Melanocortin-4 Receptor Messenger Ribonucleic Acid Expression in ...

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Endocrinology 144(12):5488 –5496 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0570

Melanocortin-4 Receptor Messenger Ribonucleic Acid Expression in Rat Cardiorespiratory, Musculoskeletal, and Integumentary Systems KATHLEEN G. MOUNTJOY, C.-S. JENNY WU, LAURENCE M. DUMONT,

AND

J. MARTIN WILD

Departments of Physiology (K.G.M., C.-S.J.W.), Molecular Medicine and Pathology (K.G.M.), Anatomy (J.M.W.), and Research Centre for Developmental Medicine & Biology (L.M.D.), University of Auckland, Auckland 1, New Zealand We determined melanocortin-4 receptor (MC4-R) mRNA ontogeny in the rat using in situ hybridization and a rat MC4-R riboprobe and showed numerous peripheral sites of expression for MC4-R. The developing heart showed MC4-R mRNA expression as early as embryonic day (E) 14. In the lungs of E16 –E20 fetuses, the cells surrounding developing bronchi expressed relatively strong in situ signal. Muscles associated with the respiratory system such as diaphragm and intercostal muscle expressed MC4-R mRNA as early as E14. Occipital and tongue muscles, in particular the genioglossus, showed diffuse signal at E15–E20. In the eye, a discrete signal was

detected in an outer neuroblastic layer which may correspond to retina or extraocular muscle. Developing limb buds expressed relatively strong signal at E14, whereas skull bone and joint capsules of the paw of the forelimb showed signal at E18 –E20. Using RT-PCR and ribonuclease protection assays, we determined that MC4-R mRNA is also expressed in adult rat heart, lung, kidney, and testis. The expression of the MC4-R in cardiorespiratory, musculoskeletal, and integumentary systems supports functional roles for the MC4-R in addition to its roles in appetite, weight control, and regulation of linear growth. (Endocrinology 144: 5488 –5496, 2003)

M

␣-MSH in late gestational pregnant rats results in fetal growth retardation (13), whereas melanocortin peptides are potent growth stimulating factors in the central and peripheral nervous systems during ontogeny. Melanocortin peptides also exert trophic effects at the developing neuromuscular junction in the rat (14). POMC-containing neurons are one of the earliest peptidergic systems to arise in the developing hypothalamus; the onset of POMC expression in the ventral diencephalon occurs on the same day as the appearance of arcuate neurons appear in mouse and rat (15–17). In the rat, POMC immunoreactivity is detected in the arcuate nucleus on embryonic day (E) 12 of gestation, in the anterior and intermediate lobes of the pituitary on E15 and E16 respectively, and in the perikarya of the nucleus tractus solitarius on E17 (16). Both quantitative and qualitative changes occur in POMC processing during development. Processing of POMC in the anterior lobe of the pituitary is different between the newborn and adult rat, while processing in the intermediate lobe is similar in newborns and adults (18). In the newborn rat, 10% of the ACTH-related material in the anterior lobe is desacetyl-␣-MSH whereas in the adult, less than 1–2% of the ACTH-related material is desacetyl-␣-MSH sized. Proteolytic cleavage of ACTH1–39 to ACTH1–13 occurs to a much greater extent in newborn than in adult rat anterior pituitary. The mechanisms through which POMC-derived peptides influence development are poorly understood, but the MC4-R clearly plays a role. ACTH, ␣-MSH, and desacetyl␣-MSH similarly activate the MC4-R in vitro (9, 16, 19, 20) and MC4-R mRNA is expressed from E14 onwards in the developing rat central and autonomic nervous systems (11). To further our understanding about developmental roles for POMC-derived peptides and MC4-R, we have under-

