Brain Research 986 (2003) 1–11www.elsevier.com/ locate/brainres

Research report

M elanocortin receptor signaling through mitogen-activated proteinkinase in vitro and in rat hypothalamus

a , c a ,1 a*Derek Daniels , Caroline S. Patten , Jonathan D. Roth , Daniel K. Yee ,a,b,cSteven J. Fluharty

aDepartment of Animal Biology, University of Pennsylvania, 3800 Spruce Street, 254E, Philadelphia, PA 19104,USAbDepartment of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104,USA

cInstitute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104,USA

Accepted 11 June 2003

Abstract

The central melanocortin system has emerged as a potential regulator of food intake. This action of melanocortins appears to occurthrough intrahypothalamic, melanocortin-containing projections, including those from the arcuate to the paraventricular nucleus (PVN).Although the complexity of feeding behavior and the long duration of the effects of melanocortins on food intake suggest changes in geneexpression, the mechanism by which such changes occur has been elusive. In the present report, we describe experiments using in vitroand in vivo approaches to demonstrate melanocortin-induced phosphorylation (activation) of members of the mitogen-activated proteinkinase (MAPK) family of transcription factors. First, application of the melanocortin agonist MTII to COS-1 cells resulted in an increasein phosphorylated MAPK after the cells were transfected with the melanocortin type 4 receptor (MC4-R), but not the type 3 receptor.Formation of cAMP, however, was observed when either receptor subtype was transfected. Subsequent experiments revealed that theeffect of MTII on MAPK activation in MC4-R-transfected cells was dose-dependent and was maximal after 10 min of MTII exposure.Second, central injections of MTII increased the number of phospho-MAPK-immunoreactive cells in the rat PVN compared tovehicle-injected animals. When coupled with immunohistochemical identification of PVN neurons containing oxytocin, a clear segregationwas apparent, allowing for a precise anatomical description of the pattern of activated MAPK within the PVN. These data are the first tosuggest a differential coupling of MC4-R and may describe a mechanism through which the long-term and persistent behavioral actions ofmelanocortins are mediated. 2003 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Ingestive behaviors

Keywords: cAMP; Melanocortin receptor; MTII; Mitogen-activated protein kinase; Paraventricular hypothalamus; Transfection; Signal transduction

1 . Introduction arcuate nucleus[29]. One putative target of these neuronsis a population of melanocortin receptor (type 4; MC4-R)-

The central melanocortin system has been implicated in containing neurons within the hypothalamic paraventricu-the control of food intake and energy homeostasis. En- lar nucleus (PVN)[1,15,30].dogenously, this action of melanocortins is thought to Ample behavioral evidence exists for the role ofoccur downstream of the leptin receptor through in- melanocortins in the regulation of food intake. For in-trahypothalamic, melanocortin-containing projections from stance, central administration of melanocortin receptorleptin receptor-containing neurons within the hypothalamic agonists, including the endogenously expresseda-

melanocyte stimulating hormone (a-MSH) and the syn-thetic MTII, decreased food intake[8,11,23,25],an effect

*Corresponding author. Tel.:11-215-898-9149; fax:11-215-573-that appears to be dependent on the expression of MC4-R5186.[16]. Conversely, central administration of MC3/4-RE-mail address: derekd@vet.upenn.edu(D. Daniels).

1Present address: Amylin Pharmaceuticals, San Diego, CA 92121, USA. antagonists, including the endogenously expressed agouti

0006-8993/03/$ – see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0006-8993(03)03162-7

2 D. Daniels et al. / Brain Research 986 (2003) 1–11

related protein (AgRP) and the synthetic SHU9119, in- Following 48 h differentiation of N1E-115 cells or isola-creased feeding and/or blocked melanocortin- or leptin- tion of adult male rat hypothalamus, total RNA wasinduced decreases in feeding[8,11,25,30,35]. isolated using TRIZOL, and cDNA was generated by

