Portal de Periódicos da CAPES

Collagen Metabolism Is a Novel Target of the Neuropeptide α-Melanocyte-stimulating Hormone

2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês

10.1074/jbc.m312549200

1083-351X

Markus Böhm, Michael Raghunath, Cord Sunderkötter, Meinhard Schiller, Sonja Ständer, Thomas Brzoska, Thomas Cauvet, Helgi B. Schiöth, Thomas Schwarz, Thomas A. Luger,

Dermatologic Treatments and Research

Suppression of collagen synthesis is a major therapeutic goal in the treatment of fibrotic disorders. We show here that α-melanocyte-stimulating hormone (α-MSH), a neuropeptide well known for its pigment-inducing capacity, modulates collagen synthesis and deposition. α-MSH in vitro suppresses the synthesis of collagen types I, III, and V and down-regulates the secretion of procollagen type I C-terminal peptide (PICP) in human dermal fibroblasts treated with the fibrogenic cytokine transforming growth factor-β1 (TGF-β1). α-MSH did not interfere with TGF-β1 signaling, because TGF-β1-induced expression of collagen mRNA was not affected, implying a posttranscriptional mechanism. Human dermal fibroblasts in vitro express a high affinity binding site for MSH, which was identified by reverse transcription PCR and immunofluorescence analysis as the melanocortin-1 receptor (MC-1R). Immunohistochemical studies on normal adult human skin confirmed MC-1R expression in distinct dermal fibroblastic cells. The MC-1R on fibroblasts appears to be functionally relevant because α-MSH increased the amount of intracellular cAMP, and coincubation with a synthetic peptide corresponding to the human Agouti signaling protein abrogated the inhibition of TGF-β1-induced PICP secretion by α-MSH. To assess the in vivo relevance of these findings, a mouse model was used in which dermal fibrosis was induced by repetitive intracutaneous injections with TGF-β1. The inductive activity of TGF-β1 on collagen deposition and the number of dermal cells immunoreactive for vimentin and α-smooth muscle actin was significantly suppressed by injection of α-MSH. Melanocortins such as α-MSH may therefore represent a novel class of modulators with potential usefulness for the treatment of fibrotic disorders. Suppression of collagen synthesis is a major therapeutic goal in the treatment of fibrotic disorders. We show here that α-melanocyte-stimulating hormone (α-MSH), a neuropeptide well known for its pigment-inducing capacity, modulates collagen synthesis and deposition. α-MSH in vitro suppresses the synthesis of collagen types I, III, and V and down-regulates the secretion of procollagen type I C-terminal peptide (PICP) in human dermal fibroblasts treated with the fibrogenic cytokine transforming growth factor-β1 (TGF-β1). α-MSH did not interfere with TGF-β1 signaling, because TGF-β1-induced expression of collagen mRNA was not affected, implying a posttranscriptional mechanism. Human dermal fibroblasts in vitro express a high affinity binding site for MSH, which was identified by reverse transcription PCR and immunofluorescence analysis as the melanocortin-1 receptor (MC-1R). Immunohistochemical studies on normal adult human skin confirmed MC-1R expression in distinct dermal fibroblastic cells. The MC-1R on fibroblasts appears to be functionally relevant because α-MSH increased the amount of intracellular cAMP, and coincubation with a synthetic peptide corresponding to the human Agouti signaling protein abrogated the inhibition of TGF-β1-induced PICP secretion by α-MSH. To assess the in vivo relevance of these findings, a mouse model was used in which dermal fibrosis was induced by repetitive intracutaneous injections with TGF-β1. The inductive activity of TGF-β1 on collagen deposition and the number of dermal cells immunoreactive for