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Endocrinology, doi:10.1210/en.2006-0984
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Endocrinology Vol. 147, No. 12 5948-5955
Copyright © 2006 by The Endocrine Society

Regulation of Endogenous Melanocortin-4 Receptor Expression and Signaling by Glucocorticoids

Julien A. Sebag and Patricia M. Hinkle

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642

Address all correspondence and requests for reprints to: Patricia M. Hinkle, Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642. E-mail: Patricia_Hinkle{at}urmc.rochester.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The melanocortin-4 (MC4) receptor plays a pivotal role in regulating food intake and energy expenditure, and obesity results from mutations that interfere with the MC4 receptor pathway. We investigated the effect of glucocorticoids on endogenous MC4 receptors expressed in GT1-1 cells, an immortalized hypothalamic neuronal cell line. Dexamethasone (Dex) caused a 5- to 10-fold increase in the cAMP response to the MC4 receptor agonist, NDP-{alpha}MSH. The stimulatory effect of Dex reached a maximum within 24 h and was blocked by the glucocorticoid antagonist RU486. This glucocorticoid effect was specific for the MC4 receptor and not a result of up-regulation of another component of the cAMP cascade, because the response to endogenous ß-adrenergic receptor stimulation was not altered by Dex. Dex also potentiated NDP-{alpha}MSH-mediated ERK1/2 activation. After 12 h, Dex caused a 3- to 5-fold increase in [125I]NDP-{alpha}MSH binding, which was maintained for at least 48 h and prevented by RU486. Dex withdrawal caused a rapid return of MC4 receptor concentration to the basal level. Dex-mediated increases in MC4 receptor concentration resulted from a rapid but transient increase in MC4 receptor mRNA. This regulation apparently requires genomic regulatory sequences because Dex did not increase MC4 receptor expression or signaling in CHO cells expressing the MC4 receptor under the control of a cytomegalovirus promoter. We conclude that in GT1-1 hypothalamic neurons, glucocorticoids increase the amplitude of MC4 receptor signaling. This regulation may serve as a control to limit the effects of glucocorticoids on food intake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MELANOCORTIN-4 RECEPTOR (MC4 receptor) is a central player in weight regulation because activation of this receptor decreases food intake and increases energy expenditure (1, 2). Mutations in the MC4 receptor are the most common cause of hereditary early onset obesity known, accounting for up to 6% of cases (3). The MC4 receptor controls not only food intake but also energy expenditure and caloric efficiency, because targeted deletion of the MC4 receptor (4) and inhibition of the receptor with the natural antagonist agouti-related protein and synthetic antagonists increase adiposity in pair-feeding paradigms where food intake is not altered (5, 6, 7). In addition to its role in feeding behavior, the MC4 receptor has been reported to play a role in regulation of the hypothalamic-pituitary-adrenal axis (8), sexual behaviors (9), inflammation in the brain (10), neuroprotection (11), emotional states such as anxiety and depression (12), and drug addiction (13, 14). The MC4 receptor is widely expressed in the central nervous system where the major sites of expression are in the arcuate nucleus and the paraventricular nucleus of the hypothalamus (15). Hypothalamic MC4 receptors are regulated by the agonists {alpha}MSH and possibly ßMSH (16, 17, 18), both products of the POMC gene (19), and the antagonist agouti-related protein (20, 21).

The MC4 receptor is one of five known members of the melanocortin receptor family (MC1 to MC5 receptors), seven-transmembrane receptors positively coupled to adenylyl cyclase through Gs. Activation of the MC4 receptor also triggers the phosphorylation of ERK1/2 MAPKs (22, 23). MAPK activation seems to be required for MC4 receptor regulation of food intake (24).

