The Melanocortin 5 Receptor is Expressed in Human Sebaceous Glands and Rat Preputial Cells
2000; Elsevier BV; Volume: 115; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2000.00094.x
ISSN1523-1747
AutoresDiane Thiboutot, Aruntha Sivarajah, Kathryn L. Gilliland, Zhaoyuan Cong, Gary A. Clawson,
Tópico(s)melanin and skin pigmentation
ResumoMelanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, β-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by α-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology. Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, β-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by α-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology. melanocortin 5 receptor α-melanocyte stimulating hormone proopiomelanocortin sebaceous gland Sebum production is a key factor in the development of acne. Potent androgens such as testosterone and dihydrotestosterone stimulate sebum production in both humans and rodents. In the rat, the effect of androgens on sebaceous glands (SGs) and preputial glands is augmented by the melanocortin, α-melanocyte stimulating hormone (α-MSH) (Thody and Shuster, 1975Thody A.J. Shuster S. Control of sebaceous gland function in the rat by alpha-melanocyte-stimulating hormone.J Endocrinol. 1975; 64: 503-510Crossref PubMed Scopus (61) Google Scholar;Thody et al., 1976Thody A.J. Cooper M.F. Bowden P.E. Meddis D. Shuster S. Effect of alpha-melanocyte-stimulating hormone and testosterone on cutaneous and modified sebaceous glands in the rat.J Endocrinol. 1976; 71: 279-288Crossref PubMed Scopus (47) Google Scholar). Melanocortins are post-transcriptionally derived from the 31 amino acid peptide proopiomelanocortin (POMC), which is secreted by the pituitary. These peptides are now known to regulate immune functions, lipid metabolism, and feeding behaviors in rodents in addition to their well-known roles in pigmentation and in neuroendocrine pathways (Wintzen and Gilchrest, 1996Wintzen M. Gilchrest B. Prioopiomelanocortin, its derived peptides, and the skin.J Invest Dermatol. 1996; 106: 3-10Crossref PubMed Scopus (123) Google Scholar;Huszar et al., 1997Huszar D. Lynch C. Fairchild-Huntress V. et al.Targeted disruption of the melanocortin-4 receptor results in obesity in mice.Cell. 1997; 88: 131-141Abstract Full Text Full Text PDF PubMed Scopus (2444) Google Scholar;Luger et al., 1997Luger T. Scholzen T. Grabbe S. The role of α-melanocyte-stimulating hormone in cutaneous biology.J Invest Dermatol Symp Proc The. 1997; 2: 87-93Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Five different melanocortin receptor subtypes (MC-R) designated MC1-R through MC5-R have been cloned and characterized (Chhajlani and Wikberg, 1992Chhajlani V. Wikberg J. Molecular cloning and expression of the human melanocyte receptor stimulating hormone receptor cDNA.FEBS Lett. 1992; 309: 417-420Abstract Full Text PDF PubMed Scopus (567) Google Scholar;Mountjoy et al., 1992Mountjoy K. Robbins L. Mortrud M. Cone R. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1421) Google Scholar;Chhajlani et al., 1993Chhajlani V. Muceniece R. Wikberg J. Molecular cloning of a novel human melanocortin receptor.Biochm Biophys Res Comm. 1993; 195: 866-873Crossref PubMed Scopus (312) Google Scholar;Gantz et al., 1993aGantz I. Konda Y. Tashiro T. et al.Molecular cloning of a novel melanocortin receptor.J Biol Chem. 1993; 268: 8246-8250Abstract Full Text PDF PubMed Google Scholar). Melanocortin receptors belong to a group of heterodimeric guanine nucleotide-binding protein (g-protein)-coupled receptors, each characterized by the presence of seven transmembrane domains (Luger et al., 1997Luger T. Scholzen T. Grabbe S. The role of α-melanocyte-stimulating hormone in cutaneous biology.J Invest Dermatol Symp Proc The. 1997; 2: 87-93Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Engagement of the receptor appears to occur in a calcium-dependent manner. After binding of the ligand, signal is transmitted via activation of adenyl cyclase and intracellular cAMP is elevated. The MC1-R and MC2-R represent the classical melanocytic α-MSH and adrenocortical (ACTH) receptors, respectively. Expression of MC3-R occurs mainly in the brain, although low levels of expression have been reported in placenta and gut tissues (Gantz et al., 1993aGantz I. Konda Y. Tashiro T. et al.Molecular cloning of a novel melanocortin receptor.J Biol Chem. 1993; 268: 8246-8250Abstract Full Text PDF PubMed Google Scholar). Messenger RNA for the MC4-R appears to be restricted to the nervous system (Gantz et al., 1993bGantz I. Miwa H. Konda Y. Shimoto Y. Waston S. DelValle J. Molecular cloning, expression and gene localization of a fourth melanocortin receptor.J Biol Chem. 1993: 15174-15179Google Scholar). MC5-R is the only receptor subtype for which widespread mRNA expression has been detected among peripheral tissues, thus identifying this receptor as a candidate mediator of many previously recognized peripheral melanocortin actions (Gantz et al., 1994Gantz I. Shimoto Y. Konda Y. Miwa H. Dickinson C. Yamada T. Molecular cloning, expression, and characterization of a fifth melanocortin receptor.Biochem Biophys Res Comm. 1994; 200: 1214-1220Crossref PubMed Scopus (280) Google Scholar;Labbe et al., 1994Labbe O. Desarnaud F. Gerick D. Vassart G. Parmentier M. Molecular cloning of a mouse melanocortin 5 receptor gene widely expressed in peripheral tissues.Biochemistry. 1994; 33: 4543-4549Crossref PubMed Scopus (185) Google Scholar;van der Kraan et al., 1998van der Kraan M. Adan R. Entwistle M. Gispen W. Burbach P. Tatro J. Expression of melanocortin-5 receptor in secretory epithelia supports a functional role in exocrine and endocrine glands.Endocrinol. 1998; 139: 2348-2355Crossref PubMed Scopus (94) Google Scholar). Targeted disruption of the MC5-R in transgenic mice results in multiple exocrine deficiencies including markedly reduced sebum production (Chen et al., 1997Chen W. Kelly M. Opitz-Araya X. Thomas R. Low M. Cone R. Exocrine gland dysfunction in MC5-R deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides.Cell. 1997; 91: 789-798Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Human sebum secretion is stimulated by androgens, but a role for melanocortins such as MSH and ACTH in this process has not been demonstrated. The goal of this study is to determine if melanocortin receptors are present in human SGs and rat preputial cells where they may play a role in regulating sebum production. Samples of normal human skin were obtained from routine surgeries performed in the Section of Dermatology at The Pennsylvania State University's Hershey Medical Center under a protocol approved by the Institutional Review Board. Samples were transported to the laboratory on ice. SGs were dissected from the skin for use in reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and cell culture as previously described (Thiboutot et al., 1995Thiboutot D. Harris G. Iles V. Cimis G. Gilliland K. Hagari S. Activity of the type 1, 5α-reductase exhibits regional differences in isolated sebaceous glands and whole skin.J Invest Dermatol. 1995; 105: 209-214Crossref PubMed Scopus (202) Google Scholar). Briefly, glands were visualized under a dissecting microscope and dissected from the dermis using fine forceps, scissors, and a 14 gauge needle. Collagen fibers were gently detached from each gland until it was visually clear of fibers. Preputial glands were removed from male Sprague Dawley rats sacrificed by CO2 narcosis and placed in cold Hanks' balanced salt solution. Glands were trimmed of fat and the capsule was excised (Laurent et al., 1992Laurent S. Mednieks M. Rosenfield R. Growth of sebaceous cells in monolayer culture in vitro.Cell Dev Biol. 1992; 28A: 83-89Crossref Scopus (22) Google Scholar). Extracts of human SGs and rat preputial glands were prepared for use in Western blotting. Tissues were homogenized using Teflon glass homogenizers in Nonidet P-40 buffer (50 mM Tris HCl pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM ethylenediamine tetraacetic acid (EDTA), phenylmethylsulfonyl fluoride, 1 μg per ml aprotinin, 1 μg per ml leupeptin, and 1 μg per ml pepstatin) at 4°C, lyzed on ice for 30 min, and centrifuged at 12,000g for 15 min at 4°C. Total protein concentrations of the supernatants were determined according to the method of Bradford (Bradford, 1976Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205559) Google Scholar). The following primers were chosen for each of the five melanocortin receptors from published sequences (Chhajlani and Wikberg, 1992Chhajlani V. Wikberg J. Molecular cloning and expression of the human melanocyte receptor stimulating hormone receptor cDNA.FEBS Lett. 1992; 309: 417-420Abstract Full Text PDF PubMed Scopus (567) Google Scholar;Mountjoy et al., 1992Mountjoy K. Robbins L. Mortrud M. Cone R. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1421) Google Scholar;Chhajlani et al., 1993Chhajlani V. Muceniece R. Wikberg J. Molecular cloning of a novel human melanocortin receptor.Biochm Biophys Res Comm. 1993; 195: 866-873Crossref PubMed Scopus (312) Google Scholar;Gantz et al., 1993aGantz I. Konda Y. Tashiro T. et al.Molecular cloning of a novel melanocortin receptor.J Biol Chem. 1993; 268: 8246-8250Abstract Full Text PDF PubMed Google Scholar; 1993b): MC1-R (Genbank #NM-002386) FP, 5′-ATCCCCCAGCTGGGGCTGGC-3′ MC1-R RP, 3′-CTTGAAG-ATGCAGCCGCACGT-5′ MC2-R (Genbank #NM_000529) FP, 5′-CAACGTGGCAGTTTTGAAAC-3′ MC2-R RP, 3′-GGAGATCT-TCCTGGTGTGGGA-5′ MC3-R (Genbank #L06155) FP, 5′-GCCCTCACCTTGATCGTGGC-3′ MC3-R RP, 3′-CTGTGGGG-CCACCCCGTCGGC-5′ MC4-R (Genbank #L08603) FP, 5′-TACT-CTGATGGAGGGTGCTA-3′ MC4-R RP, 3′-TTGGCGGAT-GGCACCAGTGCC-5′ and MC5-R (Genbank #U08353) FP, 5′-GAGGGCAACCTTTCAGGACC-3′ MC5-R RP, 3′-GCCGCAGCC-CGTGCAGAAAGC-5′. The lengths of the melanocortin DNA segments to be amplified were 733 base pairs, 304 base pairs, 178 base pairs, 568 base pairs, and 460 base pairs, respectively, for each of the types 1–5 melanocortin receptors. Primer pairs amplifying a 250 bp segment of the human β-actin gene (Genbank #NM_001101) were chosen as a positive control for the reverse transcriptase reaction. An additional positive control consisted of RNA provided in the reverse transcriptase polymerase chain reaction (RT-PCR) kit. The primers used in this reaction were designed to amplify a 500 bp segment. Total RNA was isolated from samples of SGs pooled from facial areas of 20 subjects (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar). Ten micrograms of SG RNA was treated with DNase I (GibcoBRL, Gaithersburg, MD) for 15 min and inactivated with EDTA. First strand cDNA synthesis was performed using a SUPERSCRIPT TM First-Strand Synthesis System for RT-PCR kit (GibcoBRL). As one of the positive controls, RNA supplied in the kit was also subjected to reverse transcription and PCR using primers designed to amplify a 500 bp segment. Briefly, two 5 μm aliquots of DNase-treated human SG RNA were mixed with 10 mM dNTP mix, oligo(dT)12-18 (0.5 μg per μl), in a final volume of 10 μl of DEPC-treated water, heated to 65°C for 5 min and cooled on ice. Reverse Transcriptase 50 units (SUPERSCRIPT II RNase H-reverse transcriptase) was added to one aliquot and 1 μl of water was added to the other aliquot (negative control). Samples were incubated for 50 min at 42°C. Reactions were terminated at 70°C for 15 min, and then chilled on ice. The PCR was initiated as follows. The reaction product (2 μl) was aliquoted into six sample tubes (β-actin positive control and each of the five melanocortin receptors). Two microliters from the negative control (no reverse transcriptase) and from the positive control sample from the kit were each aliquoted into separate tubes. Each sample was then heated to 95°C for 5 min and placed on ice. Taq polymerase 2.5 units (Promega) and 50 pmol each of forward and reverse primers were added in a final volume of 50 μl of sterile water. Annealing conditions were based on the average Tm of the oligonucleotide primer pairs (which ranged from 58°C to 69°C) and were generally chosen 3°C below the Tm. Reactions were run for 30 cycles. Final extension was performed at 72°C for 10 min. The annealing temperature used for the type 1 primer was 58°C. One-half of the PCR products were separated by electrophoresis on a 1.5% agarose (TAE) gel. Bands from the melanocortin reactions were cut from the