vimentin and α-smooth muscle actin was significantly suppressed by injection of α-MSH. Melanocortins such as α-MSH may therefore represent a novel class of modulators with potential usefulness for the treatment of fibrotic disorders. Fibrotic and sclerotic diseases comprise a large and heterogeneous group of inflammatory, idiopathic, toxic, hereditary, and pharmacologically induced disorders such as hypertrophic scars, keloids, localized scleroderma, systemic sclerosis, sclerodermic graft versus host disease of the skin, cirrhosis of the liver, idiopathic and bleomycin-induced lung fibrosis, or cyclosporine-induced nephropathy. The therapeutic options are limited, and treatment of these disabling disorders is still a challenge. A key feature of fibrotic disorders is excessive production of extracellular matrix, mainly type I collagen, followed by a gradual loss of organ function which, in some cases, can be fatal. In recent years it became apparent that transforming growth factor-β1 (TGF-β1), 1The abbreviations used are: TGF-β1, transforming growth factor-β1; α-MSH, α-melanocyte-stimulating hormone; α-SMA, α-smooth muscle actin; ASIP, Agouti signaling protein; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HDF, human dermal fibroblasts; MC-R, melanocortin receptor; MC-1R, melanocortin-1 receptor; NDP-MSH, [Nle4,d-Phe7]α-MSH; PBS, phosphate buffered saline; PICP, procollagen I C-terminal peptide; POMC, pro-opiomelanocortin; RT, reverse transcription. a multifunctional cytokine, is crucially involved in the pathogenesis of fibrotic disorders (1Kawakami T. Ihn H. Xu W. Smith E. LeRoy C. Trojanowska M. J. Investig. Dermatol. 1998; 110: 47-51Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 2Tuan T.L. Nichter L.S. Mol. Med. Today. 1998; 4: 19-24Abstract Full Text PDF PubMed Scopus (374) Google Scholar, 3Khalil N. Greenberg A.H. CIBA Found. Symp. 1991; 157: 194-207PubMed Google Scholar, 4Shihab F.S. Semin. Nephrol. 1996; 16: 536-547PubMed Google Scholar, 5Czaja M.J. Weiner F.R. Flanders K.C. Giambrone M.A. Wind R. Biempica L. Zern M.A J. Cell Biol. 1989; 108: 2477-2482Crossref PubMed Scopus (383) Google Scholar). It induces fibrosis by various ways (reviewed in Ref. 6Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3004) Google Scholar). It enhances the expression of several collagens including types I, III, and V. TGF-β1 decreases the production of matrix-degrading proteases and enhances the synthesis of inhibitors of such proteases. TGF-β1 also increases extracellular cross-linking of collagen by enhancing the expression and the activity of lysyl oxidase (7Feres-Filho E.J. Choi Y.J. Han X. Takala T.E. Trackman P.C. J. Biol. Chem. 1995; 270: 30797-30803Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These multiple activities explain the potent fibrotic effect of TGF-β1. Therefore, strategies aimed at antagonizing the strong profibrotic effect of TGF-β1 are regarded as providing a promising approach to preventing excessive collagen accumulation in fibrotic disorders (8McCormick L.L. Zhang Y. Tootell E. Gilliam A.C. J. Immunol. 1999; 163: 5693-5699PubMed Google Scholar, 9Yamamoto T. Takagawa S. Katayama I. Nishioka K. Clin. Immunol. 1999; 92: 6-13Crossref PubMed Scopus (114) Google Scholar, 10Hill C. Flyvbjerg A. Rasch R. Bak M. Logan A. J. Endocrinol. 