Numerous hormones play a role in appetite and metabolism regulation, but no hormonal regulation of MC4 receptor has been reported. Glucocorticoids, synthesized by the adrenal glands in response to ACTH stimulation of the MC2 receptor, have a major impact on food intake (25, 26, 27). Glucocorticoid deficiency is characterized by anorexia that is readily reversed by steroid replacement (28, 29). When glucocorticoids are used therapeutically at high concentrations, usually for their antiinflammatory properties, long-term treatment causes weight gain due to increased food intake (30). Stressful stimuli cause glucocorticoid release by the adrenal glands (31) and in some cases decreased food intake (32, 33), although no clear link between increased plasma glucocorticoid level and stress-induced diminution in food intake has been reported. Because glucocorticoid levels influence feeding behavior, we investigated the effect of glucocorticoids on expression and signaling of endogenous MC4 receptors in GT1-1 cells, an immortalized GnRH-secreting mouse hypothalamic neuronal cell line (34, 35).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection
GT1-1 cells were provided by Dr. Richard I. Weiner (University of California at San Francisco, San Francisco, CA). GT1-1 and CHO cells were grown in DMEM/F12 medium supplemented with 5% fetal bovine serum at 37 C in a humidified 5% CO2 incubator. CHO cells were transfected with a plasmid encoding a hemagluttinin (HA)-tagged human MC4 receptor with a C-terminal green fluorescent protein (GFP) in pEGFP-N1 (provided by Dr. Jeffrey Flier, Harvard Medical School, Boston, MA) using Lipofectamine (Invitrogen, Carlsbad, CA), and a pooled stable line was selected with 1 mg/ml G418. Experiments were scheduled such that all incubations ended at the same time.

cAMP assay
To measure cAMP, GT1-1 cells were grown in 12-well plates and unless noted were incubated with Nle4-D-Phe7-{alpha}MSH (NDP-{alpha}MSH) or vehicle in the presence of 100 µM 3-isobutyl-1-methylxanthine (IBMX) in Optimem I for 20 min when dishes were placed on ice. Cells were washed, and cAMP was extracted and quantified using the cAMP EIA direct assay kit from Assay Design (Ann Arbor, MI) following the manufacturer’s instructions.

MAPK phosphorylation
GT1-1 cells were plated in 6-cm culture dishes in DMEM/F12 supplemented with 5% fetal bovine serum. Cells were incubated with dexamethasone (Dex) or vehicle for 24 h, the last 12 h in serum-free medium, and then incubated with NDP-{alpha}MSH or vehicle for various times from 1 to 30 min. Dishes were then placed on ice and rinsed twice with PBS, and cells were scraped in 2x SDS-PAGE sample buffer. Proteins were resolved by SDS-PAGE, and phospho-ERK1/2 and total ERK1/2 were identified by immunoblotting with antibodies from Cell Signaling Technology (Beverly, MA) at 1:5000 dilution using chemiluminescence detection from PerkinElmer Life Sciences (Boston, MA).

MC3 receptor and MC4 receptor mRNA analysis
Confluent 25-cm2 flasks of GT1-1 cells were treated as described and then trypsinized and resuspended in serum-free DMEM/F12. Cells were pelleted for 5 min at 5000 rpm, and total mRNA was extracted using the RNeasy minikit from QIAGEN Inc. (Valencia, CA) following the manufacturer’s instructions. Mouse brain tissue (30 mg) was harvested and ground in RNeasy lysis buffer, and total mRNA was extracted as described for cells. Total mRNA extracts were treated with DNase I [Invitrogen DNase I amplification grade (1U/µl)] for 15 min at 25 C. The total mRNA was reverse transcribed using M-MLV reverse transcriptase from Invitrogen. For each sample a control reaction was carried out omitting the reverse transcriptase to rule out genomic DNA contamination. PCR was then performed on the RT products using MC4 receptor primers (sense, 5'-CTT TTA CGC GCT CCA GTA CC-3'; antisense, 5'-CCA ATC AGG ATG GTC AAG GT-3'), MC3 receptor primers (5' CAT GTA CTT CTT CCT GTG CAG C and 3' TGC TCT CGG AGT AGA TGA TGA A), or GAPDH primers (5' GCC AAA AGG GTC ATC ATC TC and 3' GGC CAT CCA CAG TCT TCT) as control. PCR was performed using recombinant Taq polymerase (Invitrogen). Amplification of MC3 and MC4 receptor cDNA was carried out for 40 cycles and GAPDH cDNA for 25 cycles.