ELANOCORTIN PEPTIDES, DERIVED from the large precursor protein, proopiomelanocortin (POMC), and melanocortin receptors are involved in a host of biological activities including pigmentation, stress, energy homeostasis, cardiovascular regulation, and immune function (1). Over the last 5 yr, much attention has been focused on the melanocortin-4 receptor (MC4-R) and body weight regulation because both the MC4-R knockout mouse (2) and the POMC knockout mouse (3) develop obesity. The significance of the melanocortin peptidergic axis to human obesity was realized when mutations in the human MC4-R (4, 5) and human POMC gene (6) were each shown to cause obesity. MC4-Rs in the hypothalamus regulate food intake (7) and metabolism (8), but much less is known about the functional roles for MC4-R mRNA expression in other regions of the central (9, 10) and autonomic nervous systems (11, 12). MC4-R mRNA is expressed at low levels throughout all regions of the brain and in the spinal cord, and also in organs innervated by the sympathetic nervous system such as penis, kidney and adrenal medulla (11). In a study to determine the ontogeny of the MC4-R in rats using in situ hybridization, we discovered that MC4-R mRNA is expressed in numerous peripheral tissues during development. These novel sites of expression could indicate developmental roles for the MC4-R. Although roles for melanocortin peptides in developmental processes and postnatal growth were indicated three decades ago, it is still not understood what these roles are, let alone the molecular mechanisms behind them. The administration of antibodies to Abbreviations: AGRP, Agouti gene-related peptide; CNS, central nervous system; d-FEN, d-fenfluramine; DNase, deoxyribonuclease; E, embryonic day; MC4-R, melanocortin-4 receptor; POMC, proopiomelanocortin; r, rat; RNase, ribonuclease; RPA, ribonuclease protection assays.

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Mountjoy et al. • Peripheral Sites for MC4-R mRNA Expression

taken a study to describe the spatio-temporal pattern of expression of MC4-R mRNA in peripheral tissues during rat fetal development. To determine whether the same peripheral tissues that express MC4-R mRNA during development also express MC4-R in adult rats, we used RT-PCR and ribonuclease protection assays (RPA) to identify MC4-R mRNA expression in the adult rat cardiorespiratory system, and in situ hybridization to localize MC4-R mRNA expression in testis. Materials and Methods Collection of rat fetuses Wistar rats were time mated and pregnant females were either anesthetized with halothane/O2 and killed by cervical dislocation, or euthanized using CO2. All animal procedures undertaken were approved by the Animal Ethics Committee of the University of Auckland. Rats between E14 and E20 of gestation (d 22 ⫽ term) were used. Fetuses from a minimum of three mothers at each gestational age were fixed in ice-cold 4% paraformaldehyde. Analyses of between five and nine fetuses at each gestational age were used for this study. They were stored at 4 C for a minimum of 1 d. Sucrose (10% wt/vol) was added 16 h before freezing the fetuses in OCT (Sakura, Torrance, CA) embedding medium. Fetuses were then stored at ⫺80 C until they were cut on the cryostat for in situ hybridization.

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sense rat (r) MC3-R riboprobe and control adult tissues hybridized with a sense rMC4-R riboprobe. The antisense rMC4-R riboprobe is specific for the MC4-R and does not cross-react with the rMC3-R (9, 11). Sections were hybridized in 65% formamide in 0.26 m NaCl, 1.3⫻ Denhardt’s, 13 mm Tris HCl (pH 8), 1.3 mm EDTA, 13% dextran sulfate at 60– 65 C for 18 h. Sections were washed and coated with emulsion for autoradiography. Following the developing of these slides, the sections were stained with hematoxylin and eosin and photographed under darkfield on a Leica (Leitz) (Global Science, Auckland, New Zealand) microscope. One series of sections from each case was not subjected to in situ hybridization but was counterstained with hematoxylin and eosin and used for the identification of structures, outlines of which are shown schematically in Figs. 1 and 2.

RNA preparation Total RNA was extracted from adult rat adrenal, heart, kidney, lung, mesenteric fat, muscle, spleen, and testis tissues using the guanidinium thiocyanate method (21). Rat tissue poly (A)⫹ (polyadenylated) mRNA was isolated using the PolyATract mRNA isolation system (Promega, Madison, WI).