2001; 170: 647-651Crossref PubMed Scopus (83) Google Scholar). α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide generated from pro-opiomelanocortin (POMC) by proteolytic cleavage (reviewed in Ref. 11Slominski A. Wortsman J. Luger T.A. Paus R. Solomon S. Physiol. Rev. 2000; 80: 979-1020Crossref PubMed Scopus (636) Google Scholar). It was originally isolated from the pituitary gland and characterized as a pigment-inducing factor regulating the coat color of many vertebrate species, but it turned out to regulate many other biological activities with regard to the skin (reviewed in Refs. 11Slominski A. Wortsman J. Luger T.A. Paus R. Solomon S. Physiol. Rev. 2000; 80: 979-1020Crossref PubMed Scopus (636) Google Scholar and 12Böhm M. Luger T.A. Horm. Res. 2000; 54: 287-293Crossref PubMed Scopus (30) Google Scholar). The biological activities of α-MSH are mediated by a family of structurally related receptors that are known as the melanocortin receptors (MC-Rs). They belong to the superfamily of G protein-coupled receptors with seven trans-membrane domains, and they activate adenylate cyclase after ligand binding. Five MC-R subtypes have been cloned that differ in their relative affinities to α-MSH and the other melanocortins (13Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. Science. 1993; 257: 1248-1251Crossref Scopus (1458) Google Scholar, 14Cone R.D. Lu D. Koppula S. Vage D.I. Klungland H. Boston B. Chen W. Orth D.N. Pouton C. Kesterson R.A. Recent Prog. Horm. Res. 1996; 51: 287-318PubMed Google Scholar). Here we show that, in addition to its multiple biological effects, α-MSH suppresses TGF-β1-induced collagen synthesis by human dermal fibroblasts (HDF) in vitro. This effect is mediated via the MC-1R. α-MSH also exerts its anti-fibrogenic activity in vivo, because injection of α-MSH into mice reduces TGF-β1-induced fibrosis. Our data establish a role for melanocortins in fibroblast biology and point toward a therapeutic potential of α-MSH and its analogues in the treatment of fibrotic and sclerotic diseases. Cells and Culture Conditions—HDF from neonatal foreskin and adult skin as well as normal human melanocytes were purchased from Cell Systems, St. Katharinen, Germany. The human fibrosarcoma cell line HT-1080 was obtained from the American Type Culture Collection (ATCC). Fibroblasts were routinely cultured in RPMI 1640 (PAA, Cölbe, Germany), 1% glutamine, 1% penicillin/streptomycin (both from PAA), and 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) in a humidified atmosphere of 5% CO2 at 37 °C. Normal human melanocytes were cultured in MBM2 medium plus MGM-3 aliquots as indicated by the manufacturer (Clonetics, Walkersville, MD). RNA Extraction, RT-PCR, and Sequencing—Total RNA was isolated from cells using a commercial purification kit (Promega, Madison, WI). After DNA digestion, 1 μg of total RNA was reverse transcribed with 15 units of avian myeloblastosis virus reverse transcriptase (Promega). The resulting cDNA was amplified with 2.5 units of Taq polymerase (Promega) and MC-R primers under conditions identical to those described previously (15Hartmeyer M. Scholzen T. Becher E. Bhardwaj R.S. Schwarz T. Luger T.A. J. Immunol. 1997; 159: 1930-1937PubMed Google Scholar, 16Bhardwaj R. Becher E. Mahnke K. Hartmeyer M. Schwarz T. Scholzen T. Luger T.A. J. Immunol. 1997; 158: 3378-3384PubMed Google Scholar, 17Böhm M. Schiller M. Li Z. Ständer S. Metze D. Schiöth H.B. Skottner A. Seiffert K. Zouboulis C.C. Luger T.A. J. Investig. Dermatol. 2002; 118: 533-539Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Primer sequences and the sizes of their amplification products are given in Table I. Only RNA samples that did not yield amplification products were reverse transcribed. For some positive controls, genomic DNA from HDF was prepared by routine protocols. Amplicons were separated in 1.5% agarose gels. The resulting MC-1R-related band in HDF was purified using a gel extraction kit (Qiagen, Santa Clarita, CA), cloned into pGEM-T easy vectors (Promega), and sequenced (4base lab GmbH, Reutlingen, Germany).Table IPrimer sets used for RT-PCR analysis of MC-RsGene accession numberGene productPrimer (F, forward; B, backward)aSequences