In addition to behavioral studies, anatomical investiga- first-strand synthesis using random primers. The MC4-Rtions have been consistent with this proposed function of was cloned following two rounds of PCR with the firsthypothalamic melanocortin neurons. Specifically, neurons round employing the primers F1 (forward/sense primerwithin the arcuate nucleus that co-express leptin receptor 59-GGTTGGATCAGTTCAAGGAGGAC-39) and R1 (re-and either AgRP or thea-MSH precursor, pro- verse/antisense primer 59-GCATTACACAGAAGAGGC-opiomelanocortin (POMC), have been observed[4,36]. AGCTG-39), followed by a second round using the primersFurthermore, terminal fields from AgRP- or POMC-con- F2 (forward/sense primer 59-CAAATCCAGCTGCTGCA-taining projections and melanocortin receptors, especially GGAAG-39) and R2 (reverse/antisense primer 59-GGTA-MC4-R, have been found in the PVN[1,15,20], a brain TCTACCTAGTTTGCACTC-39). The MC3-R was clonedarea in which robust melanocortin-induced Fos expression using similar procedures using the primers F1 (forward/has been observed[2,17]. Thus, these data strongly sense primer 59-GAATTTTCCCCAGCAGCTTGC-39) andimplicate the PVN as a target and potential mediator of R1 (reverse/antisense primer 59-GCACAGTTCCAGCTC-leptin-related, melanocortin actions. CTAAC-39), followed by a second round using the primers

Although the complexity of feeding behavior and the F2 (forward/sense primer 59-GGGACTGGACCTGCTGT-long duration of the effects of melanocortins on food TAACC-39) and R2 (reverse/antisense primer 59-CTGGA-intake suggest changes in gene expression, the mechanism TGTGGAACACCTCCAC-39). All PCR conditions wereby which such changes occur has been elusive. Melanocor- 30 cycles of 948C (1 min), 608C (1 min), and 728C (1tin receptors are members of the superfamily of G-protein min), followed by a 10-min extension step at 728C. Thecoupled receptors (GPCRs), whose ‘traditionally’ ascribed resultant PCR products were subcloned into the vectoreffectors, e.g., cAMP or inositol trisphosphate, do not pCR2.1 (Invitrogen, Carlsbad, CA, USA) for subsequentdirectly modulate gene expression. However, recent reports propagation and DNA sequencing. DNA sequencing ver-describe the ability of several GPCRs to signal through the ified that the isolated MC3- and MC4-R cDNAs weremitogen-activated protein kinase (MAPK) pathway, a identical to other previously reported murine MC3- andsignaling mechanism that does directly alter gene expres- MC4-R cDNAs[40,41], respectively. The isolated MC3-sion in cells[22]. In the present experiments, we used two and MC4-R cDNAs were later subcloned into the expres-approaches to provide evidence that melanocortin applica- sion vector pCR3 (Invitrogen) for use in all COS-1 celltion results in the phosphorylation (activation) of the transfections.transcription factors p42 and p44 MAPK. These factors,also known as extracellular signal related protein kinase 1 2 .2. Cell culture and transfectionsand 2 (ERK1 and ERK2), are abundant, ubiquitous, andhave recently become recognized for their role in signal COS-1 cells were grown in polystyrene tissue culturetransduction[22]. First, although formation of cAMP was flasks in medium consisting ofD-MEM (Gibco, Gaithers-observed when COS-1 cells were transfected with either burg, MD, USA) supplemented with 10% fetal bovineMC3- or MC4-R, increased activation of MAPK only serum (FBS),L-glutamine, and penicillin–streptomycin inoccurred in MC4-R-transfected cells. Second, we observed a humidified atmosphere of 5% CO and 95% O at 378C.2 2

MAPK phosphorylation in the PVN, a known terminal MC3-R or MC4-R was later introduced into the COS-1field of POMC- or AgRP-containing neurons[1], after cells by transfection with LipofectAMINE (Gibco) for 5 h,acute lateral ventricular injections of MTII into male rats. after which the transfection medium was removed andTaken together, these experiments provide evidence that replaced with normal growth medium. Additional cellsactivation of MC4-R results in the phosphorylation of were used that were untransfected or transfected with theMAPK and present the novel finding of different signaling vector only. Drug treatments occurred approximately 72 hcapabilities of the two receptor subtypes. post-transfection.

2 .3. In vitro drug treatments2 . Methods

MC3-R-, MC4-R-, or control-transfected cells were2 .1. Cloning of MC3- and MC4-R treated with vehicle [D-MEM with 4-(2-hydroxyethyl)-1-

piperazine ethanesulfonic acid (HEPES),L-glutamine, and4 5Full-length clones of MC3- or MC4-R were obtained penicillin–streptomycin] or MTII [(Ac–Nle ,Asp ,D–

7 10through reverse transcriptase polymerase chain reaction Phe ,Lys )-cyclo-a-MSH (4–10) amide; Bachem Bio-(rt-PCR) from differentiated murine neuroblastoma cells science, King of Prussia, PA, USA] 18 h following a(N1E-115), which have been shown previously to express medium change toD-MEM supplemented with HEPES,MC3-R RNA [26], or rat hypothalamus, respectively. L-glutamine, and penicillin–streptomycin. After a pre-de-

D. Daniels et al. / Brain Research 986 (2003) 1–11 3

termined time of exposure, the cells were rinsed with ice reactions according to manufacturer’s instructions (West-cold phosphate-buffered saline (PBS), lysed, homogenized, ern Lightning kit; Perkin-Elmer Life Sciences, Boston,and centrifuged to remove the membrane fraction. Protein MA, USA).concentrations of the remaining lysates were quantified bybicinchoninic acid (BCA) assay (Pierce, Rockford, IL,

2 .6. Animals and surgical proceduresUSA) according to manufacturer’s instructions.