[125I]NDP-{alpha}MSH binding
[125I]NDP-{alpha}MSH was prepared by iodinating NDP-{alpha}MSH (Phoenix Pharmaceuticals, Inc., Belmont, CA) to a specific activity of between 300 and 1000 Ci/mmol using the chloramine T method. GT1-1 or CHO cells seeded in six-well plates were treated as described, and the cells were rinsed once with saline and then incubated with [125I]NDP-{alpha}MSH (250,000 to 1,000,000 cpm/ml) in binding buffer (Dulbecco’s PBS with 20 mM HEPES, 0.1% BSA; pH 7.5) for 1 h at 37 C. Nonspecific binding was determined in wells incubated with radioligand and 100 nM unlabeled NDP-{alpha}MSH and has been subtracted. Dishes were placed on ice and washed three times with ice-cold saline to remove unbound labeled ligand. Cells were lysed with 0.1% SDS in PBS.

Other methods
A plasmid-encoding firefly luciferase under the control of the MMTV promoter was provided by Dr. Chawnshang Chang (University of Rochester, Rochester, NY). Luciferase activity was measured in cell lysates using the dual-luciferase reporter assay system from Promega (Madison, WI) with pRLSV40 renilla luciferase as a control following the manufacturer’s instructions.

Experiments shown in Figs. 1CGo, 2BGo, 8AGo, and 9AGo were performed three times and all others twice. Binding data show the mean and range of duplicate points, and cAMP measurements are the mean ± SE of triplicates except Figs. 5Go and 9BGo, which were in duplicate. Luciferase activity measurements present the mean ± SE of triplicates. The statistical significance of differences was analyzed by Student’s unpaired t test or ANOVA using Tukey’s post hoc test.


Figure 1
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  		FIG. 1. Effect of glucocorticoids on MC4 receptor signaling. A, cAMP assay. GT1-1 cells were treated with vehicle (0.01% ethanol) or 100 nM Dex for 24 h at 37 C and then incubated with the indicated concentrations of NDP-{alpha}MSH in the presence of 100 µM IBMX for 20 min when cAMP was measured. B, ERK1/2 phosphorylation. GT1-1 cells were incubated with vehicle or 100 nM Dex for 24 h at 37 C and serum-starved for the last 12 h of treatment. Cells were then incubated with 100 nM NDP-{alpha}MSH for 0, 2, 5, 10, 20, or 30 min, as indicated. Phosphorylated and total ERK1/2 were identified by Western blot. The graph represents the ratio of the intensity of the pERK2 band normalized to total ERK2 (mean ± range from two experiments). Similar results were obtained in two experiments in which cells were maintained in serum-containing medium throughout. C, Receptor binding. GT1-1 cells treated with vehicle or 100 nM Dex for 24 h were incubated with [125I]NDP-{alpha}MSH and the indicated concentrations of unlabeled NDP-{alpha}MSH for 1 h at 37 C.

 

Figure 2
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  		FIG. 2. Concentration dependence of Dex-induced MC4 receptor potentiation. GT1-1 cells were treated with Dex for 24 h at 37 C. A, Specific binding of [125I]NDP-{alpha}MSH was then measured. B, Cells were incubated with 100 nM NDP-{alpha}MSH in the presence of 100 µM IBMX for 20 min, and cAMP was measured.

