View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

The Melanocortin 5 Receptor is Expressed in Human Sebaceous Glands and Rat Preputial Cells

Diane Thiboutot,* Aruntha Sivarajah,² Kathryn Gilliland,* Zhaoyuan Cong,*² and Gary Clawson²³ *Section of Dermatology, Department of Medicine, and the ²Departments of Pathology and ³Biochemistry and Molecular Biology of The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, U.S.A.

Melanocortins regulate pigmentation, adrenal hor- RNA was isolated from human sebaceous glands and mone secretion, immune functions, lipid metabo- the reverse transcriptase polymerase chain reaction lism, and feeding behaviors in rodents. These was performed using primers speci®c for each of the peptides include adrenocorticotrophic hormone, subtypes. Transcripts were melanocyte stimulating hormone, b-lipotrophin, and detected for the melanocortin 5 receptor. A polyclo- the endorphins. Lipid metabolism in sebaceous nal chicken antihuman antibody to the melanocortin glands and preputial glands of rodents is regulated 5 receptor localized to sebaceous glands, eccrine by a-melanocyte stimulating hormone, the major glands, hair follicles, and epidermis in human skin, agonist for melanocortin receptors. Five melanocor- rat skin, cultured human sebocytes, and rat preputial tin receptor subtypes have been identi®ed that differ cells. Presence of the melanocortin 5 receptor pro- in their tissue localization and af®nities for melano- tein in human sebaceous glands and rat preputial cortin ligands. Targeted disruption of the melano- glands was further veri®ed by Western blotting. cortin 5 receptor in transgenic mice results in These data support further investigation of the role widespread dysfunction of exocrine glands, including of melanocortins in the regulation of human sebum a marked decrease in sebum production. A role for production and support the use of the rat preputial melanocortins in the modulation of human sebum system as an experimental model in production has not been established. The goal of this physiology. Key words: preputial gland/sebaceous gland/ study is to determine which melanocortin receptors sebocyte/sebum. J Invest Dermatol 115:614±619, 2000 are expressed in human sebaceous glands. Messenger

ebum production is a key factor in the development of (g-protein)-coupled receptors, each characterized by the acne. Potent androgens such as testosterone and presence of seven transmembrane domains (Luger et al, 1997). dihydrotestosterone stimulate sebum production in both Engagement of the receptor appears to occur in a calcium- humans and rodents. In the rat, the effect of androgens dependent manner. After binding of the ligand, signal is transmitted S on sebaceous glands (SGs) and preputial glands is via activation of adenyl cyclase and intracellular cAMP is elevated. augmented by the melanocortin, a-melanocyte stimulating hor- The MC1-R and MC2-R represent the classical melanocytic a- mone (a-MSH) (Thody and Shuster, 1975; Thody et al, 1976). MSH and adrenocortical (ACTH) receptors, respectively. Melanocortins are post-transcriptionally derived from the 31 amino Expression of MC3-R occurs mainly in the brain, although low acid peptide (POMC), which is secreted by levels of expression have been reported in placenta and gut tissues the pituitary. These peptides are now known to regulate immune (Gantz et al, 1993a). Messenger RNA for the MC4-R appears to be functions, lipid metabolism, and feeding behaviors in rodents in restricted to the nervous system (Gantz et al, 1993b). MC5-R is the addition to their well-known roles in pigmentation and in only receptor subtype for which widespread mRNA expression has neuroendocrine pathways (Wintzen and Gilchrest, 1996; Huszar been detected among peripheral tissues, thus identifying this et al, 1997; Luger et al, 1997). receptor as a candidate mediator of many previously recognized Five different melanocortin receptor subtypes (MC-R) desig- peripheral melanocortin actions (Gantz et al, 1994; Labbe et al, nated MC1-R through MC5-R have been cloned and character- 1994; van der Kraan et al, 1998). Targeted disruption of the MC5- ized (Chhajlani and Wikberg, 1992; Mountjoy et al, 1992; R in transgenic mice results in multiple exocrine de®ciencies Chhajlani et al, 1993; Gantz et al, 1993a, b). Melanocortin receptors including markedly reduced sebum production (Chen et al, 1997). belong to a group of heterodimeric guanine nucleotide-binding Human sebum secretion is stimulated by androgens, but a role for melanocortins such as MSH and ACTH in this process has not Manuscript received October 7, 1999; revised April 18, 2000; accepted been demonstrated. The goal of this study is to determine if for publication June 28, 2000. melanocortin receptors are present in human SGs and rat preputial Reprint requests to: Dr. Diane M. Thiboutot, Section of Dermatology, cells where they may play a role in regulating sebum production. The Pennsylvania State University College of Medicine, P.O. Box 850, Hershey, PA 17033. Email: [email protected] MATERIALS AND METHODS Abbreviations: MC5-R, melanocortin 5 receptor; a-MSH, a-melano- cyte stimulating hormone; POMC, proopiomelanocortin; SG, sebaceous Tissue specimens and processing Samples of normal human skin were gland. obtained from routine surgeries performed in the Section of Dermatology