PCR amplification of reverse transcribed mRNA (RT-PCR)

Male Wistar rats (approximately 250 –300 g) were anesthetized with halothane/O2 and killed with an overdose of pentobarbital. Heart, lung, kidney, adrenal, liver, trachea, and testis were dissected. Tissues collected for RNA isolation were snap frozen on dry ice and stored at ⫺80 C until they were processed. Tissues collected for in situ hybridization were placed immediately in ice-cold 4% (wt/vol) paraformaldehyde. They were stored at 4 C for a minimum of 3 days. Sucrose (10% wt/vol) was added 16 h before freezing the tissues in OCT freezing medium. Tissues were then stored at ⫺80 C until they were cut on a cryostat.

Poly (A)⫹ mRNA was deoxyribonuclease (DNase) treated using 10 U RQ1 ribonuclease (RNase)-free DNase (Promega Corp., Madison, WI) per microgram RNA for 40 min at 37 C. First-strand cDNA was synthesized using 1 ␮g poly (A⫹) mRNA, 200 U SuperScript II RNaseH⫺ reverse transcriptase (GIBCO BRL, Rockville, MD) and oligo (deoxythymidine)12–18 (Pharmacia Biotech AB, Uppsala, Sweden) at 42 C for 50 min in a final volume of 20 ␮l. To test for DNA contamination of the RNA, a reaction was carried out with 1 ␮g poly (A)⫹ mRNA and all the reagents but no reverse transcriptase (control reaction). The cDNA and control reaction (2 ␮l) were used as templates for PCR with rMC4-R specific oligonucleotides (5⬘-tgctgcaggaagatga-3⬘, sense and 5⬘-gacacatgaagcacacgca-3⬘, antisense) which were designed to amplify an 867-bp fragment. The PCR conditions were 94 C for 3 min, 40 cycles of 94 C for 40 sec, 55 C for 40 sec, and 72 C for 1 min, followed by 72 C for 10 min. The amplified cDNA products were separated on a 1.2% agarose gel alongside an EcoRI-HindIII-digested ␭ DNA ladder and stained with ethidium bromide.

In situ hybridization

Ribonuclease protection assay

Cross-sectional, sagittal, and longitudinal fetal sections were studied. Four series of sections (25 ␮m) from each fetus and five series of sections (20 ␮m) from adult rat heart, adrenal, kidney, testis, intestine, liver, lung, oesphagus, pituitary, spleen, and trachea were cut on the cryostat and mounted onto polysine microscope slides (Biolab Scientific, Auckland, New Zealand). Sections were hybridized with 33P labeled cRNA antisense rat MC4-R (628 bp). Control fetal sections were hybridized with an anti-

The cDNA template used to synthesize the antisense rMC4-R riboprobe was generated from a nucleotide DNA fragment spanning positions TMII and TMVII subcloned into pBKS (Stratagene). This recombinant DNA template was linearized with EcoRI and transcribed using T7 RNA polymerase in the presence of [␣-32P]uridine triphosphate (Amersham International, Buckinghamshire, UK) to generate a 32P-labeled 628-bp cRNA probe. Rat tissue poly (A)⫹ mRNA (1– 8 ␮g) was treated with 1 U RNase-

Collection of adult rat tissues

FIG. 1. Dark-field photomicrographs of MC4-R mRNA expression in the cardiorespiratory system. A, E14, heart with atrium and ventricle; B, E17, bronchioles and diaphragm (transverse or cross-sectional sections). Scale bar, 100 ␮m. Bronch, Bronchioles; Diaph, diaphragm; FLimb, forelimb; LAtr, left atrium; LLung, left lung; RAtr, right atrium; SC, spinal cord; Vent, ventricle.