 

Figure 8
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  		FIG. 8. Effect of Dex on MC3 and MC4 receptor mRNA levels. A, GT1-1 cells were treated with 100 nM Dex for 0 to 24 h. Gels show a representative PCR amplification of reverse-transcribed mRNA using MC4 receptor and GAPDH primers. The histogram shows average ± SE for MC4 receptor mRNA normalized to GAPDH mRNA from three separate experiments. *, P < 0.05 vs. zero time point. B, Representative PCR amplification of reverse-transcribed mRNA extracts from GT1-1 cells treated with 100 nM Dex or vehicle in the presence or absence of 5 µg/ml actinomycin D using MC4 receptor and GAPDH primers. C, Representative PCR amplification of reverse-transcribed mRNA extracts from GT1-1 cells treated with 100 nM Dex for the indicated time and from mouse brain using MC3 receptor and GAPDH primers. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MC3R, MC3 receptor; MC4R, MC4 receptor.

 

Figure 9
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  		FIG. 9. Reversal of Dex effects on MC4 receptors. GT1-1 cells were incubated with 100 nM Dex or vehicle for 30 h when Dex was removed from some dishes and replaced by 1 µM RU486 for different times. [125I]NDP-{alpha}MSH binding (A) and cAMP production in response to 100 nM NDP-{alpha}MSH over 20 min in the presence of 100 µM IBMX (B) were measured.

 

Figure 5
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  		FIG. 5. Effect of Dex on ß-adrenergic receptor signaling. GT1-1 cells were treated with 100 nM Dex or vehicle for 0 to 48 h and then incubated with 100 µM IBMX with or without 10 µM isoproterenol for 20 min.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of MC4 receptor signaling by glucocorticoids
To determine whether glucocorticoids regulate MC4 receptor signaling, we incubated hypothalamic GT1-1 cells, which express the MC4 receptor endogenously, with or without the potent glucocorticoid Dex for 24 h. Activation of the MC4 receptor was then achieved using the potent synthetic agonist NDP-{alpha}MSH and two responses, stimulation of cAMP production and phosphorylation of ERK1/2, were measured. We observed a dose-dependent 6-fold increase in cAMP with increasing concentrations of NDP-{alpha}MSH in control GT1-1 cells. The resting level of cAMP in the cells was not affected by Dex treatment, but the increase in cAMP stimulated by NDP-{alpha}MSH rose to 27-fold in Dex-treated cells, demonstrating a large potentiation of MC4 receptor signaling to adenylyl cyclase (Fig. 1AGo). The EC50 for NDP-{alpha}MSH stimulation of cAMP was the same, 1 nM, in Dex- and vehicle-treated cells.

We next asked whether glucocorticoids affect MC4 receptor-mediated activation of the MAPK pathway in GT1-1 cells. NDP-{alpha}MSH caused slight but detectable phosphorylation of ERK2 after 2 min in control cells. In Dex-treated cells, NDP-{alpha}MSH caused a 4-fold greater phosphorylation of ERK2, confirming glucocorticoid potentiation of MC4 receptor signaling (Fig. 1BGo). The kinetics of ERK2 phoshorylation were the same in vehicle- and Dex-treated cells, with maximal phospho-ERK2 signal at 5–10 min. NDP-{alpha}MSH caused similar ERK2 activation in serum-starved cells (Fig. 1BGo) and in cells grown in normal serum (data not shown). Phosphorylated ERK1 was not generally detectable. These kinetics of MAPK activation via the MC4 receptor are in full accord with recently reported findings in the same model system (22).

We also performed radioligand binding assays in GT1-1 cells pretreated with Dex or vehicle for 24 h (Fig. 1CGo). Dex caused a large increase in [125I]NDP-{alpha}MSH binding. [125I]NDP-{alpha}MSH binding was low and difficult to quantify in untreated cells; the increase in binding in response to Dex varied from 2- to 15-fold among experiments. We used competition displacement analysis to estimate the affinity of MC4 receptors and found no apparent difference in the IC50 values for NDP-{alpha}MSH in vehicle- and Dex-treated cells. The IC50 value was 0.9 ± 0.48 nM in Dex-treated cells, somewhat above the published value of 0.1 nM (35). Although the concentration of receptors could not be measured directly, both signaling and binding results suggest that the potentiation caused by Dex results from an increased number of receptors and not a change in the receptor’s affinity for its ligand.