0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 614 VOL. 115, NO. 4 OCTOBER 2000 MELANOCORTIN RECEPTORS IN HUMAN SEBACEOUS GLANDS AND RAT PREPUTIAL CELLS 615 at The Pennsylvania State University's Hershey Medical Center under a resistance and lacZa fragment to allow for blue-white screening. protocol approved by the Institutional Review Board. Samples were Plasmids were transformed into competent cells using the TA cloning transported to the laboratory on ice. SGs were dissected from the skin for system (Invitrogen, San Diego, CA) and colonies were selected on the basis use in reverse transcriptase polymerase chain reaction (RT-PCR), Western of ampicillin resistance and b-galactosidase activity. Plasmid DNA was blotting, and cell culture as previously described (Thiboutot et al, 1995). prepared from an overnight culture and veri®ed by automated sequencing Brie¯y, glands were visualized under a dissecting microscope and dissected (ABI Prism 377, Perkin Elmer, Foster City, CA). from the dermis using ®ne forceps, scissors, and a 14 gauge needle. Collagen ®bers were gently detached from each gland until it was visually Generation and characterization of an antibody to MC5-R Poly- clear of ®bers. Preputial glands were removed from male Sprague Dawley clonal antiserum was raised in chickens against a 13 amino acid, high rats sacri®ced by CO2 narcosis and placed in cold Hanks' balanced salt performance liquid chromatography analyzed, lyophilized peptide. The solution. Glands were trimmed of fat and the capsule was excised (Laurent translated protein sequence from the N-terminal region of the human et al, 1992). MC5-R protein ``N''ÐLSG/PNV/KNK/SSP/CÐ``C'' was sent to Extracts of human SGs and rat preputial glands were prepared for use in Genemed Biotechnologies, South San Francisco, CA, for peptide Western blotting. Tissues were homogenized using Te¯on glass homo- synthesis. The peptide preparation (molecular weight 1331) was coupled genizers in Nonidet P-40 buffer (50 mMTris HCl pH 7.4, 1% Nonidet P- to keyhole limpet hemocyanin using Imject Activated Immunogen 40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM Conjugation Kit (Pierce, Rockford, IL) and subsequently sent to ethylenediamine tetraacetic acid (EDTA), phenylmethylsulfonyl ¯uoride, Cocalico Biologicals (Reamston, PA) for antiserum production. The 1 mg per ml aprotinin, 1 mg per ml leupeptin, and 1 mg per ml pepstatin) at chicken was immunized on days 21, 42, 63, and 84. Test bleeds were 4°C, lyzed on ice for 30 min, and centrifuged at 12,000g for 15 min at 4°C. collected on days 31, 52, 73, and 94 from the time of initial inoculation. Total protein concentrations of the supernatants were determined The test bleed collected on day 52 was chosen for the characterization of according to the method of Bradford (Bradford, 1976). the antibody based on enzyme-linked immunosorbent assay (ELISA). Yolk immunoglobulin (IgY) was also obtained from Cocalico Biologicals. The Synthetic oligonucleotides The following primers were chosen for IgY was af®nity puri®ed against the peptide using a sulfolink kit (Pierce) by each of the ®ve melanocortin receptors from published sequences immobilizing the peptide to sulfolink coupling gel (Pierce). Antibody (Chhajlani and Wikberg, 1992; Mountjoy et al, 1992; Chhajlani et al, speci®city (IgY) was con®rmed by Western blotting using antibody that 1993; Gantz et al, 1993a; 1993b): MC1-R (Genbank #NM-002386) FP, was adsorbed to peptide as follows. A 1:10 dilution of IgY was incubated 5¢-ATCCCCCAGCTGGGGCTGGC-3¢; MC1-R RP, 3¢-CTTGAAG- with 15 mg per ml of peptide for 2 h at room temperature prior to ATGCAGCCGCACGT-5¢; MC2-R (Genbank #NM_000529) FP, 5¢- hybridization to the membrane containing rat preputial gland . CAACGTGGCAGTTTTGAAAC-3¢; MC2-R RP, 3¢-GGAGATCT- TCCTGGTGTGGGA-5¢; MC3-R (Genbank #L06155) FP, 5¢ - Cell culture Rat preputial glands were digested overnight at 4°C GCCCTCACCTTGATCGTGGC-3¢; MC3-R RP, 3¢-CTGTGGGG- in dispase/fetal bovine serum followed by trypsin (0.25%) digestion at CCACCCCGTCGGC-5¢; MC4-R (Genbank #L08603) FP, 5¢-TACT- 37°C, shaking for 40 min (Laurent et al, 1992). Cells were suspended in CTGATGGAGGGTGCTA-3¢; MC4-R RP, 3¢-TTGGCGGAT- Dulbecco's modi®ed Eagle's medium/Ham's F-12 