Male Sprague–Dawley rats (300–325 g) were obtained2 .4. cAMP assays

from Charles River Laboratories (Wilmington, MA, USA).Animals were housed in plastic cages in a temperature-

MC3-R-, MC4-R-, or control-transfected COS-1 cellscontrolled room (228C) with a 12 h light /dark cycle with

were incubated overnight in serum-freeD-MEM containingfood and water available ad libitum. All experimental33.0 mCi/ml of [ H]adenine. The medium was replacedprocedures were conducted between 2 and 6 h after the

with control or drug-containing medium (see In vitro druglights were illuminated. The handling and care of ex-

treatments above) with the addition of 1 mM iso-perimental animals conformed to the regulations provided

butylmethylxanthine (IBMX; Sigma–Aldrich, St. Louis,by the NIH Guide for the Care and Use of Laboratory

MO, USA), in which the cells were incubated for 10 min.Animals, and the experimental protocols were approved by

The drug treatment was removed, the cells were rinsedthe Institutional Animal Care and Use Committee of the

with ice cold PBS and terminated in 1 ml of 10%University of Pennsylvania.

trichloroacetic acid (TCA; Fisher Scientific, Pittsburg, PA,Under anesthesia with ketamine and xylazine (90 and 12

USA). The cells then were scraped and centrifuged. Themg/kg, respectively), stereotaxically guided injections of

resultant pellet was solublized for 24–48 h in 500ml ofvehicle (1ml saline;n55) or MTII (1 nmol in 1ml; n55)

1% sodium dodecyl sulfate (SDS) in 0.1 M NaOH andwere made into the lateral ventricle (0.9 mm posterior and

assayed for protein content by BCA assay (Pierce) accord-1.4 mm lateral to Bregma; 3.4 mm ventral to dura). Ten3ing to manufacturer’s instructions. [ H]cAMP was isolatedminutes after the injection, animals were perfused with

and measured by passing the supernatant from the cen-saline followed by 4% paraformaldehyde in 0.1 M phos-

trifuged lysates through sequential Dowex and aluminaphate buffer (PB). Additional rats (n53) were perfused

columns before elution into scintillation vials, the radioac-without receiving injections. The brains were removed,

tivity of which were quantified. Calibration curves of thepostfixed for no more than 24 h, submerged in 20%3 3elution profiles of [ H]adenine and [ H]cAMP were usedsucrose in 0.1 M PB for at least 48 h, and a block of tissue

to predetermine which fractions of eluent were collectedcontaining the full extent of the hypothalamus was cut into3for analysis. All relative measures of [ H]cAMP werefour sets of 30mm coronal sections on a freezing mi-

expressed and analyzed as counts per minute (cpm) per mgcrotome. Sections were either processed immediately or

of protein.stored in cryoprotectant[34] before being processedimmunohistochemically as described below.

2 .5. Western blotting for total and phosphorylatedMAPK

2 .7. Phospho-MAPK immunohistochemistryA 15–20-mg amount of protein from each sample were

run on a 10% SDS–polyacrylamide gel electrophoresis One set of free-floating sections from each animal was(PAGE) gel and then subsequently transferred to a nitro- washed in Tris-buffered saline (TBS; pH 7.4) and incu-cellulose membrane. Membranes were pre-blocked with bated for 15 min in TBS containing H O (1:100) before2 2