Dose dependence of MC4 receptor regulation by Dex
The effect of Dex concentration on [125I]NDP-{alpha}MSH binding and NDP-{alpha}MSH-stimulated cAMP production in GT1-1 cells is shown in Fig. 2Go. The maximal potentiation of MC4 receptor surface expression and signaling was reached at 100 nM Dex. In this experiment, Dex caused a maximal 4-fold increase in radiolabeled ligand-receptor binding and a 10- to 12-fold increase in MC4 receptor-mediated cAMP production. The EC50 values for the two Dex responses were the same, with 15 ± 4 nM Dex needed for a half-maximal increase in [125I]NDP-{alpha}MSH binding and 11 ± 4 nM Dex for a half-maximal cAMP response to NDP-{alpha}MSH (mean ± SE, n = 3). The ability of 100 nM Dex to increase MC4 receptor binding and signaling was prevented by the glucocorticoid receptor antagonist RU486 at 1 µM, whereas RU486 had no effect by itself (Fig. 3Go). The concentration dependence and ability of RU486 to antagonize the effect of Dex both suggest mediation by the nuclear glucocorticoid receptor.


Figure 3
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  		FIG. 3. Effect of RU486 on Dex potentiation of MC4 receptor responses. GT1-1 cells were treated with 100 nM Dex alone, 100 nM Dex plus 1 µM RU486, 1 µM RU486, or vehicle for 24 h at 37 C when specific [125I]NDP-{alpha}MSH binding (A) or cAMP after 20-min incubation with 100 µM IBMX with or without 100 nM NDP-{alpha}MSH (B) was measured. *, P < 0.001 vs. vehicle control.

 
Rate of MC4 receptor potentiation by Dex
The rate of Dex induction of MC4 receptors and responses was also measured in GT1-1 cells. We observed a 14-fold increase in binding as well as a 5-fold increase in MC4 receptor-mediated cAMP production after 12 h of treatment (Fig. 4Go, A and B). This up-regulation was significant within 6 h and was maintained for at least 72 h when Dex was kept in the media (Fig. 4Go, and data not shown).


Figure 4
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  		FIG. 4. Kinetics of MC4 receptor potentiation by Dex. GT1-1 cells were treated with 100 nM Dex or vehicle for 0 to 72 h. A, Specific binding of [125I]NDP-{alpha}MSH was determined. B, Cells were incubated with 100 nM NDP-{alpha}MSH and 100 µM IBMX for 20 min when cAMP was measured.

 
Specificity of glucocorticoid effect on MC4 receptor pathway
To confirm that the Dex-induced increase in MC4 receptor-mediated cAMP production was due to a specific regulation of the MC4 receptor itself and not to effects on downstream signaling elements, we tested whether glucocorticoids potentiated signaling via the ß-adrenergic receptor which, like the MC4 receptor, is positively coupled to adenylyl cyclase and expressed endogenously in GT1-1 cells. GT1-1 cells were incubated for different periods with or without Dex and then stimulated with the ß-adrenergic receptor agonist, isoproterenol. Isoproterenol caused a 5-fold increase in cAMP, and treatment with Dex for as long as 48 h did not alter ß-adrenergic receptor signaling (Fig. 5Go).

Lack of effect of Dex on transfected MC4 receptor
To determine whether Dex potentiation of signaling was limited to the MC4 receptor in its natural genomic setting, we stably expressed the receptor under the control of the strong constitutive cytomegalovirus promoter in CHO cells, which do not express MC4 receptor endogenously. Glucocorticoids had no effect on either [125I]NDP-{alpha}MSH binding or NDP-{alpha}MSH-stimulated cAMP production in a pool of stably transfected CHO cells (Fig. 6Go, A and B). To verify that these CHO cells were capable of mounting glucocorticoid response, we showed that Dex caused a 3-fold increase in luciferase activity driven by the MMTV promoter, a classical glucocorticoid receptor target (Fig. 6CGo).