are as deposited in the National Center for Biotechnology Information (NCBI) data base under the given accession number.SizebSize denotes the number of base pairs of the amplicon.ReferenceMC1R/AF326275MC-1RF: 5′-GCCACCATCGCCAAGAACC-3′B: 5′-ATAGCCAGGAAGAAGACCA-3′41615, 17MC2/Z25470cmRNA sequence for MC-2R is a co-linear but truncated form of the MC-5R.MC-2RF: 5′-CTGCATTTCTTGGATCT-3′B: 5′-AAGCTGCACATGGATGC-3′38015, 17MC3/NM_019888MC-3RF: 5′-CGGTGGCCGACATGCTGGTAAGTG-3′B: 5′-TGAGGAGCATCATGGCGAAGAACA-3′36615, 17MC4/NM_005912MC-4RF: 5′-CAATAGCCAAGAACAAGAATC-3′B: 5′-GACAACAAAGACGCCAATCAG-3′56615, 17MC5/NM_005913cmRNA sequence for MC-2R is a co-linear but truncated form of the MC-5R.MC-5RF: 5′-CATTGCTGTGGAGGTGTTTCT-3′B: 5′-GCCGTCATGATGTGGTGGTAG-3′35715-17a Sequences are as deposited in the National Center for Biotechnology Information (NCBI) data base under the given accession number.b Size denotes the number of base pairs of the amplicon.c mRNA sequence for MC-2R is a co-linear but truncated form of the MC-5R. Open table in a new tab Quantitative Real Time PCR—Quantification of mRNA levels of the various procollagen chains was carried out by real time fluorescence detection as described previously (18Müller C. Readhead C. Diederichs S. Idos G. Yang R. Tidow N. Serve H. Berdel W.E. Koeffler H.P. Mol. Cell. Biol. 2000; 20: 3316-3329Crossref PubMed Scopus (72) Google Scholar). cDNA was prepared amplified by PCR in the ABI Prism 7700 sequence detector (PE Biosystems, Foster City, CA). Primer and probe sequences were designed by the Primer Express software (PE Biosystems) or supplied by PE Biosystems (glyceraldehyde-3-phosphate dehydrogenase) (18Müller C. Readhead C. Diederichs S. Idos G. Yang R. Tidow N. Serve H. Berdel W.E. Koeffler H.P. Mol. Cell. Biol. 2000; 20: 3316-3329Crossref PubMed Scopus (72) Google Scholar). The primers used were as follows: COL(I)α1 sense, 5′-CAGCCGCTTCACCTACAGC-3′, COL(I)α1 antisense, 5′-AATCACTGTCTTGCCCCAGG-3′, and COL(I)α1 probe, 5′-ACTGTCGATGGCTGCACGAGTCACAC-3′; COL(I)α 2 sense, 5′-GATTGAGACCCTTCTTACTCCTGAA-3′, COL(I)α 2 antisense, 5′-GGGTGGCTGAGTCTCAAGTCA-3′, and COL(I)α 2 probe, 5′-TCTAGAAAGAACCCAGCTCGCACATGC-3′; and COL(III)α 1 sense, 5′-TCCAACTGCTCCTACTCGCC-3′, COL(III)α 1 antisense, 5′-GAGGGCCTGGATCTCCCTT-3′, and COL(III)α 1probe 5′-CCTAATGGTCAAGGACCTCAAGGCCC-3′. Probes were labeled at the 5′-end with the reporter dyes 6-carboxyfluorescein or VIC and at the 3′-end with the quencher dye 6-carboxy-tetramethyl-rhodamine. The 5′-nuclease activity of the Taq polymerase (Applied Biosystems) cleaved the probe and released the fluorescent dyes, which were detected by the laser detector of the sequence detector. After the detection threshold was reached, the fluorescence signal was proportional to the amount of PCR product generated. The initial template concentration could be calculated from the cycle number when the amount of PCR product passed a threshold set in the exponential phase of the PCR. Relative gene expression levels were calculated using standard curves generated by serial dilutions of cDNA from HT1080 cells. The relative amounts of gene expression were calculated by using the expression of glyceraldehyde-3-phosphate dehydrogenase as an internal standard. Expression of each gene was assessed by three independent PCR analyses and calculation of the mean ± S.E. Data were analyzed by the Student's t test. Binding Studies—[Nle4,d-Phe7]α-MSH (NDP-MSH) (Bachem, Bubendorf, Switzerland) was radioiodinated by the chloramine T method and purified by high pressure liquid chromatography. Binding studies were performed as described previously (19Schiöth H.B. Muceniece R. Wikberg J.E.S. Chhajlani V. Eur. J. Pharmacol. 