blotto (5% nonfat dried milk in PBS, 0.05% thimerosal) being incubated overnight at room temperature in mousebefore a 75-min incubation with a polyclonal antibody anti-phospho-p44/42 MAPK (1:500; Cell Signaling Tech-directed against p44 MAPK that cross-reacts with p42 nology) diluted in TBS containing 0.2% Triton X-100 andMAPK (1:5000; Santa Cruz Biotechnology, Santa Cruz, 3% normal donkey serum (NDS). Sections then wereCA, USA) or a monoclonal antibody directed against washed in TBS and incubated for 2 h with biotinylatedphospho-p44/42 MAPK (1:1000; Cell Signaling Technol- donkey anti-mouse IgG (1:1000; Jackson Immuno-ogy, Beverly, MA, USA). After being washed with PBS research). After a brief wash with TBS, sections werecontaining 0.05% Tween (TPBS; Sigma–Aldrich), the incubated for 1 h in an avidin–biotin–peroxidase complexmembranes were incubated for 45 min with peroxidase- (1:333; Elite Kit; Vector Laboratories, Burlingame, CA,conjugated goat anti-rabbit IgG (1:5000; Jackson Immuno- USA). Sections were washed with TBS and then with 50research Laboratories, West Grove, PA, USA) or goat mM Tris (pH 7.4) before immunoreactivity was visualizedanti-mouse IgG (1:2000; Jackson Immunoresearch Lab- by reacting the sections with 3,39-diaminobenzidine (0.2oratories), respectively. The blots were washed extensively mg/ml) and 0.025% H O in 50 mM Tris for 5 min. The2 2

with TPBS, followed by a single wash with PBS before reaction was stopped with TBS washes, after which theimmunoreactivity was detected using chemiluminescence sections were floated onto gelatinized slides, dehydrated

4 D. Daniels et al. / Brain Research 986 (2003) 1–11

with increasing concentrations of alcohol followed by 3 . ResultsHemo-De (Fisher), and coverslipped with Permount(Fisher). 3 .1. MTII-induced formation of cAMP in transfected

COS-1 cells

2 .8. Oxytocin and phospho-MAPK double-labeling To confirm the expression and presence of functionalreceptors in MC3-R- and MC4-R-transfected COS-1 cells,

Sections from a subset (n53) of the animals described we tested for MTII-induced formation of cAMP, a memberabove were stained for both phosphorylated MAPK and of a well-established signal transduction pathway, in cellsoxytocin. Free-floating sections were washed in TBS expressing MC3- or MC4-R[9,10]. As illustrated inFig. 1,before being incubated overnight at room temperature in in each of three independent experiments, exposure to 10mouse anti-phospho-p44/42 MAPK (1:100) and guinea nM MTII for 10 min increased cAMP formation in cellspig anti-oxytocin (1:2500; Peninsula Laboratories, Bel- transfected with either MC3-R or MC4-R, but not in cellsmont, CA, USA) diluted in TBS containing 0.2% Triton transfected with the vector only. A two-way ANOVAX-100 and 3% NDS. Sections then were washed in TBS revealed statistically significant main effects of transfectionand incubated for 2 h with Cy2-conjugated donkey anti- condition (F 512.4, P50.001), drug treatment (F 52,12 1,12

guinea pig (1:100; Jackson Immunoresearch) and Cy3- 37.4,P,0.001), and the interaction of the two (F 58.4,2,12

conjugated donkey anti-mouse (1:100; Jackson Immuno- P50.005). Student–Newman–Keuls method for multipleresearch). Sections were washed with TBS, floated onto comparisons revealed that the MTII-treated, MC3-R- andgelatinized slides, and coverslipped with Vectashield (Vec- MC4-R-transfected groups did not differ from each other,tor Laboratories). but were greater than all other groups.

2 .9. Data analysis 3 .2. MTII-induced activation of MAPK in transfectedCOS-1 cells

Digital images of Western blots were analyzed bydensitometry using Scion Image (Scion Image version An initial set of experiments (data not shown) was4.0.2; Scion, Frederick, MD, USA). When appropriate, designed to test for differences in the baseline levels ofvariance in optical density between experiments was phosphorylated MAPK when cells were transfected withcontrolled for by the use of a repeated measures analysis of MC3- or MC4-R compared to control transfection con-variance (ANOVA) and the dose–response analysis was ditions. To this end, in a total of six experiments, COS-1performed using Prism 3.0 (GraphPad Software, San simian kidney cells that were transfected with MC3- orDiego, CA, USA). Tissue sections throughout the rostral-caudal extent of the hypothalamus were analyzed by lightor fluorescent microscopy. For the quantification of acti-

vated MAPK in singly labeled tissue, six digital images ofPVN hemisections were captured from each animal. Thenumber of phospho-MAPK-immunoreactive neurons ineach image was manually counted on a printed version ofeach image with the treatment condition and animalnumber unknown until after quantification. Both lightlyand darkly stained cells were counted. Neuroanatomicalanalysis of the PVN was conducted by comparing land-marks within the section to atlas drawings[31] and theapproximate distance of each section from Bregma wascalculated based primarily by comparison with Paxinosand Watson[21]. For the rostral-caudal analysis of phos-pho-MAPK-immunoreactive neurons, one section (twohemisections) per animal for each level was used. Addi-tional qualitative anatomical analysis was conducted insections stained for both oxytocin and activated MAPK