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FIG. 2. Dark-field photomicrographs of MC4-R mRNA expression in the musculoskeletal system and integumentary tissues showing sagittal sections in A, B, C, and D, cross-sections in E, F, G, H, N, O, and P, and longitudinal sections in I, J, K, L, and M. A, E18, forelimb; B, E20, whisker follicles; C, E18, tongue; D, E20, tail; E, E15, neck muscles; F, E17, hindlimb; G, E16, abdominal muscles; H, E20, head muscle; I, E20, whisker follicles; J, E15, eye/retina; K, E20, facial muscle; L, E20, teeth; M, E15, limb buds, and spinal cord; N, E20, skull bone; O, E14, head of humerus; P, E14, intercostal muscle. Scale bar, 100 ␮m. The inset is an enlargement of the forelimb schematic shown in A showing the region where the in situ signal was observed. Adr, Adrenal; Atr, atrium; Cx, cortex; Diaph, diaphragm; FLimb, forelimb; HLimb, hindlimb; LLung, left lung; LV, lateral ventricle; SC, spinal cord; ST, sympathetic trunk; Tong, tongue; Vent, ventricle. Vert, vertebrae; Wh, whisker follicles.



FIG. 2. Contiuned. free DNase I (Roche Molecular Biochemicals, Indianapolis, IN) at 37 C for 50 min followed by the addition of 5 ⫻ 105 cpm of 32P-labeled riboprobe and the RNA probe mixture was precipitated. The RNA probe pellet was resuspended in 20 ␮l hybridization buffer [80% formamide, 40 mm PIPES (pH 6.4), 400 mm NaCl, 1 mm EDTA), denatured at 85 C for 5 min and hybridized at 45 C overnight. The hybridized RNA was digested with 100 U RNase T1 at 37 C for 50 min. The protected RNA fragments were precipitated and analyzed on a 6% denaturating polyacrylamide gel alongside a

32

P-labeled 123-bp DNA ladder (105 cpm). A digital image of 32P-labeled fragments was obtained using a Storm imaging system.

Results

Widespread expression of MC4-R mRNA was identified throughout the rat embryonic development period. This sig-

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TABLE 1. Tissue distribution of MC4-R in situ hybridization signal in developing rat fetus Tissue

Cardiorespiratory system

Musculoskeletal and integumentary systems

Examples of fetal tissues with no expression

Localization

Qualitative relative intensity

Atrial wall Ventricle wall Lung/bronchioles Diaphragm Occipital muscles of neck Tongue/genioglossus muscle specifically Whisker follicles Eye/retina/lens/extraocular muscle Facial/mylohyoid muscle Teeth Jaw Nasal epithelium Head of humerus Forelimb muscle/extensor muscle Limb buds Joint capsules Abdominal wall muscle Intercostal muscle Hindlimb muscle Dermis of tail Skull bone Liver Spleen Pancreas Stomach

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹ ⫹

Expression in the central and autonomic nervous systems has previously been described (11) and is not included in this table. Estimates of the in situ signals after viewing 35– 45 fetuses under dark-field microscopy are indicated: ⫹ (weak); ⫹⫹ (moderate); ⫹⫹⫹ (strong).

nal is specific to MC4-R because the pattern of expression using an antisense rat MC3-R probe was very different. Table 1 summarizes the MC4-R in situ signal detected in the organogenetic, early and late fetal growth periods in tissues outside the central and autonomic nervous systems. Expression of MC4-R mRNA in the developing rat cardiorespiratory system

The cardiorespiratory system showed some of the earliest expression of MC4-R mRNA (Fig. 1, Table 1). The developing heart showed MC4-R mRNA expression as early as E14 (Fig. 1A), and this expression continued throughout E18. We did not observe MC4-R mRNA expression in the developing hearts of E19 and E20 fetuses suggesting that this expression may be transient. The hematoxylin eosin staining was relatively weak following the in situ hybridization and thus it was not possible to determine the cell type that was expressing the MC4-R. In the lungs of E16 through E20 fetuses, the cells surrounding the developing bronchi expressed relatively strong in situ signal for this receptor mRNA (Fig. 1B). MC4-R mRNA is expressed in developing rat adrenal and kidney medulla (11) which are innervated by the sympathetic nervous system. In one fetus (E17) only, relatively strong expression was observed in what appeared to be either the ureter or renal nerve.