1995; 288: 311-317Crossref PubMed Scopus (116) Google Scholar). In short, cells were washed with binding buffer and distributed into 96-well plates. Cells were then incubated for 2 h at 37 °C with 50 μl of binding buffer in each well containing a constant concentration of 0.2 nm125I-NDP-MSH and appropriate concentrations of unlabeled ligand. After incubation, the cells were washed with 0.2 ml of ice-cold binding buffer and detached with 0.2 ml of 0.1 n NaOH, and the radioactivity was counted in each well. The binding assays were performed in duplicate wells. Radioactivity was determined by a gamma counter (Wallac, Wizard Automatic), and data were analyzed with a software package for radioligand binding analyses. Data were analyzed by fitting it to formulas derived from the law of mass action by the method generally referred to as computer modeling. Immunofluorescence—HDF were seeded into chamber slides and fixed with methanol for 30 min at -20 °C or, for surface staining, with 4% paraformaldehyde for 30 min at room temperature. Nonspecific binding was blocked with 5% goat/donkey serum for 1 h at room temperature. Cells were then incubated for 1 h with a rabbit polyclonal antibody against the human MC-1R (1 μg/ml). Production and characterization of the anti-human MC-1R is described in detail elsewhere (20Böhm M. Brzoska T. Schulte U. Schiller M. Kubitscheck U. Luger T.A. Ann. N. Y. Acad. Sci. 1999; 885: 372-382Crossref PubMed Scopus (19) Google Scholar, 21Böhm M. Metze D. Schulte U. Becher E. Luger T.A. Brzoska T. Exp. Dermatol. 1999; 8: 453-461Crossref PubMed Scopus (55) Google Scholar). In some experiments, double staining with a monoclonal antibody against protein disulfide isomerase (1:100; Dako, Hamburg, Germany), a cytoplasmic marker (22Vaux D. Tooze J. Fuller S. Nature. 1990; 345: 495-502Crossref PubMed Scopus (172) Google Scholar, 23Munro S. Pelham H.R. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1058) Google Scholar), was performed. Bound antibodies were visualized with a donkey anti-rabbit antibody coupled to Texas Red (1:100; Dianova, Hamburg, Germany) and a goat anti-mouse antibody coupled to fluorescein isothiocyanate (1:100; Dako). After mounting, specimens were examined with a confocal laser-scanning microscope (TCS E, Leica, Heidelberg, Germany). Determination of cAMP—For intracellular cAMP measurements, 2 × 104 HDF were seeded into 96-well tissue culture plates. The next day, the routine culture medium was changed to RPMI 1640 containing 1% FCS. Cells were cultured for additional 24 h followed by stimulation with α-MSH as indicated for 20 min in the presence of 0.1 mm isobutyl methylxanthine. 0.1-5 μm forskolin was used as a positive control. After incubation, supernatants were removed, and cells were lysed. cAMP levels in the lysates were determined by a specific enzyme immunoassay according to the instructions of the manufacturer (Amersham Biosciences). Triplicate wells were used for each individual treatment, and statistical analysis was performed using the Student's t test. Collagen Analysis—HDF were seeded into 6-well tissue culture plates (250,000 per well) and allowed to attach and grow for 16 h. Subconfluent cell monolayers were then incubated with minimal essential medium containing 0.5% FCS and 50 μg/ml l-ascorbic acid for 24 h in the presence of TGF-β1 (10 ng/ml), α-MSH (10-6m), or a combination of both substances. HDF were rinsed and depleted for 1 h in methionine/cysteine-free minimal essential medium (ICN, Costa Mesa, CA). Cells were labeled with [35S]methionine/cysteine mix (50 μCi/ml) for 16 h in the presence of ascorbate and the above agents. Media were collected, cell cultures rinsed three times with ice-cold Tris-buffered saline, and cells were scraped into 1% Nonidet P-40 in Tris-buffered saline with a rubber