Fig. 1. MTII-induced formation of cAMP in control-, MC3-R-, or MC4-using the well-documented distribution of oxytocin withinR-transfected COS-1 cells. Asterisks indicate groups that were statistical-

the PVN [27] for reference. All statistical comparisons ly significant from unmarked groups (P,0.05) using a two-way ANOVAwere made using Sigma Stat (version 2.03; SPSS, Chicago,with Student–Newman–Keuls method of multiple comparisons. The dataIL, USA). from three independent experiments are expressed here as mean6S.E.M.

D. Daniels et al. / Brain Research 986 (2003) 1–11 5

MC4-R were vehicle-treated and cell lysates were assayedfor phosphorylated MAPK. Two types of control transfec-tion conditions were used: untreated cells or cells that weretransfected with the vector only. A statistical analysisfailed to reveal differences between the two controltransfection conditions (one-way ANOVA,F 50.003,1,4

P50.96). Accordingly, these groups were collapsed forfurther analysis. Western blot analysis of phosphorylatedp42/44 MAPK also failed to show differences in thebaseline levels of phosphorylated MAPK after vehicletreatment when cells had been control-transfected ortransfected with MC3- or MC4-R (one-way repeatedmeasures ANOVA,F 50.35, P50.71). Measures of2,10

total MAPK did not show differences between the treat-ment groups.

To further investigate the nature of the activation ofMAPK through MC3- or MC4-R, experiments were con-ducted to examine the dose–response relationship of theactivation. Dose–response profiles were generated througha series of 3–4 independent experiments for each of thereceptor subtypes using 10 min exposures of a range of six

212 26MTII concentrations (1310 to 1310 M), as well as avehicle-only group (Fig. 2). Concentrations of MTII werechosen to be comparable with those used in the present andprevious reports to demonstrate activation of cAMP[9,10,13,14,28].Analysis of variance using a two-waydesign revealed statistically significant main effects oftransfection condition (F 5121.42, P,0.001), dose1,40

(F 57.414,P,0.001), as well as a statistically signifi-7,40

cant interaction (F 56.145, P,0.001). Comparison of7,40

the MTII dose–response profile within the MC3-R- orMC4-R-transfected conditions using Dunnett’s method ofmultiple comparisons revealed that 10 min of MTII-treat-

Fig. 2. Dose–response analyses of MAPK activation in COS-1 cells thatment failed to reliably increase activation of MAPK abovewere transfected with MC3-R or MC4-R and subsequently exposed tovehicle at any concentration in MC3-R-transfected cells. InMTII for 10 min. Panels A and B show representative immunoblots ofcontrast, when cells were transfected with MC4-R, 10 mindose–response analyses of phosphorylated (top) or total MAPK (bottom)210exposure to concentrations of MTII ranging from 1310 in cells transfected with MC3-R (panel A) or MC4-R (panel B). Panel C

26to 1310 M was associated with increases in phos- illustrates the data from experiments examining phosphorylated MAPK incells transfected with MC3-R (circles;n54) or MC4-R (squares;n53).phorylated MAPK (EC 50.3060.18 nM;Fig. 2), without50The data are expressed as percent of the mean response of the vehicle-associated changes in total MAPK.treated cells within each transfection condition (mean6S.E.M.). StatisticalTo further examine the effect of MTII on MAPKanalysis using a two-way ANOVA with Dunnett’s method of multiple

activation through MC4-R, a time course analysis was comparisons failed to show MTII-induced increases in phospho-MAPK inperformed. This analysis included a total of four experi- MC3-R-transfected cells; however, increasing concentrations of MTII

28 were associated with increased phospho-MAPK in MC4-R-transfectedments in which the cells were exposed to MTII (1310cells. Asterisks indicate doses that differed reliably from vehicle-treatedM) for 0, 0.5, 1, 5, 10, 30, or 60 min. As shown inFig. 3,cells within the same transfection condition.maximal levels of phosphorylated MAPK were achieved

when MC4-R-transfected COS-1 cells were exposed toMTII for 10 min. Statistical analysis of these data using arepeated measures one-way ANOVA revealed a statistical-3 .3. Intraventricular application of MTII in vivoly significant effect of MTII dose (F 54.49,P50.006).6–18

