by NDP-a-MSH in a dose-dependent manner, causing equal fold increases in phosphorylated p42 and p44. p42/p44 MAPK activation was maximal by 10 min, but did not rapidly desensitize, remaining threefold basal levels 45 min after stimulation. The activation of p42/p44 MAPK is a PI3K-dependent event as shown by lack of activation by NDP-a-MSH following pre-treatment with LY294002 and wortmannin. p42/p44 MAPK activation is not altered by PKA inhibition caused by Rp-cAMPS. Previous studies of GPCR-regulated p42/p44 MAPK activation implicate either Ga or Ghg [17]. It would seem reasonable to speculate that Ghg activate PI3K in the heterologous cell-based system we employed, though further studies are required to unambiguously establish this point. Since the experiments were performed in CHO cells, due caution must be taken when extrapolating these results to neurons expressing endogenous MC4-R though it is worth noting that stimulation of MC4-R does activity p42/p44 MAPK in the paraventricular nucleus of the hypothalamus [18]. Mountjoy et al. [15] showed that agonist treatment of exogenously expressed melanocortin receptors, including MC4-R, caused cholera toxin-sensitive increases in intracellular calcium. However, this calcium flux was not modulated by increased IP3 concentrations, implying that this is a separate phenomenon from the PI3K-mediated mechanism described here. Konda et al. [22] found that MC3-R activation caused increased in IP3 accumulation and, in turn, increased intracellular calcium concentrations when the receptor was expressed in Hepa, L-cells and CHO cells. p42/p44 MAPK activity was not examined in those experiments. Niswender et al. [23,24] demonstrated that leptin’s and insulin’s anorectic action can be ameliorated the by ICV injection of wortmannin or LY294002 prior to ICV leptin, implicating a role for PI3K in the anoretic actions of leptin and insulin. Administration of these PI3K inhibitors did not alter a-MSH-induced feeding reduction over a 4-h period [24]. Other effectors such as PDE3B and AKT are modu-
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lated by leptin and insulin through PI3K so it is unclear what downstream process was altered in the Niswender studies. Future studies that include treatment with the MEK inhibitors such as PD98059, preventing the ultimate step in p42/p44 MAPK activation, would clarify this point. Since Daniels et al. [18] clearly demonstrate MAPK activation in the hypothalamus in areas accessible to ICV administered PI3K inhibitors, this suggests that the role of MAPK in MC4-R-regulated feeding remains to be determined. Recently, activation of the MC4-R receptor was shown to be the melanocortin receptor responsible for facilitating penile erection [25]. Additional studies should investigate the relative contribution of cAMP accumulation and p42/p44 MAPK activation to this event. Acknowledgements We thank Drs. David H. Weinberg, Chun-Pyn Shen, and Marc Reitman for helpful discussions. References [1] Wikberg JES. Melanocortin receptors: perspectives for novel drugs. Eur J Pharmacol 1999;375:295 – 310. [2] MacNeil DJ, Howard AD, Guan XM, Fong TM, Nargaund RP, Bednarek MA, et al. The role of melanocortins in body weight regulation: opportunities for the treatment of obesity. Eur J Pharmacol 2002; 450:93 – 109. [3] Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD. Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 1994;8: 1298 – 308. [4] Huszar D, Lynch CA, Fairchildhuntress V, Dunmore JH, Fang Q, Berkemeier LR, et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 1997;88:131 – 41. [5] Chen AS, Metzger JM, Trumbauer ME, Guan XM, Yu H, Frazier EG, et al. Role of the melanocortin-4 receptor in metabolic rate and food intake in mice. Transgenic Res 2000;9:145 – 54. [6] Vaisse C, Clement K, GuyGrand B, Froguel P. A frameshift mutation in human MC4R is associated with a dominant form of obesity. Nat Genet 1998;20:113 – 4. [7] Gu W, Tu ZM, Kleyn PW, Kissebah A, Duprat L, Lee J, et al. Identification and functional analysis of novel human melanocortin4 receptor variants. Diabetes 1999;48:635 – 9. [8] Hinney A, Schmidt A, Nottebom K, Heibult O, Becker I, Ziegler A, et al. Several mutations in the melanocortin-4 receptor gene including a nonsense and a frameshift mutation associated with dominantly inherited obesity in humans. J Clin Endocrinol Metab 1999;84: 1483 – 6. [9] Vaisse C, Clement K, Durand E, Hercberg S, Guy Grand B, Froguel P. Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. J Clin Invest 2000;106:253 – 62. [10] Farooqi IS, Keogh JM, Yeo GSH, Lank EJ, Cheetham T, O’Rahilly S. Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. N Engl J Med 2003;348:1085 – 95. [11] Sebhat IK, Martin WJ, Ye ZX, Barakat K, Mosley RT, Johnston DBR, et al. Design and pharmacology of N-[(3R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)2-[4-cyclohexyl-4(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1), a potent, selective, melanocortin subtype-4 receptor agonist. J Med Chem 2002;45:4589 – 93.