3:1, fetal bovine serum GGCACCAGTGCC-5¢; and MC5-R (Genbank #U08353) FP, 5¢- 5%, adenine 1.8 3 10±4 M, hydrocortisone 0.4 mg per ml, insulin 5 mg GAGGGCAACCTTTCAGGACC-3¢; MC5-R RP, 3¢-GCCGCAGCC- per ml, epidermal growth factor 10 ng per ml, cholera toxin 1.2 3 10±10 M, CGTGCAGAAAGC-5¢. The lengths of the melanocortin DNA segments antibiotics (1003) containing keratinocyte growth factor 10 ng per ml, and to be ampli®ed were 733 base pairs, 304 base pairs, 178 base pairs, 568 base plated with mitomycin C (6 3 105) inactivated 3T3 ®broblasts. The pairs, and 460 base pairs, respectively, for each of the types 1±5 primary culture was subcultured using trypsin 0.05%/0.02% EDTA to melanocortin receptors. Primer pairs amplifying a 250 bp segment of the chamber slide system (Nalge Nunc International, Naperville, IL). About 20 human b-actin gene (Genbank #NM_001101) were chosen as a positive human SGs per sample were digested with 0.125% trypsin, 0.01% EDTA at control for the reverse transcriptase reaction. An additional positive control 37°C for 30 min. The trypsin/EDTA mixture was removed and plated consisted of RNA provided in the reverse transcriptase polymerase chain onto a 60 mm tissue culture plate in the above medium with mitomycin C reaction (RT-PCR) kit. The primers used in this reaction were designed to inactivated 3T3 ®broblasts. The remaining SGs were digested three more amplify a 500 bp segment. times and each harvest was plated on separate tissue culture plates. Primary cultures were subcultured using trypsin 0.05%/0.02% EDTA to chamber RT-PCR Total RNA was isolated from samples of SGs pooled from slide system in the same medium. facial areas of 20 subjects (Chomczynski and Sacchi, 1987). Ten micrograms of SG RNA was treated with DNase I (GibcoBRL, Immunohistochemistry Both secondary rat preputial and human Gaithersburg, MD) for 15 min and inactivated with EDTA. First strand sebocytes on chamber slides were ®xed in 100% ethanol. Cryostat cDNA synthesis was performed using a SUPERSCRIPT TMFirst-Strand sections of rat skin and human facial skin were ®xed in cold acetone Synthesis System for RT-PCR kit (GibcoBRL). As one of the positive (20 min). Immunoperoxidase staining was carried out at room temperature controls, RNA supplied in the kit was also subjected to reverse using the avidin-biotin complex method (ABC kit, Vector Laboratories, transcription and PCR using primers designed to amplify a 500 bp Burlingame, CA). The slides were ®rst washed in phosphate-buffered saline segment. Brie¯y, two 5 mm aliquots of DNase-treated human SG RNA (PBS) (5 min), followed by normal goat serum (20 min) and the af®nity puri®ed polyclonal antibody (IgY) at a 1:25 dilution in PBS (30 min) and were mixed with 10 mM dNTP mix, oligo(dT)12±18 (0.5 mg per ml), in a ®nal volume of 10 ml of DEPC-treated water, heated to 65°C for 5 min and PBS wash (23). Subsequent reaction steps were performed using cooled on ice. Reverse Transcriptase 50 units (SUPERSCRIPT II RNase biotinylated goat antichicken IgG (8 mg per ml) (Vector Laboratories). H-reverse transcriptase) was added to one aliquot and 1 ml of water was Sigma Fast 3,3¢-diaminobenzidine tetrahydrochloride with metal enhancer added to the other aliquot (negative control). Samples were incubated for tablet sets (Sigma, St. Louis, MO) were used as precipitating substrate for 50 min at 42°C. Reactions were terminated at 70°C for 15 min, and then the localization of peroxidase activity, which was visualized as a distinctive chilled on ice. The PCR was initiated as follows. The reaction product intense, dark blue to bluish-black reaction product. Negative controls (2 ml) was aliquoted into six sample tubes (b-actin positive control and each consisted of skin sections incubated with nonimmune serum in place of the of the ®ve melanocortin receptors). Two microliters from the negative primary antibody. control (no reverse transcriptase) and from the positive control sample from the kit were each aliquoted into separate tubes. Each sample was then Western blots Ten percent acrylamide gels were prepared and run heated to 95°C for 5 min and placed on ice. Taq polymerase 2.5 units according to the method of Laemmli (Laemmli, 1970). Samples were (Promega) and 50 pmol each of forward and reverse primers were added in heated (2 min, 90°C) in a solution (1:4 vol/vol) containing glycerol (10% a ®nal volume of 50 ml of sterile water. Annealing conditions were based on vol/vol), 2-mercaptoethanol (5% vol/vol), and SDS (3% wt/vol) in Tris the average Tm of the oligonucleotide primer pairs (which ranged from buffer with bromophenol blue as tracking dye and electrophoresed using 58°Cto69°C) and were generally chosen 3°C below the Tm. Reactions mighty small II mini gel system (Amersham Pharmacia Biotech, Piscataway, were run for 30 