gel, and DNA was extracted using Gene Clean Kit (Bio 101, La Jolla, CA) and quantified by dot analysis. DNA was ligated (overnight at 14°C) into a pCR-2.1 vector (Invitrogen, Carson City, CA) containing an ampicillin resistance gene and lacZα fragment to allow for blue-white screening. Plasmids were transformed into competent cells using the TA cloning system (Invitrogen, San Diego, CA) and colonies were selected on the basis of ampicillin resistance and β-galactosidase activity. Plasmid DNA was prepared from an overnight culture and verified by automated sequencing (ABI Prism 377, Perkin Elmer, Foster City, CA). Polyclonal antiserum was raised in chickens against a 13 amino acid, high performance liquid chromatography analyzed, lyophilized peptide. The translated protein sequence from the N-terminal region of the human MC5-R protein ''N''–-LSG/PNV/KNK/SSP/C–-''C'' was sent to Genemed Biotechnologies, South San Francisco, CA, for peptide synthesis. The peptide preparation (molecular weight 1331) was coupled to keyhole limpet hemocyanin using Imject Activated Immunogen Conjugation Kit (Pierce, Rockford, IL) and subsequently sent to Cocalico Biologicals (Reamston, PA) for antiserum production. The chicken was immunized on days 21, 42, 63, and 84. Test bleeds were collected on days 31, 52, 73, and 94 from the time of initial inoculation. The test bleed collected on day 52 was chosen for the characterization of the antibody based on enzyme-linked immunosorbent assay (ELISA). Yolk immunoglobulin (IgY) was also obtained from Cocalico Biologicals. The IgY was affinity purified against the peptide using a sulfolink kit (Pierce) by immobilizing the peptide to sulfolink coupling gel (Pierce). Antibody specificity (IgY) was confirmed by Western blotting using antibody that was adsorbed to peptide as follows. A 1:10 dilution of IgY was incubated with 15 μg per ml of peptide for 2 h at room temperature prior to hybridization to the membrane containing rat preputial gland proteins. Rat preputial glands were digested overnight at 4°C in dispase/fetal bovine serum followed by trypsin (0.25%) digestion at 37°C, shaking for 40 min (Laurent et al., 1992Laurent S. Mednieks M. Rosenfield R. Growth of sebaceous cells in monolayer culture in vitro.Cell Dev Biol. 1992; 28A: 83-89Crossref Scopus (22) Google Scholar). Cells were suspended in Dulbecco's modified Eagle's medium/Ham's F-12 3:1, fetal bovine serum 5%, adenine 1.8 × 10−4 M, hydrocortisone 0.4 μg per ml, insulin 5 μg per ml, epidermal growth factor 10 ng per ml, cholera toxin 1.2 × 10−10 M, antibiotics (100×) containing keratinocyte growth factor 10 ng per ml, and plated with mitomycin C (6 × 105) inactivated 3T3 fibroblasts. The primary culture was subcultured using trypsin 0.05%/0.02% EDTA to chamber slide system (Nalge Nunc International, Naperville, IL). About 20 human SGs per sample were digested with 0.125% trypsin, 0.01% EDTA at 37°C for 30 min. The trypsin/EDTA mixture was removed and plated onto a 60 mm tissue culture plate in the above medium with mitomycin C inactivated 3T3 fibroblasts. The remaining SGs were digested three more times and each harvest was plated on separate tissue culture plates. Primary cultures were subcultured using trypsin 0.05%/0.02% EDTA to chamber slide system in the same medium. Both secondary rat preputial and human sebocytes on chamber slides were fixed in 100% ethanol. Cryostat sections of rat skin and human facial skin were fixed in cold acetone (20 min). Immunoperoxidase staining was carried out at room temperature using the avidin-biotin complex method (ABC kit, Vector Laboratories, Burlingame, CA). The slides were first washed in phosphate-buffered saline (PBS) (5 min), followed by normal goat serum (20 min) and the affinity purified polyclonal antibody (IgY) at a 1:25 dilution in PBS (30 min) and PBS wash (2×). Subsequent reaction steps were performed using biotinylated goat antichicken IgG (8 μg per ml) (Vector Laboratories). Sigma Fast 3,3′-diaminobenzidine tetrahydrochloride with metal enhancer tablet sets (Sigma, St. Louis, MO) were used as precipitating substrate for the localization of peroxidase activity, which was visualized as a distinctive intense, dark blue to bluish-black reaction product. Negative controls consisted of skin sections incubated with nonimmune serum in place of the primary antibody. Ten percent acrylamide gels were prepared and run according to the method of Laemmli (Laemmli, 1970Laemmli U. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202762) Google Scholar). Samples were heated (2 min, 90°C) in a solution (1:4 vol/vol) containing glycerol (10% vol/vol), 2-mercaptoethanol (5% vol/vol), and SDS (3% wt/vol) in Tris buffer with bromophenol blue as tracking dye and electrophoresed using mighty small II mini gel system (Amersham Pharmacia Biotech, Piscataway, NJ). A hundred micrograms each of rat preputial gland extract and human SG extract were loaded per well. Prestained electrophoresis standard (Diversified Biotech, Boston, MA) was run along with the extracts. The identification of the apparent molecular weight of the proteins on SDS-PAGE to which the antibodies bound was carried out by protein blot analysis. The separated protein on the 10% SDS-PAGE was transferred to nitrocellulose. The blotted membrane was blocked in Tween-20/Tris 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. 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 (Figure 1): lane 1, 500 bp band in a sample of the kit control RNA; lane 2, a 250 bp band corresponding to the β-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 the MC3-R (178 bp) were detected (Figure 1, lanes 3–5 and 7, respectively). No bands were noted in lane 8 where PCR products from the negative control reaction were run. Each of the bands noted for the MC4-R and MC5-R genes were cut from the gel. DNA was isolated, cloned into a pCR2.1 vector (Invitrogen, Carson City, CA) and sequenced, which revealed homology of only the 460 bp fragment with the target MC5-R sequence. Although the band obtained for the MC4-R PCR product was of the appropriate size, its sequence consisted of repeats of the primer sequences and was not homologous to the target sequence for the MC4-R. Specificity of the affinity purified IgY was verified in blocking experiments using a rat preputial gland extract. A 43 kDa band corresponding to the MC5-R antibody was noted using the IgY (Figure 2, lane A). When the antibody in the IgY was blocked with MC5-R peptide, the 43 kDa band was diminished (Figure 2, lane B), thus confirming specificity of the IgY used in immunohistochemistry and Western blot. The MC5-R antibody localized to the epidermis, hair follicle, SG and eccrine gland (Figure 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 (Figure 4).Figure 4MC5-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 affinity-purified 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: 75 μm [scale bar in part (B) refers to parts (A), (B), and (C)].View Large Image Figure ViewerDownload (PPT) A 43 kDa band corresponding to the MC5-R peptide was noted in extracts of both human SGs and rat preputial glands (Figure 5). Apart from isotretinoin, there are no effective agents that significantly reduce the sebum production associated with acne. Advances in this area have been hampered by a lack of understanding of the mechanisms involved in the regulation of human SGs. Androgens stimulate sebum production in humans, yet in the majority of acne patients, serum levels of androgens are within the normal range. This clinical observation suggests that additional factors may regulate sebum production either directly or indirectly by mediating the local effects of androgens on the SG. In rodents, the SG response to androgens is augmented by α-MSH, a melanocortin peptide that has been referred to as a ''sebotrophic factor'' (Ebling et al., 1969Ebling F. Ebling E. Skinner J. The influence of pituitary hormones on the response of the sebaceous glands of the male rat to testosterone.J Endocrinol. 1969; 45: 245-256Crossref PubMed Scopus (39) Google Scholar;Ebling et al., 1975Ebling F. Ebling E. Randall V. Skinner J. The synergistic action of α-melanocyte-stimulating hormone and testosterone on the sebaceous, prostate, preputial, harderian and lachrymal glands, seminal vesicles and brown adipose tissue in the hypophysectomized-castrated rat.J Endocrinol. 