Subsequent post hoc tests using Dunnett’s method of Based on the finding that MAPK activation can proceedmultiple comparisons showed that the 5, 10, 30, and 60 through the MC4-R in vitro, we examined the effect ofmin groups had greater levels of phosphorylated MAPK exogenous melanocortin application in vivo. A qualitativewhen compared with the 0 min group. Changes in total examination of the rostral-caudal extent of the hypo-MAPK were not detected. thalamus revealed that the PVN was the only site of

6 D. Daniels et al. / Brain Research 986 (2003) 1–11

Fig. 3. The time course of MTII-induced phosphorylation of MAPK after MC4-R-transfected COS-1 cells were treated with MTII (10 nM). Representativeimmunoblots showing activated (top) and total (bottom) MAPK are shown in panel A. The quantification of four independent experiments is shown inpanel B. Repeated measures one-way ANOVA revealed a statistically significant effect of MTII dose (F 54.485,P50.006). Asterisks indicate time6,18

points that differed reliably from 0 min using Dunnett’s method of multiple comparisons. For clarity, the data are shown here as the percent of the meanresponse of the 0 min group (mean6S.E.M.).

MTII-induced changes in MAPK activation. Phos- (F 50.277,P50.8908) was detected. A subsequent one-4,30

phorylated MAPK-immunoreactivity was not readily de- way ANOVA with Student–Newman–Keuls post hoc teststected in other melanocortin receptor-expressing hypo- using the average number of immunoreactive cells perthalamic areas such as the ventromedial, arcuate, or section, without consideration of rostral-caudal distribu-dorsomedial nuclei. tion, confirmed the effect of MTII on the number of

Quantitative examination of the number of phospho- phosphorylated MAPK-immunoreactive neurons (F 51,11

MAPK-immunoreactive neurons in the PVN did not reveal 5.87,P50.034;Fig. 5B).differences between the saline-injected or the non-injected An initial attempt to define the neurochemical phenotypecontrols (F 50.839, P50.40). As such, these animals of the PVN neurons with MTII-induced phosphorylated1,6

were combined for further analysis.Fig. 4 shows the MAPK employed double-label immunohistochemistry tophospho-MAPK-immunoreactivity observed in coronal test for co-localization of phospho-MAPK and oxytocin. Asections from representative rats treated for 10 min with subset (n53) of the animals that were injected with MTIIeither saline (1ml; Fig. 4A and B) or MTII (1 nmol in 1ml were used in these experiments. Qualitative analysis of thesaline; Fig. 4C and D). A qualitative comparison of the tissue from these animals revealed virtually no double-distribution of phospho-MAPK-immunoreactive neurons in labeling of phosphorylated MAPK and oxytocin. Somethe PVN with the subdivisions of the PVN as described by yellow fluorescence, indicative of double-labeling, wasSwanson[31] suggested that MTII activation of PVN found to occur, but further examination revealed that thisneurons occurred within most subdivisions of the PVN, was from overlapping portions of phospho-MAPK andwith intense labeling within the dorsal and medial par- oxytocin neurons, with no instances of double-labeledvocellular subdivisions of the PVN. As shown inFig. 5A, neurons. In fact, the anatomical distribution of the twothe number of phosphorylated MAPK-immunoreactive labels, phospho-MAPK and oxytocin, demonstrated aneurons was greater across the rostral caudal extent of the nearly complete segregation (Fig. 6). The well-describedPVN in MTII-treated animals compared to controls. distribution of oxytocin[27], however, served as a land-Quantification and statistical analysis of these data using a mark with which to compare the distribution of the MTII-two-way ANOVA (drug treatment3rostral caudal location; induced phosphorylation of MAPK, allowing for a moreone section per animal per rostral caudal level) with precise qualitative anatomical description of the pattern ofStudent–Newman–Keuls post hoc tests revealed a statisti- activated MAPK. Specifically, cells with activated MAPKcally significant main effect of drug treatment (F 5 were detected in most PVN subdivisions, with the highest2,30

7.873, P50.0018). However, neither a rostral caudal density of labeling appearing to occur in the dorsal portiondifference (F 50.637, P50.5360) nor an interaction of the medial parvocellular subdivision. We also found a2,30

D. Daniels et al. / Brain Research 986 (2003) 1–11 7

Fig. 4. Digital micrographs of coronal sections including the PVN from representative rats 10 min after an injection of vehicle or MTII (1 nmol) into thelateral ventricle. Images from rats injected with 1ml saline (A) or 1 nmol MTII (C) are shown. The boxed areas in A and C are shown at highermagnification in B and D, respectively. A and C: bar5200 mm; B and D: bar580 mm; 3V5third ventricle.

notable and almost complete absence of labeled cells in the MC3- or MC4-R. Subsequent studies used immunohisto-ventral part of the medial parvocellular subdivision of the chemistry to demonstrate that the melanocortin agonistPVN. MTII can activate the same transcription factors in the rat

PVN, a brain area that receives POMC and AgRP projec-tions, in which MC4-R is richly expressed, and in which

4 . Discussion robust melanocortin agonist-induced Fos expression hasbeen observed[1,2,15,17,20].