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[12] Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Watson SJ, et al. Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J Biol Chem 1993;268:15174 – 9. [13] Adan RAH, Cone RD, Burbach JPH, Gispen WH. Differential effects of melanocortin peptides on neural melanocortin receptors. Mol Pharmacol 1994;46:1182 – 90. [14] Khong K, Kurtz SE, Sykes RL, Cone RD. Expression of functional melanocortin-4 receptor in the hypothalamic GT1-1 cell line. Neuroendocrinology 2001;74:193 – 201. [15] Mountjoy KG, Kong PL, Taylor JA, Willard DH, Wilkison WO. Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells. Physiol Genomics 2001;5:11 – 9. [16] Gutkind JS. Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors. Sci Signal Transduct Knowl Environ 2000;Re1. http://www.stke.org/cgi/content/full/ OC_sigtrans;2000/40/re1. [17] Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity. Cell Signal 2001;13: 777 – 85. [18] Daniels D, Patten CS, Roth JD, Yee DK, Fluharty SJ. Melanocortin receptor signaling kinase in vitro and through mitogen-activated protein in rat hypothalamus. Brain Res 2003;986:1 – 11. [19] Bednarek MA, Macneil T, Kalyani RN, Tang R, Van der Ploeg LHT,
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Weinberg DH. Analogs of MTII, lactam derivatives of alpha-melanotropin, modified at the N-terminus, and their selectivity at human melanocortin receptors 3, 4, and 5. Biochem Biophys Res Commun 1999;261:209 – 13. Nijenhuis WA, Oosterom J, Adan RA. AgRP(83 – 132) acts as an inverse agonist on the human-melanocortin-4 receptor. Mol Endocrinol 2001;15:164 – 71. Haskell Luevano C, Monck EK. Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor. Regul Pept 2001;99:1 – 7. Konda Y, Gantz I, DelValle J, Shimoto Y, Miwa H, Yamada T. Interaction of dual intracellular signaling pathways activated by the melanocortin-3 receptor. J Biol Chem 1994;269:13162 – 6. Niswender KD, Morrison CD, Clegg DJ, Olson R, Baskin DG, Myers MG, et al. Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus—a key mediator of insulin-induced anorexia. Diabetes 2003;52:227 – 31. Niswender KD, Morton GJ, Stearns WH, Rhodes CJ, Myers Jr MG, Schwartz MW. Intracellular signalling. Key enzyme in leptin-induced anorexia. Nature 2001;413:794 – 5. Martin WJ, McGowan E, Cashen DE, Gantert LT, Drisko JE, Hom GJ, et al. Activation of melanocortin MC4 receptors increases erectile activity in rats ex copula. Eur J Pharmacol 2002;454:71 – 9.