1975; 66: 407-412Crossref PubMed Scopus (36) Google Scholar;Thody and Shuster, 1975Thody A.J. Shuster S. Control of sebaceous gland function in the rat by alpha-melanocyte-stimulating hormone.J Endocrinol. 1975; 64: 503-510Crossref PubMed Scopus (61) Google Scholar;Cooper et al., 1976Cooper M.F. Bowden P.W. Meddis D. Thody A.J. Shuster S. Effects of testosterone and alpha-melanocyte-stimulating hormone on preputial-gland (sebaceous) activity.Biochem Soc Transactions. 1976; 4: 798-800Crossref PubMed Scopus (4) Google Scholar;Thody et al., 1976Thody A.J. Cooper M.F. Bowden P.E. Meddis D. Shuster S. Effect of alpha-melanocyte-stimulating hormone and testosterone on cutaneous and modified sebaceous glands in the rat.J Endocrinol. 1976; 71: 279-288Crossref PubMed Scopus (47) Google Scholar). More recently, the importance of melanocortins in regulating sebum production in rodents has been confirmed in studies of transgenic mice lacking the MC5-R (Chen et al., 1997Chen W. Kelly M. Opitz-Araya X. Thomas R. Low M. Cone R. Exocrine gland dysfunction in MC5-R deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides.Cell. 1997; 91: 789-798Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). These mice had hypoplastic SGs and preputial glands and reduced levels of sebum and preputial gland secretion. Whether α-MSH or melanocortin receptors play a role in locally modulating the action of androgen hormones on human SGs is worthy of investigation. The family of melanocortin peptides include α-MSH, ACTH, β-lipotrophin, and endorphin. These peptides are derived from the tissue-specific cleavage of POMC that is secreted primarily by the pituitary gland. In rodents, α-MSH circulates in higher levels and is thought to play a key role in pigmentation, feeding behavior, lipid metabolism, cardiovascular tone, and immune functions (Wintzen and Gilchrest, 1996Wintzen M. Gilchrest B. Prioopiomelanocortin, its derived peptides, and the skin.J Invest Dermatol. 1996; 106: 3-10Crossref PubMed Scopus (123) Google Scholar;Chen et al., 1997Chen W. Kelly M. Opitz-Araya X. Thomas R. Low M. Cone R. Exocrine gland dysfunction in MC5-R deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides.Cell. 1997; 91: 789-798Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar;Huszar et al., 1997Huszar D. Lynch C. Fairchild-Huntress V. et al.Targeted disruption of the melanocortin-4 receptor results in obesity in mice.Cell. 1997; 88: 131-141Abstract Full Text Full Text PDF PubMed Scopus (2444) Google Scholar;Luger et al., 1997Luger T. Scholzen T. Grabbe S. The role of α-melanocyte-stimulating hormone in cutaneous biology.J Invest Dermatol Symp Proc The. 1997; 2: 87-93Abstract Full Text PDF PubMed Scopus (127) Google Scholar;van der Kraan et al., 1998van der Kraan M. Adan R. Entwistle M. Gispen W. Burbach P. Tatro J. Expression of melanocortin-5 receptor in secretory epithelia supports a functional role in exocrine and endocrine glands.Endocrinol. 1998; 139: 2348-2355Crossref PubMed Scopus (94) Google Scholar). Because circulating levels of α-MSH are low in humans, little emphasis has been placed on its potential role in mediating similar functions in humans. The local production and metabolism of hormones within endocrine tissues such as the skin, however, has recently been recognized as an important principle in the cell-type- and tissue-specific regulation of hormone action (Labrie, 1991). This principle is particularly applicable to the skin where a variety of steroid hormone metabolizing enzymes are active. Despite low circulating levels of MSH in humans, the local production of melanocortin peptides may be important in regulating processes in skin (Schauer et al., 1994Schauer E. Trautinger F. Kock A. Bhardwaj R. Ansei J. Schwarz T. Luger T. Proopiomelanocortin-derived peptides are syntheaized and released bu human keratinocytes.J Clin Invest. 1994; 93: 2258-2262Crossref PubMed Scopus (310) Google Scholar;Wintzen and Gilchrest, 19
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