The present experiments were designed to test the The in vitro experiments in the present report usedhypothesis that MC3- or MC4-R are able to signal through transfected COS-1 cells to reveal MTII-induced MAPKmembers of the MAPK family of transcription factors activation when the cells were transfected with MC4-R,when activated by MTII. In order to explore this novel but not when MC3-R was transfected. MTII was highlysignaling capability of these receptors, we initially used in potent in activating MAPK in MC4-R-transfected cellsvitro approaches, specifically COS-1 cells transfected with (EC50.3060.18 nM; seeFig. 2), whereas MTII did not50

8 D. Daniels et al. / Brain Research 986 (2003) 1–11

Fig. 6. A representative micrograph of oxytocin- (green) and phospho-MAPK- (red) immunoreactivity within a coronal section containing thePVN of a male rat treated with 1 nmol MTII is shown in panel B. Panel Acontains a line drawing with a boxed area showing the approximatelocation from which the micrograph in panel B was taken. Abbreviationsnot in the text: 3V, third ventricle; f, fornix; ot, optic tract. Scale bar550mm.

finding, taken together with the identical transfectionprotocol used for either receptor subtype, strongly suggeststhat fully functional receptors were expressed by thetransfected cells for both receptor subtypes and that the

Fig. 5. Quantification of the number of phospho-MAPK-immunoreactiveobserved differences in MTII-induced MAPK activationcells in the PVN. Panel A: Analysis of phospho-MAPK-immunoreactivityreflect differences in the molecular properties of MC3- andacross the rostral-caudal axis of the PVN revealed a statistically signifi-

cant increase in the number of activated cells after MTII injections (n55) MC4-R, not differences in the expression of the differentcompared with controls (n58; vehicle- or non-injected; two-way ANOVA receptors.with Student–Newman–Keuls post hoc tests;P,0.05) without a rostral- In addition to the findings from in vitro experiments, thecaudal difference. Data are shown as mean6S.E.M. with the data from

present data show an increase in the number of cells in thethe most rostral sections to the left. Panel B: Subsequent analysis usingrat PVN that are immunoreactive for phosphorylatedthe mean number of labeled cells per hemisection without parsing the

data based on rostral caudal location within the PVN confirmed the effect (activated) MAPK when rats were treated with a dose ofof MTII on the number of phospho-MAPK-immunoreactive neurons MTII that was shown previously to substantially decrease(one-way ANOVA,F 55.87, P50.03).1,11 food intake in rats or mice[11,16]. The activation of PVN

neurons by MTII is consistent with earlier reports ofactivate this signaling pathway in cells transfected with melanocortin agonist-induced Fos expression in this brainMC3-R, even when high, saturating concentrations of the area[2,17]. With respect to these data and the presentlyagonist were used. The finding that transfection of either reported activation of MAPK, previous observations ofreceptor subtype permitted MTII-induced formation of MC4-R in the PVN[15,20] and the present in vitrocAMP is consistent with previous reports of this signaling experiments suggest that at least a subset, if not all of theresponse for both of these receptor subtypes[9,10]. This neurons containing MTII-induced activated MAPK were

D. Daniels et al. / Brain Research 986 (2003) 1–11 9

activated by the direct activation of MC4-R, rather than to the dorsal portion of the medial parvocellular subdivi-transsynaptically through an intermediary set of MC4-R- sion of the PVN. Notably absent, however, was labeling inexpressing cells. In fact, the distribution of MTII-induced the ventral portion of the medial parvocellular subdivision.activation of MAPK appears to be more limited than the Although not explicitly stated, a similar pattern of labelingactivation of Fos[2], perhaps reflecting a greater spe- throughout the PVN, including the lack of labeling in thecificity of the MAPK response. The possibility remains, ventral medial parvocellular PVN, is apparent in thehowever, that some of the activated MAPK observed in representative micrographs included in previous descrip-these experiments resulted from indirect (i.e., transsynap- tions of immunostaining for either AgRP fibers[1] or celltic) activation. The finding that PVN neurons that exhibit bodies containing corticotropin releasing factor (CRF)MTII-induced MAPK activation express MC4-R would [19], which, like the melanocortin system, has beenstrongly support the notion of a direct coupling between implicated in the control of food intake[3,12]. AlthoughMC4-R and MAPK. This issue, however, remains to be the physiological significance of the potential overlap ofaddressed empirically. AgRP-containing fibers, CRF, and melanocortin-induced