policeman. Cell lysates were centrifuged, and aliquots of the supernatant were subjected to liquid scintillation counting to determine cell mass. Media and combined cell lysates/scrapings were treated with pepsin to destroy non-collagenous proteins (24Raghunath M. Bruckner P. Steinmann B. J. Mol. Biol. 1994; 236: 940-949Crossref PubMed Scopus (116) Google Scholar). Proteins in either fraction were precipitated using methanol/chloroform and processed for SDS-PAGE (5% acrylamide; acryl/bisacryl, 37.5:1). All loaded aliquots were calibrated for cell mass so that all slots contained pepsin-treated collagen derived from the same amount of cells. Slab gels were fixed, and radiolabeled collagens were detected by autoradiography (25Raghunath M. Steinmann B. Delozier-Blanchet C. Extermann P. Superti-Furga A. Pediatr. Res. 1994; 36: 441-448Crossref PubMed Scopus (47) Google Scholar). Representative gels were subjected to densitometry using the Biostep Phoretix Grabber (Biostep, Jahnsdorf, Germany) Determination of Procollagen I C-terminal Peptide—The amounts of procollagen I C-terminal peptide used as a marker for procollagen I secretion were determined using a commercially available ELISA (TaKaRa, Shiga, Japan). HDF were seeded into 12-well tissue culture plates at a density of 250,000 cells per well. Confluent HDFs were then deprived of FCS for 2 days and subsequently stimulated with α-MSH (10-6-10-10m), TGF-β1 (10 ng/ml), or both agents in the presence of 50 μg/ml ascorbate. In some experiments, cells were coincubated with a synthetic peptide corresponding to the amino acids 87-132 of the human Agouti signaling peptide (Phoenix Pharmaceuticals, Belmont, CA) at a 10-fold molar excess. Culture supernatant were harvested after 48 h, centrifuged, and frozen at -70 °C until use. Statistical evaluation from triplicate wells was performed using the Student's t test. Mouse Model for Cutaneous Fibrosis—For in vivo evaluation of the anti-fibrogenic effect of α-MSH, a mouse model described previously by Shinozaki et al. (26Shinozaki M. Kawara S. Hayashi N. Kakinuma T. Igarashi A. Takehara K. Biochem. Biophys. Res. Commun. 1997; 240: 292-297Crossref PubMed Scopus (86) Google Scholar) was used with slight modifications. Accordingly, cutaneous fibrosis was induced by intracutaneous injections of 800 ng of TGF-β1 into the neck of newborn Balb/c mice on three consecutive days. Treatment groups (four groups of three mice each) consisted of mice injected with TGF-β1, α-MSH (25 μg), TGF-β1 plus α-MSH, and the solvent (0.1% BSA in PBS) in which TGF-β1 had been solubilized. On day 4, mice were sacrificed, and 4-mm punch biopsies were taken from the sites of injection for immunohistochemical analysis. Immunohistochemistry—After fixation in 4% paraformaldehyde and embedment in paraffin, biopsies from mouse skin were processed with the following stains: (i) hematoxylin and eosin; (ii) van Gieson stain, in which collagen appears red; and (iii) resorcin-fuchsin stain, according to Weigert, in which elastic tissue appears black. For collagen staining, the sections were treated with 1 mg/ml pepsin (Sigma) in 0.5 m acetic acid, washed, and incubated with a rabbit antibody against collagen type I (1:100; DPC Biermann, Bad Nauheim, Germany) for 1 h. For the staining of vimentin, sections were microwave-treated to unmask epitopes, followed by incubation with a polyclonal antibody from Abcam (Cambridge, UK) for 30 min at 37 °C. For the staining of α-smooth muscle actin, a monoclonal antibody from Dunn Labortechnik (Asbach, Germany) was incubated for 1 h at 2 μg/ml without prior unmasking. Immunohistochemistry for MC-1R in sections of normal adult human skin (n > 5) was performed exactly as outlined previously (17Böhm M. Schiller M. Li Z. Ständer S. Metze D. Schiöth H.B. Skottner A. Seiffert K. Zouboulis C.C. Luger T.A. J. Investig. Dermatol. 