Activation of MAP kinase by MC4-R through PI3 kinase Aurawan Vongs, Nicole M. Lynn, Charles I. Rosenblum * Department of Metabolic Research-Obesity, Merck Research Laboratories, Merck and Co., P.O. Box 2000, RY80M-213 Rahway, NJ 07065, USA Received 7 January 2004; received in revised form 18 February 2004; accepted 25 February 2004 Available online 2 April 2004
Abstract The melanocortin 4 receptor (MC4-R) is a Gas-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP-a-MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate. D 2004 Elsevier B.V. All rights reserved. Keywords: MC4-R; Melanocortin; p42/p44 MAPK; ERK1; ERK2; PI3K
1. Introduction 1.1. Melanocortin 4 receptor (MC4-R) introduction The MC4-R is a member of a five-member family of Gprotein-coupled receptors. Each of these receptors is modulated by one or more melanocortin peptides. Melanocortin peptides are produced by proteolytic processing of proopiomelanocortin (POMC) (reviewed in Refs. [1,2]). MC4R is widely expressed throughout the central nervous system though the highest levels expression are observed in the hypothalamus, including the paraventricular nucleus, and in the dorsal motor nucleus of the brainstem [3], implying a role in the regulation of feeding behavior. Indeed, MC4-R null mice become obese [4,5] as do humans with inactive MC4-R or diminished MC4-R function [6 –8]. Recent studies determined that 4% of severe childhood obesity is due to mutations in MC4-R [9,10]. Both null mice and humans expressing MC4-R variants show increased linear growth. While decreased MC4-R activity increases food intake, stimulation of MC4-R has the opposite effect. Treatment of normal rodents with MC4-R selective compounds lowers feed intake [11].
MC4-R is a Gas-coupled receptor, and as such, upon agonist binding, activates adenylyl cyclase; the subsequent increase in cAMP activating protein kinase A (PKA) [12 – 14]. Mountjoy et al. [15] showed that MC4-R stimulation causes cholera toxin-sensitive increases in cytosolic calcium due to release of intracellular calcium stores by an undefined mechanism. Other GPCRs are known to activate multiple intracellular signaling pathways (reviewed by Gutkind [16] and Liebmann [17]). Here, we have examined if MC4-R modulated pathways other than Gas stimulation of adenylyl cyclase. Corroborating the recent findings of Daniels et al. [18], we observed increased phosphorylation and enzymatic activity of the mitogen-activated kinases (MAPK) p42 and p44 following treatment with melanocortin agonist NDP-aMSH. Furthermore, this activation was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors but not PKA inhibitors, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.
2. Materials and methods 2.1. Chemicals
* Corresponding author. Tel.: +1-732-594-5958; fax: +1-732-5943337. E-mail address: [email protected] (C.I. Rosenblum). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.02.018
NDP-a-MSH and SHU-9119 were purchased from Bachem (King of Prussia, PA). PMSF, Tris – HCl, Earle’s
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buffered salt solution and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin was purchased from American Bioanalytical (Natick, MA). PVDF membranes were purchased from Schleicher and Schuell (Keene, NH). Wortmannin and Rp-cAMPS were purchased from Sigma-Aldrich. Sp-cAMPS was purchased from Biolog (Bremen, Germany). HEPES was purchased from Invitrogen (Carlsbad, CA). Anti-phosphorylated MAP kinase antibody (9106), LY294002 and an in vitro MAP kinase phosphorylation assay were purchased from Cell Signaling Technologies (Beverly, MA). Anti-p42/p44 MAP kinase antibody (06-182) was purchased from Upstate Biotechnology (Lake Placid, NY). A cAMP SPA assay and ECL plus chemiluminescent protein detection reagent were purchased from Amersham Biosciences (Piscataway, NJ).
jC. After thorough washing the blot was incubated with anti-mouse-HRP conjugate (1:5000) for 1 h at room temperature. Following thorough washing, the bound antibody was detected using ECL plus reagent (Amersham Bioscience) and exposed to Bio-Max film (Eastman Kodak, Rochester, NY). Antibodies were then stripped using the Amersham ECL plus protocol. Following blocking in TBST, 5% non-fat dried milk, it was incubated overnight in antiMAP kinase antibody, 1:5000 at 4 jC. After thorough washing, the blot was incubated with anti-rabbit – HRP conjugate (1:3000) for 1 h at room temperature. Following thorough washing, the bound antibody was detected using ECL plus reagent and exposed to Bio-Max film.