cycles. Final extension was performed at 72°C for 10 min. NJ). A hundred micrograms each of rat preputial gland extract and human The annealing temperature used for the type 1 primer was 58°C. One-half SG extract were loaded per well. Prestained electrophoresis standard of the PCR products were separated by electrophoresis on a 1.5% agarose (Diversi®ed Biotech, Boston, MA) was run along with the extracts. (TAE) gel. Bands from the melanocortin reactions were cut from the gel, The identi®cation of the apparent molecular weight of the proteins on and DNA was extracted using Gene Clean Kit (Bio 101, La Jolla, CA) and SDS-PAGE to which the antibodies bound was carried out by protein blot quanti®ed by dot analysis. DNA was ligated (overnight at 14°C) into a analysis. The separated protein on the 10% SDS-PAGE was transferred to pCR-2.1 vector (Invitrogen, Carson City, CA) containing an ampicillin nitrocellulose. The blotted membrane was blocked in Tween-20/Tris 616 THIBOUTOT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY buffered saline (TTBS) and incubated with 1:10 dilution of IgY in TTBS buffer. The protein recognized by antibody was revealed as dark purple bands using horseradish peroxidase linked rabbit antichicken IgG (Sigma) 1:5000 dilution in TTBS buffer, 60 min, and 4-chloro-1-naphthol (0.05%) and H2O2 (0.05%) in a 5:1 (vol/vol) mixture of Tris-buffered saline and methanol as chromogen.

RESULTS Transcripts for the 5 receptor are detected using RT-PCR (Fig 1) PCR products from the controls and the 5 melanocortin reaction were separated on a 2% agarose gel. Bands of the appropriate sizes were obtained as follows (Fig 1): lane 1, 500 bp band in a sample of the kit control RNA; lane 2, a 250 bp band corresponding to the b-actin gene; lane 6, a single band of approximately 568 bp corresponding to the MC4-R gene; lane 7,a single distinct 460 bp band corresponding to the MC5-R gene. No bands consistent with the MC1-R (733 bp), MC2-R (304 bp), or Figure 1. Ampli®ed sequence consistent with the MC5-R is the MC3-R (178 bp) were detected (Fig 1, lanes 3±5 and 7, detected in human SGs by RT-PCR. Total RNA was extracted from respectively). No bands were noted in lane 8 where PCR products microdissected human SG and treated with DNase. cDNA was synthesized from the negative control reaction were run. Each of the bands and PCR was performed for each of the ®ve melanocortin receptor noted for the MC4-R and MC5-R were cut from the gel. subtypes and positive and negative controls. The gel was stained with DNA was isolated, cloned into a pCR2.1 vector (Invitrogen, ethidium bromide. Lane M, 100 bp markers; lane 1, positive control (PCR Carson City, CA) and sequenced, which revealed homology of products from the RT-PCR reaction performed on the positive control only the 460 bp fragment with the target MC5-R sequence. RNA supplied in the kit); lane 2, positive control (PCR products using Although the band obtained for the MC4-R PCR product was primers to the human b-actin gene); lane 3, MC1-R; lane 4, MC2-R; lane 5, MC3-R; lane 6, MC4-R; lane 7, MC5-R; lane 8, negative control (no of the appropriate size, its sequence consisted of repeats of the reverse transcriptase; SG RNA, reacted with b-actin primers). Expected primer sequences and was not homologous to the target sequence sizes of ampli®ed sequences based on primer design were 500 bp for the kit for the MC4-R. positive RNA control; 250 bp for the b-actin positive control; 733 bp for MC1-R, 304 bp for MC2-R, 178 bp for MC3-R, 568 bp for MC4-R, and Veri®cation of MC5-R antibody speci®city Speci®city of the 460 bp for MC5-R. Bands consistent with positive controls, MC4-R, and af®nity puri®ed IgY was veri®ed in blocking experiments using a MC5-R are detected. Sequence veri®cation was obained for only the rat preputial gland extract. A 43 kDa band corresponding to the MC5-R. MC5-R antibody was noted using the IgY (Fig 2, lane A). When the antibody in the IgY was blocked with MC5-R peptide, the 43 kDa band was diminished (Fig 2, lane B), thus con®rming speci®city of the IgY used in immunohistochemistry and Western blot. Immunohistochemical localization of MC5-R The MC5-R antibody localized to the epidermis, hair follicle, SG and eccrine gland (Fig 3) in samples of human and rat skin incubated with primary antibody. Localization was not detected in these structures in the negative controls. Within the skin, localization was prominent in the basal layer of the SG and the outer root sheath of the hair follicle. The MC5-R antibody localized in the cytoplasm of cultured human sebocytes and rat preputial cells but not in the cytoplasm of cells in the negative control (Fig 4). Western blot A 43 kDa band corresponding to the MC5-R peptide was noted in extracts of both human SGs and rat preputial glands (Fig 5).