mRNA as early as E14 (Fig. 2P). In addition to the diaphragm and intercostal muscle, MC4-R mRNA was expressed in a number of other skeletal muscles, as well as integumentary tissues (Fig. 2 and Table 1). The developing limb buds expressed relatively strong signal for MC4-R mRNA as early as E14 and expression in the developing limbs continued through E20 (Fig. 2, M and F). It is unclear from the hematoxylin eosin staining which cell types, bone or muscle, are expressing this signal, but its position suggests the latter. In situ signal was observed in skull bone (Fig. 2N) and at the head of the humerus (Fig. 2O), areas where the signal may possibly be located in growth plates. Specific and consistent MC4-R mRNA expression was also observed on the dorsal aspect of the joint capsules of the paw of the forelimb E18, E19 and E20 (Fig. 2A) and the dermis of the tail (Fig. 2D). The neck muscles in the occipital region showed diffuse expression of MC4-R mRNA at E15, E16, E17, and E20 (Fig. 2, E and H). Muscles in the tongue, specifically the genioglossus, showed diffuse in situ signal for MC4-R mRNA at E16, E17, and E18 days of gestation (Fig. 2C). In the eye at E16 and E17, a discrete signal was detected in an outer neuroblastic layer which may correspond to retina or extraocular muscle (Fig. 2J). Two integumentary tissues, whisker follicles and teeth, showed relatively strong MC4-R mRNA signal at E20 only (Fig. 2, I and L).

Expression of MC4-R mRNA in the developing rat musculoskeletal and integumentary systems (Figs. 1 and 2 and Table 1)

Expression of MC4-R mRNA in the adult rat cardiorespiratory system

The diaphragm expressed MC4-R mRNA (Fig. 1B) at the same time (E16-E20) as expression for this receptor was observed in the lung. Another muscle associated with the cardiorespiratory system, intercostal muscle, also expressed MC4-R

MC4-R mRNA is expressed in adult rat heart and lung as observed using two different methods. First, cDNA fragments of the expected size were amplified from rat heart and lung poly (A)⫹ mRNA (Fig. 3) and second, DNA fragments


of the expected size were protected in an RPA (Fig. 4). We were unable to detect MC4-R mRNA expression in adult rat heart or lung using in situ hybridization. This is probably due to the very low expression of MC4-R mRNA in these tissues. To detect MC4-R mRNA by RPA we needed to use 6 ␮g heart poly (A)⫹ and 8 ␮g lung poly (A)⫹. Although we were able to detect MC4-R mRNA in adult rat kidney by RT-PCR (Fig. 3) and RPA (Fig. 4), we were not able to detect MC4-R mRNA in kidney sections using in situ hybridization. The expression in kidney is considerably lower than in heart or testis by both RT-PCR and RPA. One explanation, therefore, for not detecting MC4-R mRNA in kidney by in situ hybridization is that the message was too sparse to be detected. Alternatively, MC4-R mRNA may not be expressed in adult rat kidney and the signal we detected by RT-PCR and RPA might have been due to MC4-R mRNA expression in either renal nerve, ureter, or other contaminating tissue. Expression of MC4-R mRNA in the adult rat testis

Using two different methods, RPA (Fig. 4) and in situ hybridization (Fig. 5), we observed MC4-R mRNA in rat testis. Our in situ hybridization data show MC4-R mRNA is expressed at very low levels in stage-dependent germinal epithelium of the seminiferous tubules in cells that appear to be spermatocytes. No signal was observed in testis when we used a sense rat MC4-R riboprobe. We were unable to detect MC4-R mRNA by in situ hybridization or RT-PCR in adult rat adrenal, liver, trachea, esophagus, pituitary, mesenteric fat, or spleen.

FIG. 3. RT-PCR showed MC4-R mRNA expression in adult rat heart, kidney, and lung. MC4-R PCR product (867 bp) in heart (lane 2), kidney (lane 3), lung (lane 4), with brain (lane 5) as positive control. Controls of specificity were the absence of PCR products in the reactions without reverse transcriptase (heart, lane 6, kidney, lane 7, lung, lane 8). Lane 9 is the water control for PCR. The PCR products were run on a 1.2% agarose gel alongside a HindIII-EcoRI digested ␭ DNA molecular weight marker (lane 1).