2.2. Cell culture
Growth medium was removed from confluent cultures of CHO-hMC4-R or CHO-K1 cells. Cells were placed in assay solution (EBSS, 0.1% BSA, 25 mM HEPES, 5 mM MgCl2) and treated with various concentrations of NDP-a-MSH at 37jC for the length of time indicated in the Results. For studies with inhibitors of signaling pathways, cells were transferred into assay solution and treated with pathway inhibitors or vehicle for 30 min before addition of 100 nM NDP-a-MSH for 10 min. Cells were washed with ice-cold phosphate buffered saline solution and lysed with a cell lysis buffer (no. 9803, Cell Signaling Technologies) that contains phosphatase inhibitors and protease inhibitors and that was supplemented with 1 mM PMSF. Lysates were passed through a 25-gauge needle three times and centrifuged at 16,000 g at 4 jC for 15 min, and supernatants were retained. The protein concentration of lysates were determined using the Bio-Rad protein assay (Richmond, CA).
In vitro MAP kinase enzymatic activity was determined using a kit in which an Elk-1 fusion protein serves as substrate for activated MAP kinase. However, the protocol was modified from the manufacture’s to create a moderate throughput protein dot-blot assay rather than a western blot assay. 200 Ag of cell lysate was immunoprecipitated with 7 Al of anti-phospho p42/p44 MAP kinase antibody-conjugated agarose beads for 16 h at 4 jC. This material was then loaded into a Handee spin cup column (Pierce Biotechnology, Rockford, IL) and was centrifuged at 300 g for 10 s to remove supernatant. The agarose conjugate was washed once with 400 Al of cell lysis buffer and two times with 400 Al kinase reaction buffer (25 mM Tris (pH 7.5), 5 mM hglycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). Fifty microliters of kinase reaction buffer, 200 AM ATP and 2 Ag GST – Elk-1 fusion protein were added to the agarose conjugate and were incubated at 30 jC for 30 min. The reaction was terminated by centrifugation at 15,000 g for 30 s and incubation at 100 jC for 4 min. Twenty microliters of reaction product was spotted onto PVDF membrane (Schleicher and Schuell) using a 96-well vacuum manifold (Schleicher and Schuell). Immunochemistry was used to detected the phosphorylated Elk-1 using reagents and following the protocol provided with the assay kit. The amount of phosphorylation was quantitated using an Eastman Kodak Image Station 440C (Rochester, NY). Statistical analyses were performed using GraphPad Prism for Windows version 4.0 (San Diego, CA).
2.4. Western blot analysis
2.6. cAMP assays
Twenty micrograms of cell lysate was prepared for SDS –PAGE using Laemmli sample buffer (Bio-Rad) and proteins were separated on a 10% polyacrylamide gel (Invitrogen). The proteins were electrophoretically transferred onto PVDF membrane (Invitrogen) and the blot was blocked in 50 mM Tris –HCl pH 8, 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T) and 5% non-fat dried milk. The blot was incubated with anti-phospho-p42/p44 MAP kinase antibody (1:2000) in TBS-T, 5% BSA overnight at 4
CHO-MC4R cells were treated as described in the Results. cAMP accumulation was determined using an RIA [19].