2002; 118: 533-539Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 21Böhm M. Metze D. Schulte U. Becher E. Luger T.A. Brzoska T. Exp. Dermatol. 1999; 8: 453-461Crossref PubMed Scopus (55) Google Scholar). Sections were developed by the indirect immunoperoxidase technique using 3-amino-9-ethylcarbazole (Sigma) as a chromogen. Negative controls included incubation with control IgG at the same protein concentration as the primary antibody, omission of the first antibody, or pre-incubation with the immunogenic peptide in 10-fold weight excess in the case of MC-1R immunostaining. Vimentin immunostaining and α-smooth muscle actin immunostaining in sections of mouse skin were quantitatively assessed by counting the number of immunoreactive interfollicular dermal cells in three high power fields (×400). Means ± S.D. from 3-4 independent experiments were analyzed by analysis of variance. α-MSH Modulates Collagen Expression by HDF in Vitro—We addressed the question of whether α-MSH can modulate the key function of HDF, namely the expression and secretion of collagen. To this end, cultured, normal HDF from neonatal foreskin were treated with α-MSH, TGF-β1, or both substances. The amounts of collagen present in cell lysates and culture supernatants were separately determined after metabolic labeling, pepsin digestion, and SDS-PAGE. TGF-β1, a well known inducer of collagen synthesis (6Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3004) Google Scholar), increased the amount of secreted collagens in the culture medium (Fig. 1A). α-MSH alone appeared to reduce the extracellular amount of collagens I, III, and V by 50-70% as determined by densitometry (Fig. 1A, and data not shown). This reduction was not due to intracellular retention, although there was some increase in α1(I) chains that we attribute to a higher synthesis rate. The intracellular bands showed no delayed migration and, thus, excluded significant posttranslational overmodification due to abnormal intra-endoplasmatic retention (Fig. 1A). Most strikingly, α-MSH dramatically reversed the stimulatory effects of TGF-β1 on the extracellular collagen presence with the strongest effects on collagens I and III and somewhat milder effects on collagen V (Fig. 1A). In the conspicuous absence of intracellular retention, these findings implicate either intracellular or extracellular proteolytic degradation or a combination of both. To further substantiate the activity of α-MSH on collagen synthesis and/or secretion, we determined the amount of procollagen I C-terminal peptide (PICP) in the culture media of HDF stimulated with α-MSH, TGF-β1, or both agents. The addition of TGF-β1 led to a dramatic increase of PICP in the culture medium by >500%. In accordance with the modulatory effect of α-MSH on the TGF-β1-induced collagen biosynthesis and subsequent secretion, we found significantly reduced secreted amounts of PICP by HDF (677.9 ± 60.1 pg/ml versus 1313.9 ± 136 pg/ml; p < 0.005) (Fig. 1B). α-MSH alone, in contrast, did not affect the basal amounts of secreted PICP. These findings suggested either an intracellular or an extracellular cause for the reduction of secreted procollagen I. Modulation of Collagen Synthesis by α-MSH Is Not Mediated by Reduced mRNA Expression—We next wondered if the modulatory activity of α-MSH on collagen synthesis is regulated at the transcriptional level. HDF from neonatal foreskin were stimulated with α-MSH, TGF-β1, or both agents for 12 h. The relative mRNA levels for the α1(I) and α2(I) chains of collagen I (alleles COL1A1 and COL1A2, respectively) and for the α1(III) chains for collagen III (allele COL3A1) were subsequently determined by quantitative real-time PCR. TGF-β1 significantly increased the mRNA levels of collagen type I α1 and α2 as well as that of collagen type III α1 as compared with non-treated cells (Table II). The observed rate of increase in the amount of these collagens by TGF-β1 was in accordance with earlier reports (27Raghow R. Postlethwaite A.E. Keski-Oja J. Moses H.L. Kang A.H. J. Clin. Investig. 