Figure 6
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  		FIG. 6. Lack of effect of Dex on transfected MC4 receptor. A and B, CHO cells stably expressing an MC4 receptor-GFP fusion protein were pretreated with 100 nM Dex or vehicle. A, After 12 h, specific [125I]NDP-{alpha}MSH binding was measured. B, After 24 h, cells were incubated with 100 nM NDP-{alpha}MSH or vehicle in the presence of 100 µM IBMX for 20 min, and cAMP was measured. C, Cells were transiently transfected with plasmids encoding firefly luciferase with an MMTV promoter and control renilla luciferase and incubated with or without 100 nM Dex for 20 h when luciferase activities were measured. *, P < 0.05 vs. control group.

 
Effect of Dex on endogenous MC4 receptor desensitization
Most G protein-coupled receptors are desensitized after activation as a way to turn off the signaling cascade in the prolonged presence of the agonist. We investigated whether the glucocorticoid-induced increase in MC4 receptor signaling resulted in part from diminished desensitization of the activated receptor by measuring agonist-induced MC4 receptor desensitization as described by Shinyama et al. (36). Cells were pretreated with vehicle or Dex for 12 h and then exposed to NDP-{alpha}MSH or vehicle for 30 min. The cells were then washed to remove hormone, permitted to recover for 10 min, and challenged again with NDP-{alpha}MSH (Fig. 7Go). The extent to which the second response is less than the first reflects desensitization. In both cases, the first stimulation of MC4 receptor by NDP-{alpha}MSH caused a large increase in cAMP production with the expected 5-fold larger response in Dex-pretreated cells. The second stimulation with agonist caused a much smaller increase in cAMP in both vehicle- and Dex-treated cells, indicating that MC4 receptors are desensitized normally after Dex treatment. The extent of desensitization is in good agreement with published data (36).


Figure 7
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  		FIG. 7. Effect of Dex on endogenous MC4 receptor desensitization. GT1-1 cells were incubated with 100 nM Dex or vehicle for 24 h. Cells were incubated with 100 nM NDP-{alpha}MSH or vehicle for 30 min at 37 C, washed twice in Optimem I, and incubated with 100 µM IBMX for 10 min, and finally incubated with 100 µM IBMX and either 100 nM NDP-{alpha}MSH or vehicle for 20 min when cAMP was measured. Data are normalized to vehicle-treated values. *, P < 0.002 vs. first stimulation.

 
Mechanism of Dex-induced MC4 receptor up-regulation
Because it is well documented that the glucocorticoid receptor can regulate gene activity through direct binding to DNA sequences or indirect interactions with other transcription factors, we measured MC4 receptor mRNA levels by RT-PCR in GT1-1 cells pretreated with Dex for different times. As shown in Fig. 8AGo, Dex caused a transient rise in MC4 receptor mRNA level that was maximal after 3 to 6 h. MC4 receptor mRNA progressively decreased with longer times of Dex exposure. Incubation of GT1-1 cells with vehicle for the same times did not cause any modulation of MC4 receptor mRNA concentration. Inclusion of actinomycin D during the treatment with Dex caused a significant drop in MC4 receptor mRNA and prevented the Dex effect (Fig. 8BGo).

Like the MC4 receptor, the MC3 receptor is expressed in neurons, coupled to Gs and activated by NDP-{alpha}MSH. MC3 receptor mRNA has been reported in some lines of GT1 cells (35). To rule out the possibility that the Dex effects described above resulted in part from induction of MC3 receptor, we also measured MC3 receptor mRNA by RT-PCR in GT1-1 cells after different times of incubation with Dex (Fig. 8CGo). MC3 receptor mRNA was not detected in any samples, showing that the MC3 receptor is not normally expressed in this cell line and its mRNA is not induced by glucocorticoids.