Discussion

We show for the first time that MC4-R mRNA is expressed in adult rat heart, lung, kidney, and testis. These novel sites of MC4-R mRNA expression indicate that this melanocortin receptor, which is well recognized for regulating food intake, also has functions outside the central and autonomic nervous systems. Melanocortin peptides have long been known to play roles in development and to modulate early postnatal growth, but how they do this is not known. We show for the first time that MC4-R mRNA is expressed in a large number of peripheral tissues in the developing rat fetus. This expression indicates that the MC4-R is a receptor through which melanocortin peptides exert many of their developmental roles. The MC4-R, when expressed in heterologous human kidney 293 cells, couples to adenylyl cyclase in response to ␣-MSH, desacetyl-␣-MSH and ACTH with similar potencies (9) and therefore could be activated by endogenous ligands as soon as the receptor protein is available. MC4-R mRNA is expressed in the developing rat fetus on E14; the earliest time that we looked for it. POMC mRNA is expressed in the rat arcuate nucleus on E12 and melanocortin peptides are produced shortly after. The timing of POMC and MC4-R expression in the embryonic and fetal brain suggests they have important functions during development and brain maturation (11). Targeted deletion of MC4-R identified this receptor as a gene involved with appetite control and weight regulation as well as with increased linear growth (2). The mechanism whereby the MC4-R affects feeding is unknown. Feeding is a complicated activity having somatomotor and autonomic components and involving the orofacial and tongue musculature, as well as the gustatory and olfactory systems. The sequence of events during feeding is coordinated by neuronal networks in both the brain stem and spinal cord. Basic activity patterns generated by these neuronal networks can be modified by sensory inputs from the mouth and/or descending commands from the forebrain. MC4-R mRNA is expressed in the VMH (9)—an area of the brain known to be involved in the regulation of feeding; the trigeminal nerve— the main sensory nerve of the head that transmits afferent information from the tooth pulp, gingiva and periodontal membrane; the medulla oblongata and pons— both of which play an essential role in feeding; and regions of the cortex involved in olfactory responses such as tenia tecta and ol-

FIG. 4. Ribonuclease protection assay showed MC4-R mRNA expression in adult rat brain, heart, kidney, lung, and testis, but not in adrenal, liver, or spleen. The amounts of poly (A⫹) mRNA used for each reaction are: lane 1, 5 ␮g rat brain; lane 2, 1 ␮g adrenal; lane 3, 6.5 ␮g heart; lane 4, 7.5 ␮g kidney; lane 5, 3.8 ␮g liver; lane 6, 8.1 ␮g lung; lane 7, 3 ␮g spleen; lane 8, 8.14 ␮g testis; lane 9, 5 ␮g brain. The labeled fragments were run on a 6% polyacrylamide gel alongside a radiolabeled 123-bp DNA ladder. The full-length antisense MC4-R riboprobe is 628 bp and the specific rMC4-R protected fragment is 553 bp.

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FIG. 5. Dark-field and bright-field photomicrographs of MC4-R mRNA expression in adult rat testis. MC4-R mRNA is expressed in stage-dependent germinal epithelium of the seminiferous tubules shown in the dark-field photomicrograph (right) and in the bright-field photomicrograph (left) which is an enlargement of the boxed area shown in B. The arrows indicate silver grains.