1987; 79: 1285-1288Crossref PubMed Scopus (368) Google Scholar). Despite some variation, neither α-MSH alone nor coincubation of α-MSH and TGF-β1 caused a significant reduction in the relative levels of the collagen mRNAs (Table II). Similar results were obtained when HDFs were treated with TGF-β1 and α-MSH for 24 h (data not shown). These findings show that α-MSH does not interfere with TGF-β1 signaling and that α-MSH may affect collagen expression at the posttranscriptional level.Table IIα MSH does not suppress collagen synthesis at the transcriptional level in HDFGene productRelative mRNA expressionaRelative mRNA levels were measured by real-time PCR and normalized for GAPDH. Data are means ± S.E. from three independent experiments.N/AMTT + MCol α1(I)22.2 ± 1.122.9 ± 2.755.6 ± 9bp < 0.05 versus untreated cells.55.5 ± 2.3Col α2(I)81.6 ± 9.695.2 ± 14.4149.2 ± 20.3bp < 0.05 versus untreated cells.135.2 ± 23.9Col α1(III)28.8 ± 2.828.6 ± 3.876.2 ± 14.8bp < 0.05 versus untreated cells.52.3 ± 3.8a Relative mRNA levels were measured by real-time PCR and normalized for GAPDH. Data are means ± S.E. from three independent experiments.b p < 0.05 versus untreated cells. Open table in a new tab Detection of High Affinity Binding Sites for MSH on HDF—The identified effects of α-MSH on the amount of extracellular collagen suggested the presence of specific binding sites in HDF. Therefore, we examined HDF from neonatal foreskin for competitive radioligand binding using an iodinated synthetic α-MSH analogue, NDP-MSH. Displacement was performed with an unlabeled ligand at varying concentrations, and COS-1 cells were used as a negative control. HDF exhibited a specific and saturable binding kinetic with 125I-NDP-MSH. The affinity of the radioligand was similar to COS-1 cells transfected with the human MC-1R (Fig. 2). The Ki values were 0.058 ± 0.012 nm for the HDF and 0.086 ± 0.033 nm for COS-1 cells transfected with the human MC-1R, the latter value being similar to previous studies (19Schiöth H.B. Muceniece R. Wikberg J.E.S. Chhajlani V. Eur. J. Pharmacol. 1995; 288: 311-317Crossref PubMed Scopus (116) Google Scholar). HDF, therefore, exhibited similar affinity but slightly lower expression levels of high affinity MSH binding sites than did COS-1 cells transfected with MC-1R. These data strongly suggested that α-MSH binds to specific surface receptors on the surface of HDF, which appear to mediate its biological action. Expression of MC-1R in HDF in Vitro and in Situ—To investigate in detail the expression of MC-Rs in HDF, we performed RT-PCR analysis using primers against all known MC-Rs (Table I). MC-1R was the only MC-R expressed in HDF derived from neonatal foreskin (Fig. 3A). Similarly, HDF derived from adult human skin expressed MC-1R at the RNA level (data not shown). The MC-1R amplification product of HDF comigrated exactly with that of normal human melanocytes used as a positive control (Fig. 3A). The identity of the amplification product in HDF (416 bp) was determined by DNA sequencing and found to be identical with the mRNA sequence of MC-1R as deposited in the National Center for Biotechnology Information (Table I, and data not shown). In contrast to MC-1R, no other MC-R was expressed in HDF as shown by RT-PCR (Fig. 3A). The amplification products of the positive controls were all of the expected size (Fig.