Reversal of Dex effects
Because glucocorticoid concentrations vary physiologically, it was of interest to determine how rapidly MC4 receptor levels would decline when Dex was withdrawn. To examine this, cells were incubated with Dex for 30 h, when Dex was removed and the glucocorticoid receptor antagonist RU486 was added to prevent rebinding of dissociated hormone. The amount of [125I]NDP-{alpha}MSH bound and the cAMP response to NDP-{alpha}MSH both fell promptly, returning halfway toward baseline after approximately 8 h (Fig. 9Go). These results suggest a fairly rapid turnover of the MC4 receptor in the absence of glucocorticoids.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MC4 receptor plays a critical role in regulating food intake and energy expenditure. In vivo, signaling through the MC4 receptor pathway will depend on the relative concentrations of the natural agonist, {alpha}MSH, and the endogenous inverse agonist, agouti-related protein, as well as signals from any intersecting pathways. Receptor activity will also depend on the concentration of receptor expressed on the plasma membrane of hypothalamic neurons. The density of receptors appears to be critical, because haploinsufficiency of MC4 receptors is sufficient to cause severe obesity (3, 37). In this study, we have shown that glucocorticoids induce expression of functional MC4 receptor in a hypothalamic neuronal cell line expressing the receptor endogenously. This model system has allowed us to study modulation of receptor activity without the bias of overexpression in a situation where the MC4 receptor gene is under the control of its natural promoter. An important limitation of the model is that GT1-1 is a GnRH-secreting cell line, not a direct model for studying mechanisms regulating appetite.

Glucocorticoids increased the cAMP response to activation of endogenous MC4 receptors but did not alter the response to isoproterenol, which acts on endogenous ß-adrenergic receptors. This shows that glucocorticoids do not exert general effects on common downstream signaling components such as Gs, adenylyl cyclase, or cyclic nucleotide phosphodiesterases. Dex also increased the ability of NDP-{alpha}MSH to activate ERK1/2, a response that is reported to be mediated by protein kinase A when MC4 receptor agonists are administered in vivo (24), to involve phosphatidylinositide 3 kinase and not protein kinase A in transfected CHO cells (23), and to be mediated by protein kinase C in GT1-1 cells (22).

The ability of glucocorticoids to increase MC4 receptor signaling can be attributed to an increase in the concentration of functional MC4 receptors on the plasma membrane. The IC50 value for NDP-{alpha}MSH binding to MC4 receptors in GT1-1 cells, 0.9 nM, is close to the EC50 of 1 nM for NDP-{alpha}MSH-stimulated cAMP production. The similar concentration dependence of binding and second messenger generation implies that there is not a large population of spare receptors and that changes in receptor concentration should alter the efficacy but not the potency of agonists.

The fact that Dex has no effect on binding or signaling of transfected MC4 receptors suggests that glucocorticoids act by regulating transcription from the endogenous promoter. In agreement with previous findings, we found that signaling in GT1-1 cells was strongly desensitized (36); a second stimulation with NDP-{alpha}MSH produced a much lower response than the initial stimulation. Because desensitization was not changed by Dex treatment, it is unlikely that altered desensitization contributes to potentiation of MC4 receptor signaling.

Glucocorticoids caused a transient increase in mRNA encoding the MC4 receptor that peaked at 3–6 h, earlier than the increase in MC4 receptor protein. The increase in mRNA averaged 3-fold in different experiments and was sufficiently large to account for much or all of the increase in MC4 receptor. The rapid fall in MC4 receptor mRNA after actinomycin D addition suggests that the message is short lived, and the ability of actinomycin D to block induction by Dex points to increased transcription rather than message stabilization as the mechanism of the Dex effect. The increase in receptor mRNA was transient, whereas the increase in receptor protein was sustained, indicating that the receptor is very stable when Dex is present. MC4 receptor binding and signaling returned to basal levels within 12 h after Dex withdrawal, indicating that the protein may be less stable in the absence of glucocorticoids.