The creation and characterization of CHO-K1 cells expressing human MC4-R (CHO-hMC4-R) was previously described [19]. Cells were cultured in Iscove’s modified Dulbecco medium supplemented with 10% fetal bovine serum, 1 mg/ml G418, (Invitrogen) and 100 AM sodium hypoxanthine and 160 AM thymidine. 2.3. Cell treatment
2.5. In vitro MAP kinase assays
3. Results To assess whether stimulation of the MC4-R receptor modulates p42/p44 MAPK, CHO-hMC4-R cells were
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treated with the MC4-R agonist NDP-a-MSH for 10 min. p42 (ERK2) and p44 (ERK1) MAPK phosphorylation was assessed by western blot (Fig. 1A). Both p42 and p44 were found to be phosphorylated following NDP-aMSH treatment. Though the absolute amount of phosphorylated p42 was greater than that of p44, the relative increases in phosphorylated p42 and p44 were found to be equal as measured by image quantitation of the blot itself. We used an in vitro substrate phosphorylation assay to measure enzymatically activated p42/p44 MAPK. In this assay, phosphorylated p42/p44 MAPK is immunoprecipitated and phosphorylation of Elk-1, a substrate of p42/p44, is determined by immunoblot analysis. We modified a commercial kit to create a moderate throughput assay utilizing spin columns as reaction vessels and dot blotting, eliminating PAGE and a protein transfer step. Utilizing a dot-blot manifold permitted spotting of up to 96 samples per membrane. In order to determine if p42/p44 MAPK activation was occurring via MC4-R activation, wild-type CHO-K1 cells and CHO-hMC4-R cells were treated with NDP-a-MSH. p42/p44 MAPK activation, as measured by Elk-1 phosphorylation, did not occur in the CHO-K1 cells lacking hMC4-R (Fig. 1B). In another experiment, pre-treatment of the CHOhMC4-R cells with SHU-9119, a potent MC4-R antagonist, inhibited NDP-a-MSH-mediated p42/p44 MAPK activity (Fig. 1C). Together, these observations conclusively demonstrate that stimulation of MC4-R causes phosphorylation and activation of p42 and p44. 3.1. Time course of activation We studied p42/p44 MAPK activation by treating hMC4-R CHO cells with 100 nM NDP-a-MSH for various periods of time. Substantial phosphorylation of the Elk-1 substrate was observed by 5 min of stimulation. Activity was maximal by 10 min of treatment (Fig. 2). Interestingly,
Fig. 1. (A) NDP-a-MSH treatment causes phosphorylation of p42 and p44 in CHO-K1 cells expressing human MC4-R. Cells were treated with 0, 100 nM or 1 AM NDP-a-MSH for 10 min. Phosphorylated p42/p44 MAPK was determined by western blot analysis using anti-phospho-p42/p44 MAPK. Blots were stripped and blotted with anti-p42/p44 MAPK to determine total p42/p44 MAPK. (B) p42/p44 MAPK activation is mediated through MC4R. Wild-type and CHO-hMC4-R were treated with 100 nM NDP-a-MSH for 10 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Open bars: wild-type CHO-K1 cells; solid bars: CHO-hMC4-R. Results represent the mean F S.E.M. of three independent measurements. An asterisk indicates a statistically significant difference from unmarked bars ( p < 0.01) by one-way ANOVA. (C) p42/p44 MAPK activation is blocked by inhibitors of MC4-R. Prior to treatment with 100 nM NDP-a-MSH for 10 min, CHO-hMC4-R were incubated 5 AM SHU9119 for 30 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent measurements. An asterisk indicates a statistically significant difference from unmarked bars ( p < 0.05) by one-way ANOVA.
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though activity declined, at 45 min, Elk-1 phosphorylation remained threefold the basal level. In subsequent experiments, cells were treated with agonist for 10 min. 3.2. Dose response To determine if p42/p44 MAPK activation occurred in a dose responsive manner, cells were treated with increasing amounts of NDP-a-MSH (Fig. 3). The EC50 of NDP-aMSH was determined to be approximately 1 nM, very similar to that observed for hMC4-R stimulated cAMP production (Fig. 4B).
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Fig. 2. Time course of NDP-a-MSH-induced p42/p44 MAPK activity. CHOhMC4-R cells were treated with 100 nM NDP-a-MSH for the indicated period of time. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent measurements.