factory bulb (9, 11). We now show for the first time that MC4-R mRNA is expressed in tongue (specifically the genioglossus muscle), facial muscle, jaw, and lower incisors of the developing rat fetus, all of which are tissues or organs involved in feeding behavior postnatally. Interestingly, MC4-R mRNA is expressed in both tongue and neck muscles and these are derived from the same myotome. Until now, MC4-R mRNA has only been shown to be expressed in the central nervous system (CNS) (9) in mammals and therefore the function(s) of this receptor in brain has been the focus of understanding the mechanism causing obesity. Our study now shows that MC4-R mRNA is also expressed in fetal skeletal muscle, in particular the abdominal wall and limb muscles. Melanocortins accelerate maturation of the neuromuscular system when they are administered during development (22, 23). Therefore, there may be a role for the MC4-R in melanocortin-induced myocyte proliferation. Increased skeletal growth is associated with the obese type 2 diabetes phenotype of the yellow obese agouti mouse (24) and the MC4-R knockout mouse (2). Huszar et al. (2) hypothesized that MC4-R expression in the hypothalamus regulates growth hormone secretion, thereby altering growth. Our data show strong expression of MC4-R mRNA in the developing limb buds and skull bone, in what may be the growth plates. Therefore, it is also possible that MC4-R may have a role in directly regulating bone growth in the periphery. It is of interest that morbidly obese children with defective MC4-Rs have been found to have significantly increased bone mineral density (25). Although the mechanism for this is thought to involve hyperinsulinaemia, it is also possible that the MC4-R has a direct role in bone metabolism. We have observed MC4-R mRNA expression in organs associated with the cardiorespiratory system such as vessel walls, atria and ventricular membranes, diaphragm, and lung. POMC mRNA is also expressed in lung (26, 27) and heart (28). This is the first time that expression of a melanocortin receptor has been associated with either the developing or adult rat heart. Furthermore, whereas MC5-R has been shown using RT-PCR to be present in lung (29, 30), this is the first time that MC4-R mRNA has been observed in devel-

oping and adult rat lung. Roles for the MC4-R in cardiorespiratory control have previously been assumed based on MC4-R mRNA expression in those regions of the developing and adult rat central and autonomic nervous system that are associated with cardiorespiratory control, i.e. medulla, pons, and the dorsal motor nucleus of the vagus, in particular (9, 11). ␣-MSH and ACTH have cardiovascular actions in human (31), dogs (32), sheep (33), and rabbits (34). These actions include increases in heart rate, ventricular contractile force, ascending aorta blood flow, blood flow to the heart, adrenals, and lungs. The MC4-R may mediate some of these actions. In both the adult and fetal rat brain MC4-R mRNA is expressed in somatomotor regions, such as the caudateputamen, nucleus accumbens and red nucleus. In the developing rat fetus, we have now observed MC4-R mRNA expression in peripheral organs that may be associated with this somatomotor control, such as limbs, joint capsules, abdominal wall muscle, and the tail. The eye has previously been identified as a target for melanocortin peptide activity. Perinatal treatment of rats with melanocortin peptides was found to result in earlier eye opening and accelerated maturation of the neuromuscular system (35, 36). ␣-MSH has also been shown to enhance prostaglandin production by bovine retinal pigment epithelium (37), and it has been proposed that ␣-MSH may modulate the neuroactivity of the retina and induce increased permeability of the blood-aqueous barrier of the eye (38). In our study, we observed MC4-R mRNA in the outer neuroblastic layer of the developing eye, a region that may correspond to either retina or extraocular muscle, and therefore the MC4-R may be mediating some of these melanocortin effects on the eye. Immunoreactive POMC peptides have been found in testicular extracts and appear to be localized to the Leydig cell (39, 40). The function of melanocortin peptides in testis is unknown. We now show that MC4-R mRNA is expressed in stage-dependent germinal epithelium of seminiferous tubules, and it therefore seems likely that the MC4-R is one melanocortin receptor through which these peptides function in the testis. Interestingly, neither the yellow obese ag-