Traditionally, glucocorticoids have been thought to act through one of two nuclear receptors, glucocorticoid or mineralocorticoid receptors. More recently, it has been appreciated that steroid hormones, including glucocorticoids, can also exert direct actions at cell membranes (38). The high affinity of Dex (EC50 10–15 nM) and the ability of RU486 to reverse Dex effects suggest that the nuclear glucocorticoid receptor is involved in glucocorticoid regulation of MC4 receptor concentrations. Dex failed to modulate the expression of MC4 receptor stably expressed in CHO cells under the control of the cytomegalovirus promoter, suggesting a requirement for regulatory regions in the natural promoter. Classical glucocorticoid response elements have not been found in the proximal promoter region of the MC4 receptor gene, but SP1, AP1 and CREB sites have been identified (39, 40). The glucocorticoid receptor may be acting directly at noncanonical sites or indirectly by interacting with other transcription factors such as those binding to the SP1, AP1 or CREB sites. Alternatively, glucocorticoids may act indirectly through effects on other genes.

The physiological significance of glucocorticoid regulation of MC4 receptor signaling is uncertain. Adrenalectomized animals are anorexic, and administration of glucocorticoids promptly increases food intake in these animals (29). Adrenalectomy reverses the obesity phenotypes in several genetic models of obesity (41, 42). In intact rodents, however, centrally administered glucocorticoids cause increased food intake and weight gain, but peripherally administered glucocorticoids are catabolic (43). High doses of glucocorticoids clearly increase food intake in humans (30). Glucocorticoids blunt the ability of leptin to signal satiety, which may contribute to their hyperphagic effects (41). It has also been reported that the ability of intracerebroventricular melanotan II, an MC4 receptor agonist, to suppress food intake is larger in adrenalectomized than sham-operated animals (44).

We initiated these studies to test the possibility that the hyperphagic effects of glucocorticoids involve direct inhibition of MC4 receptor systems. Unexpectedly, we found the opposite: glucocorticoids potentiate MC4 receptor signaling, a change expected to reduce food intake. The effects of steroid hormones on MC4 receptors have not been studied previously, but adrenalectomy is reported to cause no significant change in hypothalamic MC4 receptor mRNA in rats (44). The difference between the in vivo and in vitro data could result from the different model systems or the time scale of the studies, because we found that glucocorticoid effects on MC4 receptor mRNA were transient, whereas those on protein were sustained. Intracerebroventricular injection of agouti-related protein was found to have no effect on food intake in adrenalectomized rats unless glucocorticoids were replaced (44). This could be explained if glucocorticoids are needed to maintain MC4 receptor concentrations. Glucocorticoid potentiation of MC4 receptor activity may also contribute to the anorexia seen in stressful situations in which glucocorticoids are elevated. Indeed, stress has been shown to increase hypothalamic MC4 receptor mRNA concentration in rats (45).

Interplay between glucocorticoid and melanocortin systems may also be important in inflammation. Glucocorticoids are potent antiinflammatory hormones, and activation of MC3 and MC4 receptors reduces inflammation in brain, decreasing nitric oxide production and nuclear translocation of nuclear factor-{kappa}B (10). It is plausible that the ability of glucocorticoids to increase MC4 receptors in neurons is one of the means by which glucocorticoids reduce inflammation in the brain. Although additional work is required to elucidate the physiological significance of the glucocorticoid effects we have described, our findings suggest that hormonal regulation of hypothalamic MC4 receptor signaling may contribute to the complex regulation of energy balance and to other melanocortin responses.


   Footnotes
 
This research was supported by National Institutes of Health Grant DK19974.

J.A.S. and P.M.H. have nothing to declare.

First Published Online September 14, 2006

Abbreviations: Dex, Dexamethasone; IBMX, 3-isobutyl-1-methylxanthine; MC4, melanocortin-4.

Received July 21, 2006.

Accepted for publication September 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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