DISCUSSION Apart from isotretinoin, there are no effective agents that signi®cantly reduce the sebum production associated with acne. Advances in this area have been hampered by a lack of Figure 2. MC5-R antibody speci®city is con®rmed in blocking understanding of the mechanisms involved in the regulation of experiments. Western blotting of a rat preputial gland extract was human SGs. Androgens stimulate sebum production in humans, yet performed. A 1:10 dilution of IgY was preincubated in the presence and in the majority of acne patients, serum levels of androgens are absence of the MC5-R peptide. Lane M, molecular weight markers; lane A, within the normal range. This clinical observation suggests that test IgY alone; lane B, test IgY preincubated with MC5-R peptide. The additional factors may regulate sebum production either directly or 43 kDa band representing the MC5-R was diminished by preincubation of the antibodies with the peptide, con®rming speci®city of the generated indirectly by mediating the local effects of androgens on the SG. antibodies. In rodents, the SG response to androgens is augmented by a- MSH, a melanocortin peptide that has been referred to as a ``sebotrophic factor'' (Ebling et al, 1969; 1975; Thody and Shuster, 1975; Cooper et al, 1976; Thody et al, 1976). More recently, the role in locally modulating the action of androgen hormones on importance of melanocortins in regulating sebum production in human SGs is worthy of investigation. rodents has been con®rmed in studies of transgenic mice lacking The family of melanocortin peptides include a-MSH, ACTH, the MC5-R (Chen et al, 1997). These mice had hypoplastic SGs b-lipotrophin, and endorphin. These peptides are derived from the and preputial glands and reduced levels of sebum and preputial tissue-speci®c cleavage of POMC that is secreted primarily by the gland secretion. Whether a-MSH or melanocortin receptors play a pituitary gland. In rodents, a-MSH circulates in higher levels and is VOL. 115, NO. 4 OCTOBER 2000 MELANOCORTIN RECEPTORS IN HUMAN SEBACEOUS GLANDS AND RAT PREPUTIAL CELLS 617

Figure 3. MC5-R is detected in human facial skin and sebocytes. Immunohistochemistry was performed on cryostat sections of human facial skin and human sebocytes using a 1:25 dilution of af®nity-puri®ed IgY. (A) Negative control, human facial skin incubated in the absence of primary antibody. (B) Human facial skin: MC5-R is detected in epidermis (large arrow), hair follicles, SGs, eccrine glands, and endothelial cells (small arrow). (C) Human facial skin: high power view reveals localization of MC5-R in hair follicles (large arrow), sebaceous duct (small arrow), and basal layer of the SG. (D) Negative control, human facial sebocytes incubated in the absence of primary antibody. (E) Human facial sebocytes: MC5-R is detected in sebocytes (arrow) and surrounding 3T3 ®broblasts. (F) Human facial sebocytes: high power view reveals cytoplasmic localization of MC5-R in sebocytes (arrow). Scale bars:75mm[scale bar in part (B) refers to parts (A), (B), and (E); scale bar in part (C) refers to parts (C), (D), and (F)].