Although the present analysis included the entire ros- activation of the transcription factor remains to be exploredtrocaudal extent of the hypothalamus, an MTII-induced empirically, it is tempting to conclude that these twoincrease in MAPK activation was observed only in the biochemical systems are coordinated by PVN neurons.PVN. While this finding may reflect a unique property of Of the five types of melanocortin receptors identified tothe receptors expressed by cells in the PVN, it remains date, only MC3- and MC4-R have been found primarily inpossible that this population of MC4-R-expressing neurons neural tissue[9,10,24],with MC4-R being the predominantis better situated to rapidly access MTII when injected into form in the PVN[20]. Like other melanocortin receptors,the lateral ventricle. The same argument cannot be made, MC4-R is a member of the G-protein coupled receptorhowever, for the lack of activated MAPK in the arcuate superfamily that, consistent with the present data, mediatesnucleus, because of its similar proximity to the ventricular the production of cAMP through the coupling to thesystem. The difference in the MAPK activation in the PVN G-protein G . The novel finding that MTII induced MAPKs

and the arcuate nucleus, which predominantly express activation through MC4-R, but not MC3-R, adds to theMC4-R or MC3-R, respectively[1,15,20,24],is consistent growing list of differences between these receptor sub-with the present experiments using transfected cell models. types. For example, anatomical evidence demonstratingAs such, the present in vitro and in vivo data provide co-localization of POMC or AgRP with MC3-R, but notstrong support for the novel finding that MC4-R is able to MC4-R[1] as well as evidence that MC3-R, but notsignal through MAPK while MC3-R is not. Nevertheless, MC4-R acts to regulate POMC neurons[7] illustrates thefuture research is required to examine the lack of MTII- markedly different roles of these receptor subtypes. Con-induced activation of MAPK in other hypothalamic brain sistent with these differences is the present finding ofareas that are known to express MC4-R[15]. These data, MTII-induced MAPK activation in the PVN, in whichhowever, warrant future examination of MTII-induced MC4-R is the predominant receptor subtype[15,20], and aactivation of MAPK in MC4-R-containing ex- lack of similar changes in MC3-R-containing hypothalamictrahypothalamic areas[15], including brainstem sites that areas such as the arcuate nucleus.have been identified as targets of melanocortins with In addition to the activation of cAMP[9,10,13,14,28],respect to the regulation of food intake[11,35]. the present data show that melanocortin treatment resulted

The present data demonstrated a nearly complete ana- in the phosphorylation of MAPK, a diverse protein thattomical segregation of MTII-induced phosphorylated alters transcription to affect many cellular functions includ-MAPK and oxytocin within the PVN. The lack of co- ing proliferation, differentiation, and the biosynthesis oflocalization of MC4-R and oxytocin implied by these data inflammatory cytokines[5]. The mechanism through whichis consistent with a recent report that failed to co-localize MTII treatment resulted in the phosphorylation of MAPKoxytocin with melanocortin antagonist-induced Fos expres- is not clear. In general, the activation of MAPK occurs assion in the PVN[39]. Nevertheless, the finding that AgRP part of a three-kinase cascade in which MAPK is activatedattenuated LiCl-induced Fos expression in oxytocin-con- by a MAP/ERK kinase (MEK), which is activated by ataining neurons of the PVN, while partially blocking LiCl- MEK kinase (MEKK). The best characterized cascade isinduced conditioned taste aversion[37] suggests a con- that activated by members of the Raf family of proteins,nection between melanocortin receptors and oxytocin which are, in turn, activated by members of the Ras proteinwithin the PVN that was not apparent in the present family[5,6]. Activation of this cascade, however, wasanalysis. The nearly complete segregation of MTII-induced inhibited by PKA[18,33,38]. Because PKA is activatedphosphorylated MAPK and oxytocin, coupled with the downstream of cAMP[10], one might predict the inhibi-well-known distribution of oxytocin in the PVN[27] tion of MAPK by melanocortins acting through MC4-R;provided the opportunity for a more detailed level of however, the present demonstration that MTII activatedqualitative anatomical analysis, suggesting that the areas of MAPK only through MC4-R, but stimulated cAMP forma-intense phospho-MAPK-immunoreactivity were localized tion through either MC3- or MC4-R, suggests that cAMP

10 D. Daniels et al. / Brain Research 986 (2003) 1–11

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