3.3. Signaling pathway Some Gas-coupled GPCRs have been shown to activate p42/p44 MAPK via Gas through PKA, while others couple via Ghg activation of PI3K. PI3K, in turn, modulates a Src-mediated activation of Ras [17]. Therefore, cells were pre-treated with compounds that inhibit either PKA or PI3K. Cells were pre-incubated with Rp-cAMPS, a PKA antagonist, or the PI3K inhibitors wortmannin and LY294002. Pre-treatment of cells with 100 AM Rp-cAMPS did not inhibit NDP-a-MSH stimulated Elk-1 phosphorylation (Fig. 4A), demonstrating that p42/p44 MAPK activation is not a PKA-dependent event. Treatment of cells with the cAMP analog Sp-cAMPS (100 AM) did stimulate
Fig. 3. NDP-a-MSH treatment of CHO-hMC4-R cells causes dosedependent stimulation of p42/p44 MAPK activity. Cells were treated with various concentrations of NDP-a-MSH for 10 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent measurements.
Fig. 4. (A) Inhibitors of PI3K but not PKA block p42/p44 MAPK activity. Thirty minutes prior to treatment with NDP-a-MSH, CHO-hMC4-R cells were incubated with vehicle, 100 AM Rp-cAMPS, 50 nM wortmannin or 25 AM LY294002. Cells were then treated with assay solution with or without 100 nM NDP-a-MSH for 10 min. Cell lysates were prepared and p42/p44 MAPK enzymatic activity was measured by phosphorylation of Elk-1 substrate as described in the Materials and Methods. Results represent the mean F S.E.M. of three independent experiments. (B) PI3K inhibitors do not alter MC4-R mediated cAMP accumulation. Thirty minutes prior to assay, CHO-hMC4-R cells were incubated with vehicle, 100 AM RpcAMPS, 50 nM wortmannin or 25 AM LY294002. Cells were then treated with various amounts of NDP-a-MSH for 45 min and cAMP was measured by SPA assay. Results represent the mean F S.E.M. of three independent measurements. An asterisk indicates a statistically significant difference from unmarked bars ( p < 0.01) by one-way ANOVA.
p42/p44 MAPK indicating that PKA activation of p42/p44 MAPK is functional in CHO-K1 cells (not shown). Pretreatment of cells with either 50 nM wortmannin or 25 AM LY294002 inhibited p42/p44 MAPK activity, demonstrating that MC4-R-mediated p42/p44 MAPK activation is mediated through PI3K. It is of interest to note that the amount of Elk-1 phosphorylation appears to be diminished to below basal levels by treatment with the PI3K inhibitors. This is likely due to inhibition of constitutive MC4-R activity [20, 21]. In order to be certain that these concen-
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trations PI3K inhibitors were not altering Gas-mediated adenylyl cyclase activity, indirectly altering p42/p44 MAPK activity, cells were assayed for cAMP accumulation following MC4-R agonist stimulation following pre-treatment with the PI3K antagonists. No alteration in the efficacy or potency of NDP-a-MSH was observed following PI3K inhibitor pre-treatment (Fig. 4B).
4. Discussion We demonstrate that human MC4-R, stably expressed in CHO-K1 cells, activates p42/p44 MAPK through a PI3Kmediated pathway. We did this by modifying a commercial assay that measures p42/p44 MAPK enzymatic activity using western blotting and converted it to moderate throughput method. p42/p44 MAPK was activated by NDP-a-MSH in a dose-dependent manner, causing equal fold increases in phosphorylated p42 and p44. p42/p44 MAPK activation was maximal by 10 min, but did not rapidly desensitize, remaining threefold basal levels 45 min after stimulation. The activation of p42/p44 MAPK is a PI3K-dependent event as shown by lack of activation by NDP-a-MSH following pre-treatment with LY294002 and wortmannin. p42/p44 MAPK activation is not altered by PKA inhibition caused by Rp-cAMPS. Previous studies of GPCR-regulated p42/p44 MAPK activation implicate either Ga or Ghg [17]. It would seem reasonable to speculate that Ghg activate PI3K in the heterologous cell-based system we employed, though further studies are required to unambiguously establish this point. Since the experiments were performed in CHO cells, due caution must be taken when extrapolating these results to neurons expressing endogenous MC4-R though it is worth noting that