Figure 4. MC5-R is detected in rat skin and rat preputial sebocytes. Immunohistochemistry was performed on cryostat sections of rat skin and rat preputial sebocytes using a 1:25 dilution of the af®nity-puri®ed IgY. (A) Negative control, rat skin incubated in the absence of primary antibody. (B) Rat skin: MC5-R is detected in epidermis (large arrow), hair follicles (small arrow), SGs, eccrine glands, and endothelial cells. (C) Negative control, rat preputial sebocytes (arrow) incubated in the absence of primary antibody. (D) Rat preputial sebocytes: cytoplasmic distribution of MC5-R is detected. Scale bars:75mm [scale bar in part (B) refers to parts (A), (B), and (C)]. thought to play a key role in pigmentation, feeding behavior, lipid is particularly applicable to the skin where a variety of steroid metabolism, cardiovascular tone, and immune functions (Wintzen hormone metabolizing enzymes are active. Despite low circulating and Gilchrest, 1996; Chen et al, 1997; Huszar et al, 1997; Luger et al, levels of MSH in humans, the local production of melanocortin 1997; van der Kraan et al, 1998). Because circulating levels of a- peptides may be important in regulating processes in skin (Schauer MSH are low in humans, little emphasis has been placed on its et al, 1994; Wintzen and Gilchrest, 1996; Luger et al, 1997). In fact, potential role in mediating similar functions in humans. skin was the ®rst organ in which peripheral formation of MSH The local production and metabolism of hormones within peptides was noted. a-MSH could be detected in the skin of rats endocrine tissues such as the skin, however, has recently been whose central production of melanocortins was ablated via recognized as an important principle in the cell-type- and tissue- hypophysectomy (Thody et al, 1983; Luger et al, 1997). speci®c regulation of hormone action (Labrie, 1991). This principle Subsequently, POMC was demonstrated in human 618 THIBOUTOT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

The MC5-R is the most ubiquitous of the melanocortin receptors (Chen et al, 1997). It utilizes ACTH and each of the MSH subtypes as ligands. MC5-R has high af®nity for ACTH. This receptor has been localized to brain, adrenal gland, adipocytes, skeletal muscle, bone marrow, spleen, and thymus (Gantz et al, 1993a; Boston and Cone, 1996). Transgenic mice lacking the gene for the MC5-R knockout mice absorbed almost twice as much water through their skin during a test swim (Chen et al, 1997). The defect in water repulsion appeared to be related to a decrease in sebaceous lipids including sterol esters. Radioligand binding studies were performed in mutant mice to document functionality of melanocortin receptors in exocrine glands using 125 I-NDP-a- MSH (Chen et al, 1997). Binding was absent in the Harderian gland, preputial gland, and lacrimal gland of MC5-R mutants, suggesting an absence of signi®cant amounts of MC1-R, MC3-R, and MC4-R. These studies show that MC5-R is the only functional melanocortin receptor in exocrine glands of mice. The MC5-R is also expressed in the secretory epithelia of exocrine glands of rats including the lacrimal, Harderian, preputial, and prostate glands as well as the pancreas, adrenal gland, esophagus, and thymus (van der Kraan et al, 1998). This study demonstrates that the MC5-R is expressed at the Figure 5. MC5-R protein is detected in Western blots of human SG message and protein level in human SGs and cultured human and rat preputial gland extracts. Blots were incubated in the presence of sebocytes. In addition, immunolocalization of MC5-R antibody a 1:10 dilution of IgY. Lane M, molecular weight markers; lane A, rat was noted in human epidermis, hair follicles, and eccrine glands. preputial gland extract; lane B, human SG extract. 43 kDa bands consistent The function of melanocortins in these structures is not known. with the MC5-R antibody were detected in both rat preputial glands and Because the rat preputial gland is often used as a model for the study human SGs, con®rming the presence of MC5-R protein in each tissue. of human SGs, studies were run in parallel in rat skin, human skin, cultured rat preputial cells, and cultured human sebocytes. The expression of the MC5-R in rat skin and preputial cells paralleled skin, con®rming that this protein can be expressed in peripheral that found in human skin and sebocytes. These data support the use tissues (Slominski et al, 1995). In addition, cleavage products of of preputial sebocytes as a model for studying the effects of POMC (MSH and ACTH) are produced by human skin and melanocortin peptides on the growth and differentiation of lipid- cultured keratinocytes (Schauer et al, 1994; Wintzen and Gilchrest, producing glands such as the preputial gland and SG. 1996; Luger et al, 1997; Chakraborty et al, 