outi mouse nor the MC4-R knockout mouse have a problem with fertility (2, 41). POMC and POMC-like peptides—like the melanocortin receptors, including MC4-R— have been found in many locations outside the pituitary and CNS, albeit in much lower concentrations than in pituitary and CNS. POMC mRNA and POMC-derived peptides have been detected in the rat in the gastrointestinal and reproductive tracts, heart, liver, kidney, and pancreas (39, 42). The wide low level expression of melanocortin peptides indicates that they have paracrine and/or autocrine effects. Natural antagonists for the melanocortin receptors also exert paracrine and/or autocrine effects. Mouse agouti protein, an antagonist of the MC1-R and MC4-R, is primarily expressed in skin in wild-type mice and is temporally regulated (43). In humans, where the function of agouti protein is not known, agouti protein is expressed in testis, heart, and kidney (44). Furthermore, agouti generelated peptide (AGRP), an antagonist of MC3-R and MC4-R, has its mRNA expressed in adrenal cortex and adrenal medulla (45), and also at low levels in testis, lung, and kidney (46). Using RT-PCR it has been shown that MC4-R, POMC, and AGRP are expressed in numerous peripheral tissues in chicken (47– 49). In the chicken, MC4-R was found to be expressed in various peripheral tissues including kidney, adrenal, testis, skeletal muscle, and the eye (47, 49). The specific functions of melanocortin peptides, agouti protein and AGRP, and the melanocortin receptors they interact with in many of these peripheral tissues are unknown. However, the present identification of MC4-R mRNA in various peripheral tissues of the fetal and adult rat contributes to an appreciation of various functional roles for melanocortin peptides and MC4-R. For example, our discovery that MC4-R is expressed in developing bone led to the discoveries that MC4-R is expressed in osteoblasts (50) and melanocortin peptides may play a role in bone metabolism (51). However, no obvious defects in organogenesis have been noted in the MC4-R knockout mouse or humans with a defective MC4-R allele and therefore the MC4-R is not critical for organogenesis. This does not necessarily exclude a functional role for the MC4-R in organogenesis that may be subtle, redundant, or not yet described. Furthermore, roles for MC4-R in the modulation of fetal and postnatal development and growth suggests that serious perturbations on the health of an individual resulting from stress to a fetus or newborn, may be mediated through melanocortin peptides (including ACTH) acting on the MC4-R. The expression of the MC4-R in cardiorespiratory, musculoskeletal, and integumentary systems supports functional roles for the MC4-R in addition to its known roles in appetite, weight control and regulation of linear growth. In particular, our data support a role for the MC4-R in cardiovascular regulation. No cardiovascular abnormalities have been reported for the MC4-R knockout mouse, nor have any humans with defective MC4-R genes been reported to suffer from cardiovascular problems. Maybe other members of the melanocortin receptor family can compensate for this function when there is a loss of MC4-R. Similar to POMC and MC4-R, the potent stimulator of food intake, ghrelin and its receptor, GH secretagogues receptor, are expressed in numerous peripheral tissues including heart (52), testis (53), lung (54), and


kidney (55). The melanocortin system may be similar to ghrelin and GH secretagogues receptor and have important roles in regulating both food intake and cardiac function. Developers of antiobesity therapeutics are today targeting the MC4-R. The novel sites of MC4-R mRNA expression described here should caution them regarding the possibility of side effects arising from the use of these drugs. For example, recently a potential antiobesity drug targeting the MC4-R was discovered to have an unexpected side effect, i.e. stimulation of penile erection (56). Subsequently this group showed MC4-R expression in adult penis. This side effect could have been predicted from our previously described MC4-R mRNA expression in the developing rat autonomic nervous system and penis (11). Interestingly, d-fenfluramine (d-FEN) was an effective appetite suppressor drug but was withdrawn from the market in 1997 due to unexpected cardiac complications. The mechanism underlying d-FEN⬘s anorexic actions involves the central melanocortin system (57). Is it possible that the mechanism underlying d-FEN⬘s adverse cardiopulmonary events also involve the central melanocortin system and perhaps MC4-R expression in the heart? Acknowledgments The authors thank Miss C. Elia and Miss A. DeAth for technical assistance Ms. J. Ross for her expert assistance with image analysis. Received May 8, 2003. Accepted August 13, 2003. Address all correspondence and requests for reprints to: Kathleen G. Mountjoy, University of Auckland, Auckland 1, New Zealand. E-mail: [email protected]. This work was supported by The Wellcome Trust, Health Research Council of New Zealand, National Child Health Foundation and the NZ Lottery Board.

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