stimulation of MC4-R does activity p42/p44 MAPK in the paraventricular nucleus of the hypothalamus [18]. Mountjoy et al. [15] showed that agonist treatment of exogenously expressed melanocortin receptors, including MC4-R, caused cholera toxin-sensitive increases in intracellular calcium. However, this calcium flux was not modulated by increased IP3 concentrations, implying that this is a separate phenomenon from the PI3K-mediated mechanism described here. Konda et al. [22] found that MC3-R activation caused increased in IP3 accumulation and, in turn, increased intracellular calcium concentrations when the receptor was expressed in Hepa, L-cells and CHO cells. p42/p44 MAPK activity was not examined in those experiments. Niswender et al. [23,24] demonstrated that leptin’s and insulin’s anorectic action can be ameliorated the by ICV injection of wortmannin or LY294002 prior to ICV leptin, implicating a role for PI3K in the anoretic actions of leptin and insulin. Administration of these PI3K inhibitors did not alter a-MSH-induced feeding reduction over a 4-h period [24]. Other effectors such as PDE3B and AKT are modu-
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lated by leptin and insulin through PI3K so it is unclear what downstream process was altered in the Niswender studies. Future studies that include treatment with the MEK inhibitors such as PD98059, preventing the ultimate step in p42/p44 MAPK activation, would clarify this point. Since Daniels et al. [18] clearly demonstrate MAPK activation in the hypothalamus in areas accessible to ICV administered PI3K inhibitors, this suggests that the role of MAPK in MC4-R-regulated feeding remains to be determined. Recently, activation of the MC4-R receptor was shown to be the melanocortin receptor responsible for facilitating penile erection [25]. Additional studies should investigate the relative contribution of cAMP accumulation and p42/p44 MAPK activation to this event. Acknowledgements We thank Drs. David H. Weinberg, Chun-Pyn Shen, and Marc Reitman for helpful discussions. References [1] Wikberg JES. Melanocortin receptors: perspectives for novel drugs. Eur J Pharmacol 1999;375:295 – 310. [2] MacNeil DJ, Howard AD, Guan XM, Fong TM, Nargaund RP, Bednarek MA, et al. The role of melanocortins in body weight regulation: opportunities for the treatment of obesity. Eur J Pharmacol 2002; 450:93 – 109. [3] Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD. Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 1994;8: 1298 – 308. [4] Huszar D, Lynch CA, Fairchildhuntress V, Dunmore JH, Fang Q, Berkemeier LR, et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 1997;88:131 – 41. [5] Chen AS, Metzger JM, Trumbauer ME, Guan XM, Yu H, Frazier EG, et al. Role of the melanocortin-4 receptor in metabolic rate and food intake in mice. Transgenic Res 2000;9:145 – 54. [6] Vaisse C, Clement K, GuyGrand B, Froguel P. A frameshift mutation in human MC4R is associated with a dominant form of obesity. Nat Genet 1998;20:113 – 4. [7] Gu W, Tu ZM, Kleyn PW, Kissebah A, Duprat L, Lee J, et al. Identification and functional analysis of novel human melanocortin4 receptor variants. Diabetes 1999;48:635 – 9. [8] Hinney A, Schmidt A, Nottebom K, Heibult O, Becker I, Ziegler A, et al. Several mutations in the melanocortin-4 receptor gene including a nonsense and a frameshift mutation associated with dominantly inherited obesity in humans. J Clin Endocrinol Metab 1999;84: 1483 – 6. [9] Vaisse C, Clement K, Durand E, Hercberg S, Guy Grand B, Froguel P. Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. J Clin Invest 2000;106:253 – 62. [10] Farooqi IS, Keogh JM, Yeo GSH, Lank EJ, Cheetham T, O’Rahilly S. Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. N Engl J Med 2003;348:1085 – 95. [11] Sebhat IK, Martin WJ, Ye ZX, Barakat K, Mosley RT, Johnston DBR, et al. Design and pharmacology of N-[(3R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)2-[4-cyclohexyl-4(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1), a potent, selective, melanocortin subtype-4 receptor agonist. J Med Chem 2002;45:4589 – 93.
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