1999). Melanocortins A role for the MC5-R in regulating human sebum production such as a-MSH and ACTH have been identi®ed in human has not yet been established. Although studies of functionality of epidermis by ligand binding assays, RT-PCR, and immunohis- the melanocortin receptors in human SGs clearly need to be tochemistry, suggesting that melanocortins may serve an autocrine performed, it is interesting to hypothesize that melanocortin or paracrine function in the skin (Schauer et al, 1994; Wintzen and peptides may mediate the hormonal responses of human SGs to Gilchrest, 1996; Luger et al, 1997). stress. Peptides such as MSH and ACTH may form the link Five distinct melanocortin receptors have been identi®ed, each between stress and the exacerbation of acne. During periods of of which exhibits varying tissue speci®city and preference for stress, high circulating levels of ACTH may act directly on the SG ligands. This ®nding further supports a role for melanocortins in to mediate steroidogenesis and sebum production. The interaction local tissue regulation. MC1-R is expressed by melanocytes, of ACTH with its receptor increases the intracellular concentration monocytes, keratinocytes, ®broblasts, and endothelial cells in the of cyclic AMP, which in turn promotes the conversion of skin (Luger et al, 1997; Chakraborty et al, 1999). It has been cholesterol to pregnenolone via the P450 side chain cleavage presumed that ACTH and MSH bind to the MC1-Rs found on enzyme, the rate limiting step in steroidogenesis. This enzyme has these cells where they play a role in the modulation of immune been identi®ed in human skin via RT-PCR (Slominski et al, 1996). function, cellular proliferation and differentiation, and response to Preliminary data (unpublished) suggest that this enzyme is present ultraviolet light. in human SGs where it may be stimulated by ACTH via an MC2-R is the ACTH receptor, found in the adrenal gland. It exocrine or paracrine mechanism. The SG possesses the enzymes binds ACTH, MSH, but not b-lipotrophins. MC2-R has been needed to subsequently convert pregnenolone to potent androgens, identi®ed in whole skin via RT-PCR suggesting that ACTH may which can stimulate sebum production. Additional mechanistic have a direct effect on the skin (Slominski et al, 1996). In rodents, studies are clearly needed to determine if melanocortin receptors MC3 and MC4 are expressed mainly in the central nervous system play a functional role in the tissue-speci®c regulation of sebum where they may regulate feeding behavior, metabolism, body production. temperature, and cardiovascular tone (Chen et al, 1997). MC3-R has also been detected in the placenta and gut. MC4-R has been localized to brain and gut (Huszar et al, 1997). MC3-R is speci®c We wish to thank Dr. Elizabeth Billingsley for assistance in obtaining skin for the heptapeptide core shared by ACTH and MSH. Each of the specimens. This work was supported by Hexal Pharmaceuticals, Inc., and NIH MSHs serves as a ligand for the MC4-R. Inactivation of MC4-R grant K08AR02018. results in mice that develop a maturity-onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia and increased linear growth that recapitulates features of the Agouti syndrome (Huszar et al, 1997). The function of MC3-R and MC4- REFERENCES R in humans has not yet been fully determined. Recent studies Boston B, Cone R: Characterization of melanocortin receptor subtype expression in suggest that haplotype insuf®ciency mutations in the MC4-R may murine adipose tissues and in the 3T3-L1 cell line. Endocrinology 137:2043± 2050, 1996 be linked to some autosomal dominant forms of human obesity Bradford M: A rapid and sensitive method for the quantitation of microgram (Hinney et al, 1999; Sina et al, 1999; Vaisse et al, 1999; Yeo et al, quantities of protein utilizing the principle of protein-dye binding. Anal 1999). Biochem 72:248±254, 1976 VOL. 115, NO. 4 OCTOBER 2000 MELANOCORTIN RECEPTORS IN HUMAN SEBACEOUS GLANDS AND RAT PREPUTIAL CELLS 619

Chakraborty A, Funasaka Y, Pawelek J, Nagahama M, Ito A, Ichihashi M: Enhanced classical and peripheral intracrine tissues. Bailliere's Clin Endocrinol Metabol expression of melanocortin-1 receptor (MC1-R) in normal human 8:451±474, 1994 keratinocytes during differentiation: evidence for increased expression of Laemmli U: Cleavage of structural proteins during the assembly of the head of the POMC peptides near suprabasal layer of epidermis. J Invest Dermatol 112:853± bacteriophage T4. Nature 227:680±685, 1970 860, 1999 Laurent S, Mednieks M, Rosen®eld R: Growth of sebaceous cells in monolayer Chen W, Kelly M, Opitz-Araya X, Thomas R, Low M, Cone R: Exocrine gland culture in vitro. Cell Dev Biol 28A:83±89, 1992 dysfunction in MC5-R de®cient mice: evidence for coordinated regulation of Luger T, Scholzen T, Grabbe S: The role of a-melanocyte-stimulating hormone in exocrine gland function by melanocortin peptides. Cell 91:789±798, 1997 cutaneous biology. J Invest Dermatol Symp Proc The 2:87±93, 1997 Chhajlani V, Wikberg J: Molecular cloning and expression of the human melanocyte Mountjoy K, Robbins L, Mortrud M, Cone R: The cloning of a family of genes that receptor stimulating hormone receptor cDNA. FEBS Lett 309:417±420, 1992 encode the melanocortin receptors. Science 257:1248±1251, 1992 Chhajlani V, Muceniece R, Wikberg J: Molecular cloning of a novel human Schauer E, Trautinger F, Kock A, Bhardwaj R, Ansei J, Schwarz T, Luger T: melanocortin receptor. Biochm Biophys Res Comm 195:866±873, 1993 Proopiomelanocortin-derived peptides are syntheaized and released bu human Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid keratinocytes. J Clin Invest 93:2258±2262, 1994 guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem Sina M, Hinney A, Ziegler A, et al: Phenotypes in three pedigrees with autosomal 162:156±159, 1987 dominant obesity caused by haploinsuf®ciency mutations in the melanocortin- Cooper MF, Bowden PW, Meddis D, Thody AJ, Shuster S: Effects of testosterone 4 receptor. Am J Human Genet 65:1501±1507, 1999 and alpha-melanocyte-stimulating hormone on preputial-gland (sebaceous) Slominski A, Ermak G, Hwang J, Chakroborty A, Mazurkiewicz J, Mihm M: activity. Biochem Soc Transactions 4:798±800, 1976 Proopiomelanocortin, corticotrophin releasing hormone and corticotrophin Ebling F, Ebling E, Skinner J: The in¯uence of pituitary hormones on the response of releasing hormone receptor genes are expressed in human skin. FEBS Lett the sebaceous glands of the male rat to testosterone. J Endocrinol 45:245±256, 374:113±116, 1995 1969 Slominski A, Ermak G, Mihm M: ACTH receptor, CYP11A1, CYP17 and Ebling F, Ebling E, Randall V, Skinner J: The synergistic action of a-melanocyte- CYP21A2 genes are expressed in skin. J Clin Endocrinol Metab 81:2746±2749, stimulating hormone and testosterone on the sebaceous, prostate, preputial, 1996 harderian and lachrymal glands, seminal vesicles and brown adipose tissue in the hypophysectomized-castrated rat. J Endocrinol 66:407±412, 1975 Thiboutot D, Harris G, Iles V, Cimis G, Gilliland K, Hagari S: Activity of the type 1, Gantz I, Konda Y, Tashiro T, et al: Molecular cloning of a novel melanocortin 5a-reductase exhibits regional differences in isolated sebaceous glands and receptor. J Biol Chem 268:8246±8250, 1993a whole skin. J Invest Dermatol 105:209±214, 1995 Gantz I, Miwa H, Konda Y, Shimoto Y, Waston S, DelValle J: Molecular cloning, Thody AJ, Shuster S: Control of sebaceous gland function in the rat by alpha- expression and gene localization of a fourth melanocortin receptor. J Biol Chem melanocyte-stimulating hormone. J Endocrinol 64:503±510, 1975 15174±15179, 1993b Thody AJ, Cooper MF, Bowden PE, Meddis D, Shuster S: Effect of alpha- Gantz I, Shimoto Y, Konda Y, Miwa H, Dickinson C, Yamada T: Molecular melanocyte-stimulating hormone and testosterone on cutaneous and modi®ed cloning, expression, and characterization of a ®fth melanocortin receptor. sebaceous glands in the rat. J Endocrinol 71:279±288, 1976 Biochem Biophys Res Comm 200:1214±1220, 1994 Thody A, Ridley K, Penny R, Chalmers R, Fisher C, Shuster S: MSH peptides are Hinney A, Schmidt A, Nottebom K, et al: Several mutations in the melanocortin-4 present in mammalian skin. Peptides 4:813±816, 1983 receptor gene including a nonsense and a frameshift mutation associated with Vaisse C, Clement K, Guy-Grans B, Froguel P: A frameshift mutation in MC4R dominantly inherited obesity in humans. J Clin Endocrinol Metabol 84:1483± associated with dominantly inherited human obesity. Nature Genet 20:111±112, 1486, 1999 1999 Huszar D, Lynch C, Fairchild-Huntress V, et al: Targeted disruption of the van der Kraan M, Adan R, Entwistle M, Gispen W, Burbach P, Tatro J: Expression melanocortin-4 receptor results in obesity in mice. Cell 88:131±141, 1997 of melanocortin-5 receptor in secretory epithelia supports a functional role in Labbe O, Desarnaud F, Gerick D, Vassart G, Parmentier M: Molecular cloning of a exocrine and endocrine glands. Endocrinol 139:2348±2355, 1998 mouse melanocortin 5 receptor gene widely expressed in peripheral tissues. Wintzen M, Gilchrest B: Prioopiomelanocortin, its derived peptides, and the skin. J Biochemistry 33:4543±4549, 1994 Invest Dermatol 106:3±10, 1996 Labrie F, Simard J, Luu-The V, Pelletier G, Belghmi K, Belanger A: Structure, Yeo G, Farooqi I, Aminian S, Stanhope R, O'Rahilly S: A frameshift mutation in regulation and role of 3b-hydroxysteroid dehydrogenase, 17b-hydroxysteroid human MC4R is associated with a dominant form of obesity. Nature Genet dehydrogenase and aromatase enzymes in the formation of sex steroids in 20:111±112, 1999